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Pathogenesis of neurofibromatosis 1 associated neurofibromas Tucker, Tracy 2006

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Pathogenesis of Neurofibromatosis 1 Associated Neurofibromas  by  Tracy Tucker B.Sc. Honours Science, Biology Option, University of Waterloo, Canada, 2000  A T H E S I S SUBMITTED IN PARTIAL F U L F I L M E N T O F THE REQUIREMENTSFOR THE D E G R E E OF  DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  T H E UNIVERSITY O F BRITISH C O L U M B I A December 2006 © Tracy Tucker, 2006  ABSTRACT Neurofibromatosis 1 (NF1) is an autosomal dominant disease. Neurofibromas, benign tumours that develop from peripheral nerves, are a hallmark feature of NF1. Malignant peripheral nerve sheath tumours (MPNSTs) are one of the leading causes of death in people with N F 1 . Clinical evidence suggests that most M P N S T s develop from preexisting plexiform neurofibromas. Most studies treat all NF1-associated neurofibromas as a single entity, ignoring important differences between pathological details, clinical presentation and natural history. I analysed clinical information on 476 probands with NF1 from the Henri Mondor database and found that individuals with subcutaneous neurofibromas were 3 times more likely to have internal plexiform neurofibromas and that individuals with internal plexiform neurofibromas were 20 times more likely to have M P N S T s than individuals without such tumours. These findings suggest that pathogenic differences in some neurofibromas may lead to different risks of progressing to malignancy. I collected formalin-fixed paraffin-embedded samples from NF1 patients and classified them histologically as nodular or diffuse neurofibromas. By using histochemistry, I found that mast cells were absent in M P N S T s and significantly more abundant in diffuse neurofibromas than in nodular neurofibromas. Mast cells were located at the periphery of nodular neurofibromas but were evenly distributed throughout diffuse neurofibromas. Double immunofluorescent staining of S100 (a marker of Schwann cells, the presumed tumour progenitor cell type) and neurofibromin (the protein product of NF1 gene) (Nf) showed that diffuse neurofibromas had significantly more S100+/Nf+ cells and fewer S100-/Nf- cells than nodular neurofibromas. Using laser microdissection of immunofluorescently stained slides, I found that some neurofibromas show evidence of clonal (presumably neoplastic) proliferation of S100+/Nf- cells while other neurofibromas appear to be neurofibromin haploinsufficient and polyclonal, and thus may be hyperplastic rather than neoplastic lesions. The results presented in this thesis support the hypothesis that neurofibromas in people with NF1 are pathogenically heterogeneous and that some kinds of neurofibromas are associated with the development of M P N S T s . These findings have important implications for the surveillance and treatment of people with N F 1 .  I  TABLE OF CONTENTS ABSTRACT  II  TABLE OF CONTENTS  Ill  LIST OF T A B L E S  VI  LIST O F F I G U R E S  VII  LIST O F A B B R E V I A T I O N S  VIII  ACKNOWLEDGEMENTS  IX  CO-AUTHORSHIP STATEMENT  X  1. INTRODUCTION  1  BACKGROUND ON NEUROFIBROMATOSIS  1  T Y P E S O F N E U R O F I B R O M A S IN NF1  3  T H E "TWO-HIT H Y P O T H E S I S "  7  Loss of Heterozygosity  8  Clonality in Neoplastic Cells  9  M O U S E M O D E L S O F NF1  13  M O L E C U L A R A N A L Y S I S IN NF1 A S S O C I A T E D N E U R O F I B R O M A S  13  M A S T C E L L S WITHIN N E U R O F I B R O M A S  18  MALIGNANT P E R I P H E R A L N E R V E S H E A T H T U M O U R S  19  RESEARCH OBJECTIVES  19  HYPOTHESES:  20  Risk of developing M P N S T s  20  Mast cell distribution in neurofibromas  20  Histologically-defined diffuse neurofibromas  20  Histologically-defined nodular neurofibromas  22  RELEVANCE  23  REFERENCES  25  2. ASSOCIATION BETWEEN BENIGN AND MALIGNANT PERIPHERAL NERVE SHEATH TUMOURS IN NF1 31 INTRODUCTION  31  METHODS  32  Subjects and Data Description  32  Statistical Analysis  33  RESULTS Prevalence of Neurofibromas  34 34  Logistic Regression  38  Survival Analysis  41  DISCUSSION  42  REFERENCES  47  3. HETEROGENETITY OF MAST CELL DISTRIBUTION IN NF1 NEUROFIBROMAS  50  INTRODUCTION  50  MATERIALS & METHODS  51  Sample Collection  51  Histopathological Classification of Neurofibromas  52  Identification of Granulated Mast Cells  52  Identification of Mast Cells and Schwann Cells  53  Tumour Characteristics  53  Statistical Analysis  54  RESULTS  54  Cellularity  55  Vascularity  57  Mast Cell Distribution Within Neurofibromas and M P N S T s  57  Distribution of Granulated and Non-granulated Mast Cells  59  DISCUSSION  60  REFERENCES  66  4. PATHOGENETIC HETEROGENEITY OF NF1 ASSOCIATED NEUROFIBROMAS  68  INTRODUCTION  68  METHODS  70  Sample Collection  .....70  Histopathological Classification of Neurofibromas  70  Tumour Characteristics  71  Immunofluorescent Staining for S100 and Neurofibromin  71  Image Acquisition  72  Image Analysis  72  Statistical Analysis  73  Method Development for Immunofluorescently Labeled-formalin Fixed Samples for Laser Microdissection  73  Tissue Preparation for Laser Microdissection  74  iv  Sample Collection by Laser Microdissection  74  DNA Purification  74  XCI Clonality Assay  75  RESULTS  75  Cellularity  78  Lymphocytic Infiltration  78  Vascularity  78  Neurofibromin & S100 Staining within Benign Neurofibromas  80  Correlations  82  Clonality of S100+/Nf+, S100+/Nf- and S100-/Nf+ Cells  83  DISCUSSION  89  REFERENCES  97  5. GENERAL DISCUSSION  101  SUMMARY  101  Risk of M P N S T Development  101  Benign Neurofibroma Development  103  H O W THIS S T U D Y FITS INTO C U R R E N T NF1 R E S E A R C H  105  F U T U R E DIRECTIONS  106  REFERENCES  108  APPENDIX A  110  R E S E A R C H ETHICS BOARD CERTIFICATES OF A P P R O V A L  v  110  LIST OF TABLES Table 1.1 National Institutes of Health diagnostic criteria for NF1  1  Table 1.2 Summary of loss of heterozygosity analysis, clonality studies and "second hit" somatic mutations in NF1-associated neurofibromas Table 2.1 Summary of Patient Data for those with M P N S T s  15 37  Table 2.2 Summary of odds ratios and 95% confidence intervals for logistic regression models  39  Table 3.1 Examples of different classifications for NF1-associated neurofibromas  50  Table 4.1 Summary of Kendall's tau correlations for pathological features in histologically-defined nodular and diffuse neurofibromas from patients with NF1 80 Table 4.2 Summary of Kendall's tau correlations for S100 and neurofibromin staining in histologically-defined nodular and diffuse neurofibromas from patients with NF1 83 Table 4.3 Summary of clonality results from histologically-defined nodular and diffuse neurofibromas and the proportion of each cell type within the neurofibromas 87  vi  LIST OF FIGURES Figure 1.1 Schematic drawing showing a normal nerve and a neurofibroma  4  Figure 1.2 Examples of histologically defined types of neurofibromas that develop as part of NF1  5  Figure 1.3 Examples of clinically defined types of neurofibromas that develop as part of NF1  6  Figure 1.4 Cartoon depicting neoplasia as a clonal expansion of one cell that underwent a stochastic somatic mutation  10  Figure 1.5 Schematic of X-Chromosome inactivation (XCI) assay with Androgen Receptor  11  Figure 1.6 Haploinsufficient model of tumour development in histologically-defined diffuse neurofibromas in NF1  22  Figure 1.7 "Second hit" model of development for histologically-defined nodular neurofibromas in NF1  23  Figure 2.1 Kaplan Meier Survival of 25 NF1 patients with M P N S T s  42  Figure 3.1 Images of typical neurofibromas in the current study that represent the majority of the tumour Figure 3.2 Distribution of pathological features of neurofibromas classified  55  histopathologically  56  Figure 3.3 Mast cell distribution within neurofibromas  58  Figure 3.4 Mast cell distribution in M P N S T s  59  Figure 3.5 Confocal image of a diffuse neurofibroma stained with S100 and c-Kit  60  Figure 4.1 Images of typical neurofibromas in the current study that represent the majority of the tumour  76  Figure 4.2 Distribution of pathological features of neurofibromas classified histopathologically  .79  Figure 4.3 Boxplot summarizing S100 and neurofibromin staining in nodular and diffuse neurofibromas  81  Figure 4.4 Images showing selective capture of single cell microdissection of S100+/Nf-negative cells from heterogeneous tissue  84  Figure 4.5 Images of Blood Vessel Microdissection  86  vii  LIST OF ABBREVIATIONS  4'6-diamidino-2-phenylindole-2HCI  (DAPI-j  Applied biosystems (ABI) Computed tomography (CT) Confidence intervals (CI) Deoxyribonucleic acid (DNA) Ethylene diamine tetraacetate (EDTA) Fluorescence in-situhybridization (FISH) Guanosine diphosphate (GDP) Guanosine triphosphatase-activatingprotein (GAP) Guanosine triphosphate (GTP) Hemotoxylin and eosin (H&E) Hydrochloric acid (HCI) Kilobase (Kb) Loss of heterozygosity (LOH) Magnetic resonance imaging (MRI) M a l i g n a n t p e r i p h e r a ln e r v e s h e a t h t u m o u r (MPNST) Matrixmetalloproteinases (MMP) Messenger ribonucleicacid (mRNA) Neurofibromatosis  type 1  (NF1)  Odds ratio (OR) Polyethylene naphtholate (PEN) Polymerase chain reaction (PCR) Sodium acetate (NaOAc) Tris (hydroxymethyl) methylamine (Tris) T u b e r o u s s c l e r o s i sc o m p l e x ( T S C ) Ultraviolet (UV) Vascular endothelialgrowth factor X-chromosome inactivation(XCI)  (VEGF)  viii  ACKNOWLEDGEMENTS  Iwould liketo thank my research supervisor and mentor, Jan Friedman, and my friend and coworker, PatriciaBirch,for theirunwavering encouragement and guidance. Thank you to Dr Carolyn Brown and Dr PierreWolkenstein, without you thisthesis would not be possible, I offermy sincere appreciation. Thank you to my supervisory committee, Dr Carolyn Brown, Dr Connie Eaves, and Dr Keith Humphries for theircontinual feedback and support. To members of the BritishColumbia Neurofibromatosis Foundation, thank y o u f o r r e m i n d i n g m e t h a t w e , a s r e s e a r c h e r s , c a n m a k e ad i f f e r e n c e a n d f o r a c o n s t a n t reminder of why we need to perform such research. Thank you to my family for theirfaithand encouragement. Finally,and most importantly, to my husband, Jordan Hood, for his companionship and help and without whose support Icould not have completed my thesis,you have my undying gratitude.  ix  CO-AUTHORSHIP STATEMENT  In Chapter 2, "Association between benign and malignant peripheral nerve sheath tumors in NF1", Iwas responsible for the design and data analysis of the research presented. Iwas responsible for the preparation of the manuscript with input from all contributing authors. P Wolkenstein was the physician who collected the data on the patients and J Revuz and J Zeller designed the database that stores the patient information. In Chapter 3, "Heterogeneity of mast cell distribution in NF1 neurofibromas", I was responsible for the design of the research. In addition I performed all the immunohistochemistry, data collectionand data analysis. VM Riccardi and J Wechsler reviewed tissue sections for this study to characterize them histologically. Together, M Sutcliff and I reviewed tissue sections to assess tumour characteristics. J Vielkind financially supported the pathological services necessary for this project. P Wolkenstein contributed some of the samples used in this study. Iwas responsible for the preparation of the manuscript with input from allcontributing authors. In Chapter 4, "Pathogenetic heterogeneity of NF1-associated neurofibromas", I was responsible for the design of the research. In addition, I performed all histochemistry, data collectionand data analysis. The majority of the molecular work was performed in the lab of, and with partial financial support by, C Brown. JFee assisted in troubleshooting laser microdissection. VM Riccardi and J Wechsler reviewed tissue sections for this study to characterize them histologically. Together, M Sutcliff and I reviewed tissue sections to assess tumour characteristics. P Wolkenstein contributed some of the samples used in this study. Iwas responsible for the preparation of the manuscript with input from allcontributing authors.  x  1. INTRODUCTION  Background on Neurofibromatosis N e u r o f i b r o m a t o s i s 1( N F 1 ) i s o n e o f t h e m o s t c o m m o n d o m i n a n t l y i n h e r i t e d d i s e a s e s , w i t h ab i r t h i n c i d e n c e o f 1 i n 3 5 0 0 p e o p l e ( 1 ) . N F 1 w a s f i r s t c l i n i c a l l y r e c o g n i z e d b y v o n R e c k l i n g h a u s e n i n 1 8 8 2 ( 2 ) , b u t i tt o o k m o r e t h a n 1 0 0 y e a r s f o r c r i t e r i af o r d i a g n o s i n g NF1 to be established (3) (Table 1.1).The disease is characterised by multiple cutaneous neurofibromas, plexiform neurofibromas, malignant peripheral nerve sheath tumours, and optic and other central nervous system gliomas, as well as by cafe-au-lait s p o t s a n d a b n o r m a l i t i e s o f t h e s k e l e t a l ,c a r d i o v a s c u l a r a n d c e n t r a l n e r v o u s s y s t e m s . Table 1.1 National Institutes of Health diagnostic criteria for NF1. Cardinal Clinical Features  ( A n yt w oo rm o r ea r er e q u i r e df o rd i a g n o  Six or more cafe-au-laitspots over 5mm in greatest diameter in prepubertal individuals or over 15mm in greatest diameter in postpubertal individuals Two or more neurofibromas of any type, or one plexiform neurofibroma Freckling in the axillary or inguinal  regions  Optic glioma Two or more Lisch nodules (iris hamartomas) A distinctiveosseous lesion such as sphenoid cortex with or without pseudarthrosis A firstdegree  dysplasia or thinning of the long  relative (parent, sibling, or offspring) with NF1  by the above  bone  criteria  T h e NF1 g e n e i s l o c a t e d i n t h e p e r i c e n t r i c r e g i o n o f t h e l o n g a r m o f c h r o m o s o m e 1 7 a n d w a s i d e n t i f i e d i n 1 9 9 0 (4, 5 ) . T h e g e n e h a s 6 0 e x o n s a n d e n c o m p a s s e s 3 5 0 k b o f genomic DNA. The large gene size may account forthe high estimated mutation rate of 1x10" per generation (6),with 50% of NF1 cases due to new mutations. There are 3 other genes embedded within intron27b that are transcribed in the opposite direction of t h e NF1 g e n e ; t w o e c t o p i c v i r a l i n t e g r a t i o n s i t e l o c i (EV12A, 4  1  EV  o l i g o d e n d r o b o n em a r r o w ,a n d E i se x p r e s s e do n t h es inactivate one or allof known.  c y t V 1 2 B u r f a these  e i se c eo gen  m y e l x p r e s s fo l i g o d es, but t  i ng l e de x c e n d r o heir rol  y g o p l u s i v c y t e s( e in the  r o t e i n (OMGP). EV12A e l y i nt h eb o n e m a r r o w( 7 ) 8 ) . M u t a t i o n sw i t h i nN F 1 pathogenesis of disease is n  i se x p r , w h i l e OM m a y ot  U n p r o c e s s e d p s e u d o g e n e st h a ts h a r em o s to ft h eN F 1 s e q u e n c eh a v eb e e ni d e n t i f i e d at 2q21, 12q12, 14q11, 15q11, 18p11, 21q11, and 22q11. Six of these regions are closely associated with centromeres, consistent with theiremergence by pericentric interchromosomal transposition (9).24 exons (exons 7-9, 10b, 11-23 and 24-27b) of the f u n c t i o n a l N F 1 g e n e h a v e h o m o l o g o u sc o u n t e r p a r t s i nt h e p s e u d o g e n e s . O n l yt h e p s e u d o g e n e o n c h r o m o s o m e 2 1 h a s a n u n i n t e r r u p t e dr e a d i n g f r a m e , a l t h o u g h pseudogenes on chromosome 2, 15 and 21 have shown some transcriptionalactivity (10).  N e u r o f i b r o m i n , t h e p r o t e i np r o d u c t o f t h e N F 1 g e n e , i s o v e r 2 2 0 k D i n s i z e ( 1 1 ) a n d i s a u b i q u i t o u s l ye x p r e s s e d p r o t e i n . T h e b e s tc h a r a c t e r i s e df u n c t i o n o f N F 1 w a sd e d f r o m ad o m a i n e n c o d e d b y e x o n s 2 1 - 2 7 a t h a t i s h o m o l o g o u s t o t h e G T P a s e - a c t i v a t i n g ( G A P ) d o m a i n o f h u m a n p 1 2 0 G A P a n d y e a s t IRA1 a n d IRA2 ( 1 2 ) f u n c t i o n s a s an e g a t i v e r e g u l a t o r o f R a s b y a c c e l e r a t i n g t h e c o n v e r s i o n o f a c t i v e R a s GTP to inactive Ras-GDP. Ras functions as part of a signal transduction pathway involvingepidermal growth factor,nerve growth factor and platelet-derived growth f a c t o r . R a s c a n s t i m u l a t e c e l l u l a rp r o l i f e r a t i o n t h r o u g h t h e R a f - M A K ( m i t o g e n a c t i v a t e d k i n a s e ) p a t h w a y ( 1 3 ) a n d c a n a l s o i n h i b i ta p o p t o s i s t h r o u g h t h e p h o s p h o i n o s i t o l 3 ' kinase (PI3 kinase) pathway (14). T h r e em a j o ra l t e r n a t i v e l ys p l i c e da n d d i f f e r e n t i a l l ye x p r e s s e d i s o f o r m so fN have been identifiedin humans. The most common isoform includes exon 23a within t h e G A P - r e l a t e d f u n c t i o n a l d o m a i n ( 1 5 ) a n d r e s u l t s i n ad e c r e a s e i n G A P a c t i v i t y ( 1 6 ) i n d i f f e r e n t i a t e dc e l l s ( 1 7 ) . A n o t h e r i s o f o r m c o n t a i n s e x o n 4 8 a a n d i s p r e f e r e n t i a l l y e x p r e s s e d i n m u s c l e ( 1 8 ) , a n d at h i r d i s o f o r m t h a t i n c l u d e s e x o n 9 a i s f o u n d e x c l u s i v e l y in the central nervous system during embryogenesis (19). A s e c o n d d o m a i n u p s t r e a m o f t h e N F 1 G A P - r e l a t e d d o m a i n h a s ar o l ei n c A M P signaling as itcontains cAMP-dependent protein kinase A (PKA) binding sites (15). The NF1 h o m o l o g u e i n Drosphila melangaster a c t s a s a n a c t i v a t o r o f t h e 2  w e l la s a b y a n N F functions that neur PKA and proteins  n e g a t i v e r e g u l a t o r o f R a s ( 2 0 ) ; m o r e o v e r ,N F 1 m u t a n t f l i e s c a n b e r e s c u e d 1 t r a n s g e n e a n d e x p r e s s i o n o f a c t i v a t e d P K A ( 2 1 ) . T h i ss u g g e s t s t h a t P K A downstream o f o r p a r a l l e l t o n e u r o f i b r o m i n i n D r o s o p h i l a . I n r a t s , i tw a s found o f i b r o m i nG A P a c t i v i t yw a s d e c r e a s e d u p o n b i n d i n g a n d p h o s p h o r y l a t i o n b y s u b s e q u e n t i n t e r a c t i o n w i t h 1 4 - 3 - 3 , ap r o t e i n k n o w n t o m o d u l a t e v a r i o u s t a r g e t (22).  Types of Neurofibromas in NF1 In this thesis, I use the words "tumour" and "neurofibroma" interchangeably of convenience. The question of whether neurofibromas are true neoplasms of my research, and my use of "tumour" in this context is not intended to im about the nature of the process that produces these lesions. Neurofibromas benign; the malignant counterpart isthe malignant peripheral nerve sheath (MPNST). In this thesis any reference to malignancy willbe explicit.  as a matter is a focus ply anything are always tumour  Neurofibromas are tumours that develop within a peripheral nerve, and sometimes individualaxons are visiblewithin the tumour. Within the peripheral nervous system, each axon is surrounded by a Schwann c e l l t o f o r m an e r v e f i b r e . S e v e r a l n e r v e f i b r e s become encased within the perineurium, a tubular structure made up of perineurial cells a n d c o l l a g e n , t o f o r m an e r v e f a s c i c l e . T h e s p a c e b e t w e e n t h e S c h w a n n c e l l s t h a t surround the individualaxons and the perineurium is the endoneurium. In larger nerves, several nerve fascicles are bundled together and are encased by a dense fibrous sheath, the epineurium (Figure 1.1). The development of a neurofibroma results in an increase in the number of Schwann cellsand disorganized nerve structure with possible breakdown of some structures (Figure 1.1).  3  Figure 1.1 Schematic drawing showing a normal nerve and a neurofibroma.  Illustrationcourtesy of Dr Gottfried. Images 2006.  published in Neurosurgery.  58(1), 1-16,  Neurofibromas vary in terms of their age of appearance, natural history,and malignant p o t e n t i a l .C o n s e q u e n t l y , s e v e r a l d i f f e r e n tc l a s s i f i c a t i o n s o f n e u r o f i b r o m a s h a v e b e e n proposed (Table 3.1).None of these classificationshas achieved universal acceptance, and the literaturecontains many examples of inconsistentuse and confusion of various c l i n i c a la n d p a t h o l o g i c a l d e s c r i p t i v e t e r m s . All neurofibromas contain Schwann cells (the suspected progenitor celltype), but fibroblasts,mast cells,lymphocytes, perineurial cells,endoneurial fibroblasts and endothelial cells also occur in varying numbers (23). Despite the similar cell types, n e u r o f i b r o m a s c a n b e c l a s s i f i e d h i s t o p a t h o l o g i c a l l yi n t o t w o c a t e g o r i e s o f d i f f u s e a n d nodular neurofibromas. Diffuse neurofibromas are predominantly located in the dermis and involve the thin terminal branches of peripheral nerves (Figure 1.2A). Diffuse neurofibromas are characterized by proliferationof cells that are not limitedby the perineurium and can encircle normal structures (exocrine glands, hairfollicles and 4  vessels) without destroying them. The proliferatingcells are irregularlydispersed within a f i b r o u s a n d / o r m y x o i d b a c k g r o u n d . M y e l i n f i b r i l sa r e r a r e o r a b s e n t . N o d u l a r n e u r o f i b r o m a s u s u a l l y i n v o l v e d e e p e r t i s s u e s , a n d t h e c e l l u l a r p r o l i f e r a t i o ni s intraneuraland enclosed within large hypertrophic nerves circumscribed by the perineurium (Figure 1.2B). Nodular tumours include both single and plexiform types. Single nodular tumours involvejust one fascicle,while nodular plexiform tumours may involve several fascicles, nerve branches or anerve plexus. Dispersed or fascicular myelin fibrilsare usually found in the central area of the nodules. On transverse section, the central area is loose with amyxoid background substance, while the peripheral area is usually more cellular. Some neurofibroma usually found in the surrounded by diffu normal pre-existing  s c o n t a i n b o t h d i f f u s ea n d n o d u l a r c o m p o n e n t s ; s u c h t u m o u r s a r e deep dermis and subcutaneous tissue.The nodular component is s e e x t r a n e u r a l c e l l p r o l i f e r a t i o n ,i n f i l t r a t i n g t h e f a t t i s s u e a n d o t h e r structures.  Figure 1.2 Examples of histologically defined types of neurofibromas that develop as part of NF1.  A. H&E with an 10.25x pointin  section at 10.25x magnification of ahistologically-defined diffuse neurofibroma arrow pointing to the skin that overlays the neurofibroma.B. H&E section at magnification of ahistologically-defined nodular neurofibromas with an arrow g to perineurium that circumscribes the neurofibroma.  Riccardi (24) has respect to their ag c l a s s i f i c a t i o nd o e s above. Riccardi's  distinguished 4types of neurofibromas clinicallythat differ with e of appearance, natural history,and malignant potential.This clinical not correlate completely with the pathological classification given cutaneous neurofibromas occur in the skin surface and develop from 5  t h e t e r m i n a l b r a n c h e s o f p e r i p h e r a l n e r v e s ( F i g u r e 1.3A). S u b c u t a n e a r e f i r m a n d l i e d e e p e r i n t h e s u b c u t a n e o u s l a y e r o f t h e s k i n ( F i g u r e 1.3B). T h e y h a v d i s c r e t e m a r g i n s b u t m a y e x t e n d a l o n g an e r v e . N o d u l a r p l e x i f o r m n e u r o f i b r o m a s a r i s e w i t h i n m a j o r p e r i p h e r a l n e r v e s a n d m a y l i ej u s t b e n e a t h t h e d e r m i s o r m u c h d e e p e r i n t h e b o d y ( F i g u r e 1.3C). T h e y c a n b e l o c a l i z e d o r e x t e n d t h e l e n g t h o f t h e n e r v e , i n an entire nerve plexus, or spinal nerve roots at multiple layers. The lasttype is diffuse plexiform neurofibromas, which are sometimes referred to as congenital plexiform n e u r o f i b r o m a s b e c a u s e t h e y a r e u s u a l l y a p p a r e n t a t b i r t h o r w i t h i n t h e f i r s tf e w y e a r s o f l i f e ( F i g u r e 1.3D). T h e s e n e u r o f i b r o m a s c a n b e s u p e r f i c i a l , i n v o l v i n g t h subcutaneous tissue,or involve deeper tissue.The tumours do not have discrete borders but invade adjacent normal tissues.Subcutaneous, nodular plexiform and diffuse plexiform neurofibromas can progress to malignancy, resultingin MPNSTs. Figure 1 . 3 Examples of clinically defined types of neurofibromas that develop as part of NF1. C i s a n M R I i m a g e o f t h e p e l v i s i n f r o n t a l s e c t i o n .  T g a a  h e h i s t o l o g i c a l a n d c l i n i c a l a p p r o a c h e s d o n o t c l a s s i f y i n d i v i d u a lt u m o u r s i n t o t h e s a m e roups. Both clinicallydefined cutaneous and diffuse plexiform neurofibromas usually re diffuse neurofibromas histologically,although some diffuse plexiform neurofibromas re histologically-mixeddiffuse and nodular neurofibromas. Tumours that are classified 6  o u sn e  v o l  es k i  clinicallyas subcutaneous  or nodular plexiform are usually nodular neurofibromas  on  histopathological exam. Most studies of neurofibroma pathogenesis treat all neurofibromas as a single class d e s p i t e t h e i r c l i n i c a la n d p a t h o g e n i c h e t e r o g e n e i t y . T h i s h e t e r o g e n e i t y i s i m p o r t a n t because some kinds of neurofibromas have little, ifany, malignant potential,while other kinds may progress to malignancy. MPNSTs are one of the leading causes of death in NF1 patients (25). There is currently no effectivetreatment known for benign neurofibromas or MPNSTs except surgicalremoval in favourable cases. The "two-hit hypothesis" In the early 1970's Alfred Knudson suggested that two mutations or "hits"were required for the development of a retinoblastoma and that the inheritance of one of these m u t a t i o n s c o u l d a c c o u n t f o r t h e e a r l i e r o n s e t a n d f r e q u e n t b i l a t e r a lo c c u r r e n c e o f t h e hereditaryform of thistumour (26).Subsequent molecular studies supported Knudson's hypothesis in both hereditary and sporadic retinoblastomas by demonstrating mutations o f b o t h a l l e l e s o f a g e n e t h a tw a s n a m e d " R B 1 " i n b o t h h e r e d i t a r y a n d s p o r a d i c retinoblastomas (27). In hereditary retinoblastoma, individuals begin lifewith a c o n s t i t u t i o n a lm u t a t i o nt h a ti n a c t i v a t e so n e a l l e l eo ft h e R B 1 g e n e .T h e " s e c o n dh i t occurs somatically and usually involves allor part of the chromosome containing the n o r m a lR B 1 a l l e l e , p o t e n t i a l l y a f f e c t i n g o t h e rg e n e s a n d g e n e t i c m a r k e r si n t h e r e g i o n t h e n o r m a lR B 1 a l l e l e . I fs o m e o f t h e s e g e n e t i c m a r k e r sh a p p e n t o b e h e t e r o z y g o u s i n a n i n d i v i d u a l , l o s so f o n e a l l e l e o n t h e s a m e c h r o m o s o m e a s t h e n o r m a l R B 1 a l l e l e produces loss of heterozygosity (LOH), a cell-specific phenotype that is relatively easy to test in tumour tissue. The "two-hit"hypothesis has subsequently been shown to apply to many other involved in many other inheritedand sporadic tumours, and functional loss of bo alleles of such genes is considered to be a principal mechanism of tumourigenesis 29). As a consequence, LOH for a particular gene within a specific tumour type become a major criteriafor the involvement of that gene in tumour pathogenesis  genes th (28, has (30).  Genes that permit tumour development when both alleles are inactivated or lost are k n o w n a s " t u m o u r s u p p r e s s o r g e n e s " ( 2 9 ) . N F 1 i s a c o n f i r m e d t u m o u r s u p p r e s s o rg e n e a s m o s t c o n s t i t u t i o n a lm u t a t i o n si n N F 1 r e s u l ti n l o s so f f u n c t i o no f t h e p r o t e i n , a n d L O 7  has been observed in pheochromocytomas (33, 34) in NF1  (31), myeloid leukemia (32) and  MPNSTs  patients. Loss of Heterozygosity  LOH analysis is performed by screening paired blood and tumour samples with polymorphic genetic markers in the region of an established or suspected tumour s u p p r e s s o r g e n e . T h e d e t e c t i o no f L O H e x p l o i t st h e p r e s e n c e o f D N A p o l y m o r p h i s m s throughout the genome to discriminate between the paternal and maternal alleles at a tumour suppressor locus. In practice,identificationof LOH is somewhat arbitrary b e c a u s e ad e c r e a s e i n t h e r e l a t i v e i n t e n s i t y , r a t h e r t h a n at o t a l l o s s o f o n e a l l e l e , i s usually observed in tumours. This is referred to as "allelicimbalance", and different s t u d i e s h a v e u s e d d i f f e r e n tc u t o f f s t o d i a g n o s e L O H . A l t h o u g h a l l e l i ci m b a l a n c e i s usually considered to be evidence of LOH, the ratioof alleles in neoplastic tissue can a l s o b e d i s t u r b e d b y o t h e r m e c h a n i s m s , i n c l u d i n gt r i s o m y a n d l o c a l D N A a m p l i f i c a t i o n (29). In some instances, however, inactivation of both alleles of a tumour suppressor gene m a y o c c u r b u t L O H m a y n o t b e d e t e c t e d .F a i l u r et o o b s e r v e L O H m a y r e s u l tf r o m t e c h n i c a l l i m i t a t i o n so f t h e a s s a y , s u c h a s i n a c t i v a t i o no f t h e n o r m a l a l l e l e o f t h e t u m o u r suppressor gene by point mutations, small deletions or epigenetic mechanisms that do not produce loss of closely linked polymorphic markers. In other circumstances, LOH w o u l d n o t b e e x p e c t e d b e c a u s e t h e n e o p l a s m a r i s e s b y am e c h a n i s m s u c h a s activatingmutations of one or more oncogenes rather than inactivationof both alleles of a tumour suppressor gene. V o g e l s t e i n e ta l . ( 3 5 )e x p l a i n e d t h e i n c r e a s population with age by means of a more complex multistep tumorigenesis model. This model describes neoplastic development as the expansion of a clone of cells that has accumulated several somatic mutations.The neoplasm may be eitherbenign or malignant, depending on whether itscells possess the abilityto metastasize. The process is seen as one of progressive selection for cells with an increasingproliferative a d v a n t a g e . T h e f i r s t m u t a t i o n i n as o m a t i c c e l l p r o v i d e s as l i g h t g r o w t h a d v a n t a g e t o i t s progeny. One of these cells acquires a further proliferative advantage through a second m u t a t i o n , a n d o n e o f i t s c e l l u l a r p r o g e n y a c q u i r e s at h i r d m u t a t i o n , p o s s i b l y f o r m i n g a benign tumour. Subsequent mutations in the descendants of these cellsmay permit 8  them to escape apoptosis, cellcycle checkpoints, or other growth control mechanisms or to become genetically unstable, increasing the likelihood for a tumour to progress. Colorectal cancer is a well characterized cancer that follows the multistep model of t u m o u r i g e n e s i s ( 3 5 ) . T u m o u r i n i t i a t i o n o c c u r s w i t h am u t a t i o n i n t h e t u m o u r suppressor g e n e , A P C ( a d e n o m a t o s i s p o l y p o s i s c o l i ) , w h i c h c a u s e s ah y p e r p r o l i f e r a t i o n o f t n o r m a l e p i t h e l i u m r e s u l t i n g i n ab e n i g n c o l o n a d e n o m a . M u t a t i o n s o f t h e A P C g e n e o c c u r s o m a t i c a l l y , c a u s i n g as i n g l e t u m o u r , o r i n t h e g e r m l i n e , r e s u l t i n g i n a predisposition that may cause the development of thousands of adenomas. A s u b s e q u e n t m u t a t i o n i n t h e RAS g e n e o c c u r s , l e a d i n g t o a f u r t h e r e x p a n s i o n o f t h e c e l and an increase in the size of the benign adenoma. Additional mutations in the DCC ( d e l e t e di nc o l o r e c t a lc a r c i n o m a )a n d P 5 3 g e n e s r e s u l ti na n i n c r e a s ei nt h ec l expansion and progression from benign adenoma to malignant carcinoma. Other alterationsoccur that permit metastasis.  he can  ls  Clonality in Neoplastic Cells  Neoplasia almost always arises through mutation, although examples of abnormal epigenetic changes have also been documented as likelycontributing events (36, 37). A acquired mutational event in somatic cells arise randomly, giving the affected cella growth advantage compared to the surrounding cells.The cell then gives rise to a g e n e t i c a l l y i d e n t i c a l c l o n e o f c e l l s t h a t , o v e r t i m e , c a n f o r m at u m o u r . A l l o f t h e c e l l s o f t h e r e s u l t i n gt u m o u r a r e c l o n a l d e r i v a t i v e s o f t h e o n e c o m m o n a n c e s t o r c e l l t h a t u n d e r w e n tt h e m u t a t i o n . S u c h t u m o u r s a r e s a i d t o b e " c l o n a l " ( F i g u r e 1 . 4 ) .  9  o n a l  Figure 1.4 Cartoon depicting neoplasia as a clonal expansion of one cell that underwent a stochastic somatic mutation.  Each dark blue circle represents anormal cell in any tissue. One cell (light blue) has a somatic mutation that gives itaproliferative advantage over all other surrounding cells. The light blue cell expands clonally to form a tumour.  Tumour  Normal Tissue  ° " undergoes a mutation n  e  Mutated cell has a growth advantage j forms a tumour  c e  a n c  M o s t n o r m a l t i s s u e s a r e s a i d t o b e " p o l y c l o n a l " , i . e . ,c o m p o s e d of amixture of cells that a r o s e f r o m m a n y d i f f e r e n t p r e c u r s o r s , n o n e o f w h i c h h a d as i g n i f i c a n tg r o w t h advantage over the others. Clonalitymay be observed among adjacent cellsin normal solid tissues because the two daughter cells produced by mitoticdivisiontend to remain close to each other in solid tissues. However, the "patches" of clonalitythat occur in normal tissues are usually small. The finding of alarge region of clonality within a tissue suggests the presence of aneoplastic process. There are some (38, 39), arising of benign tumour part of tuberous However, tubers dysplasia rather  e x c e p t i o n s t o t h i s r u l e ,f o r e x a m p l e s o m from the infection of apolyclonal set of s that do not display clonality,such as s c l e r o s i s c o m p l e x ( T S C ) , ad o m i n a n t l y - i are considered to be hamartomas that than true neoplasms.  e  neoplasms are virally induced cells. There are also examples t h e c o r t i c a lt u b e r s t h a t o c c u r a s nherited disease (40-42). a r i s e t h r o u g h ap r o c e s s o f  T h e m o s t f r e q u e n t l y - u s e d a s s a y o f t i s s u e c l o n a l i t yi n h u m a n s i s b a s e d o n t h e f a c t t h a t normal tissues in females are mosaic, being comprised of amixture of cells, some of w h i c h h a v e a n a c t i v e X - c h r o m o s o m e i n h e r i t e df r o m t h e m o t h e r , a n d o t h e r s o f w h i c h have an active X-chromosome inheritedfrom the father.Mary Lyon first hypothesized that one X-chromosome in each female cell is randomly inactivated (43), and this is true in almost allcells of human females. X-inactivation occurs in early embryogenesis, and 10  once one of the two X-chromosomes i n a n e m b r y o n i c c e l l i s i n a c t i v a t e d ,t h e s a m e X chromosome (maternal or paternal) remains inactivated in allof the clonal progeny of that cell. X-inactivation is arandom process, and on average half of the cells in any w o m a n w o u l d b e e x p e c t e d t o h a v e a n i n a c t i v ep a t e r n a l X - c h r o m o s o m e , a n d t h e o t h e r halfto have an inactive maternal X-chromosome. However, the actual ratioof the two c e l l t y p e s i n a n y n o r m a l t i s s u e f o l l o w s aG a u s s i a n d i s t r i b u t i o n w i t h am e a n o f 5 0 : 5 0 . Substantial variation occurs around this mean as aresult of the relatively small size of the cell pool present at the time of X-inactivation (43). Itis therefore not surprising that n o r m a l f e m a l e t i s s u e s o f t e n h a v e am o r e o r l e s s u n e q u a l p r o p o r t i o n o f c e l l s w i t h t h e maternal or paternal X-chromosome inactive. T h e p a t t e r n o f X - i n a c t i v a t i o n i n at i s s u e c a n b e e s t i m a t e d b y a n a l y s i s o f p o l y m o r p h i c X l i n k e d m a r k e r g e n e s ( e . g . , AR, a n d r o g e n r e c e p t o r ) t h a t c o n t a i n r e s t r i c t i o n s i t e s t h a methylated on the inactiveX-chromosome but not on the active X-chromosome (44). The marker is amplified by PCR with and without preceding digestion by a methylationsensitive restrictionenzyme. Following digestion, only the alleleon the methylated i n a c t i v e Xc h r o m o s o m e persists as an intactfragment that can be amplified by PCR (Figure 1.5). Figure 1.5 Schematic of X-Chromosome Inactivation (XCI) a s s a y with Androgen Receptor.  A . B l u e b o x r e p r e s e n t s t h e AR l o c u s p o l y m o r p h i c C A G r e p e a t o f a l l e l e A ; r e d b o x r e p r e s e n t s a l l e l e B . T h e v e r t i c a l l i n e s r e p r e s e n t Hpall r e s t r i c t i o n s i t e s a n d b l a r r o w s r e p r e s e n t t h e P C R p r i m e r s . A l l e l e s Aa n d Br e p r e s e n t t w o a l l e l e s i n a n i n d i v i d u a l h e t e r o z y g o u s a t t h e AR l o c u s . B . A f t e r g e n o m i c d i g e s t i o n w i t h Hpall, t d i f f e r e n t s c e n a r i o s : r a n d o m X C I a n d s k e w e d X C I . Hpall s i t e s t h a t a r e n o t m e t h w i l lb e c u t ( i n d i c a t e d b y a n o p e n s p a c e b e t w e e n t h e r e s t r i c t i o n s i t e s ) .M e t h y l a t e d s i t e s will remain intact. In random XCI, allele Ais cut half the time, as is allele B. In c o m p l e t e l y s k e w e d X C I , o n l y o n e a l l e l e i s c u t ( a l l e l e Bi n t h e f i g u r e ) . C . U p o n P C R o f t h e d i g e s t e d s a m p l e s , a m p l i f i c a t i o n f r o m t h e AR l o c u s w i l l o n l y o c c u r i f t h e a l l e l e h a s n o t b e e n c u t w i t h Hpall. D . T h e a l l e l e s c a n t h e n b e s e p a r a t e d b y e l e c t r o p h o r e s i s .  11  Hpall Sites  (CAG)n  Allele  A  A l l e l eB Hpall Digestion  B  Skewed XCI  Random XCI  OR  Amplification  D  V  Electrophoresis Allele  A  A l l e l eB  12  Normal polyclonal tissues in aheterozygous female have amixture of cells expressing the maternal and paternal alleleson the active X-chromosome. Such tissues usually exhibitapproximately equal expression of each alleleof the majority of X-linked genes. T h e p r e s e n c e o f t h e s a m e i n a c t i v e a l l e l e i n a l l c e l l s o f a t u m o u r f r o m aw o m a n whose normal tissues exhibit both inactive alleles in approximately equal amounts confirms the monoclonal originof the neoplasm. Demonstrating c l o n a l i t y w i t h i n at i s s u e i s c o n s i s t e n t w i t h i t b e i n g n e o p l a s t i c , b u t c l o n a l i t y analysis does not provide information on the specificmolecular events that lead to tumour initiationor progression. Clonalityonly means that one or more rare events (usuallypresumed to be mutations) have occurred during the process of tumour development. A tumour that is of clonal origin may contain several distinct subclones as a result of subsequent m u t a t i o n s . I n a d d i t i o n , at u m o u r s a m p l e m a y c o n t a i n enough non-malignant cells that it appears to represent apolyclonal population. Mouse Models of NF1  Mouse models of NF1 have been developed, but these models onlypartially r e c a p i t u l a t et h e h u m a n d i s e a s e . M i c eh e t e r o z y g o u sf o rN f 1 a r e v i a b l e b u td o n o t d e v e l o p n e u r o f i b r o m a so fa n yk i n d , a n d h o m o z y g o u sN d e f e c t s , p o i n t i n g t o ac r i t i c a lf u n c t i o n o f n e u r o f i b r o m i n i n m o u s e e m b r y o n i c development ( 4 5 ). M i c e w i t h a c o n d i t i o n a l d e l e t i o n o f b o t h N f 1 a l l e l e s r e s t r i c t e d t o S c h w a s u r v i v ew i t he n l a r g e dp e r i p h e r a ln e r v e s ,d e m o n s t r a t i n gt h a tc o m p l e t ed e f i c i e n in Schwann c e l l s i s s u f f i c i e n t t o i n i t i a t e ap r o l i f e r a t i v e a b n o r m a l i t y o f t h e S c h w a n n cell ( 4 6 ). I n t e r e s t i n g l y , t h e s e m i c e d e v e l o p n e u r o f i b r o m a s o f t h e d o r s a l r o o t g a n g l i a o n o t h e rc e l l si nt h em i c ea r eh e t e r o z y g o u sf o rN f 1- a nN f 1 h a p l o i n s environment appears necessary forthe pathogenesis of these histologically-mixed tumours (46). The neurofibromas that arise in this mouse model are histologically similarto nodular plexiform neurofibromas that occur in people with NF1. Molecular Analysis in NF1 Associated Neurofibromas  Itis widely assumed t h a t a l l b e n i g n N F 1 n e u r o f i b r o m a s d e v e l o p w h e n a" s e c o n d h i t " o c c u r ss o m a t i c a l l yi nt h e n o r m a lN F 1 a l l e l e . S c h w a n n c e l l sa r e t h es u s p e c t e dp r o g e n i i n n e u r o f i b r o m a s b e c a u s e t h e y c o n s t i t u t e al a r g e p r o p o r t i o n o f t h e t u m o u r , 4 0 - 8 0 % , and s o m e c u l t u r e s d e r i v e d f r o m N F 1 p a t i e n t n e u r o f i b r o m a s d i s p l a y l o s s o f t h e n o r m a lN allele in Schwann cells but not in fibroblasts (47-49). 13  S t u d i e s h a v e f o c u s e d o n u n c o v e r i n g s o m a t i c m u t a t i o n s o r " s e c o n d h i t s "i n N F 1 a s s o c i a t e d n e u r o f i b r o m a s . E v i d e n c e o f c l o n a l i t yo r L O H h a s b e e n o b s e r v e d i n 2 6 % o f discrete dermal neurofibromas (Table 1.2).A dermal neurofibroma reported by Sawada e ta l . ( 5 0 )w a se s p e c i a l l yi n f o r m a t i v e b e c a u s et h es o m a t i cm a l l e l ew a s i d e n t i f i e d . H o w e v e r , b o t h t h e m u t a n t a n d n o n - m u t a n t v e r s i o n o f t h e s e c o n d N F 1 a l l e l ew e r e f o u n d i n a h o m o g e n o u s - a p p e a r i n ga r e a o f t u m o u r c e l l s , s u g g e s t i n gt h a t these cells were not allclonal derivatives of the cellthat had sustained the second pathogenic mutation. This interpretationis strongly supported by studies of cultured Schwann cellsfrom 10 neurofibromas obtained from 6 NF1 patients (51) whose germl i n eN F 1 m u t a t i o n sh a d b e e n i d e n t i f i e d .T w o d i f f e r e n tp o p u l a t i o n so fS c h w a n nc e l were demonstrated in cultures from 7 of these 10 tumours; one population had a " s e c o n dh i t " s o m a t i cN F 1 m u t a t i o n , a n d t h e o t h e rd i d n o t . T h i s f i n d i n g r a i s e s t h e possibility that the "second hit" mutation occurred as a secondary event within a neurofibroma that had already developed through some polyclonal process. Alternatively,these tumours may have arisen through a "two-hit"mechanism, but the proliferatingneoplastic clone (presumably Schwann cells)stimulated invasion of the t u m o u r b y n o n - n e o p l a s t i c S c h w a n n c e l l s ,p e r i n e u r a l c e l l s ,m a s t c e l l s a n d f i b r o b l a s t s .  14  Table 1.2 Summary of loss of heterozygosity analysis, clonality studies and "second hit" somatic mutations in NF1associated neurofibromas. Plexiform  Dermal NF1 Patients Studied  Study  Tumours Studied  With "2-hit" or Clonality  14  14  "2-hit"  19  Clonality  NF1 Patients Studied  LOH Analysis  Tumours With "2-hit" or Clonality  Markers  Method  Intragenic  0  PCR  G-6-PD  19  0  Southern  8  8  8  PCR  5  22  8  PCR  1  1  1  PCR  4  20  31  0  7  7  1  PCR  4  8  8  3  5  5  1  PCR  AR  10  14  8  PCR  4  1  1  1  PCR  9  Studied  Extragenic  Primary Tumor Fialkow et al. (51a) Clonality Skuse et a/.(52) 7 PGR  Colman et a/.|(53) "2-hit" Sawada ef  ..  12  12  al.(50)  "2-hit" Daschner ef  al.(54)  "2-hit" Clonality  1  Kluwe era/.(47) LOH Eisenbarth ef al. (55) "2-hit" John ef a/.(56)  3  6  3  2  Plexiform  Dermal Study  NF1 Patients Studied  Tumours Studied  With "2-hit" or Clonality  42  74  10  LOH  5  5  "2-hit"  8  "2-hit"  NF1 Patients Studied  LOH Analysis  Tumours  Markers  Studied  With "2-hit" or Clonality  Method  Intragenic  Extragenic  3  3  1  PCR  8  5  2  9  9  3  PCR  3  3  15  2  10  10  4  PCR  10  10  2  33  13  PCR  7  4  17  80  . 13  3  3  1  PCR  9  4  4  4  0.  10  11  1  FISH  6  28  6  PCR  6  11  PCR  4  Luijten ef a/.(57)  Weist ef al.(58) "2-hit" Upadhyaya ef a/.(59) "2-hit" DeLuca ef a/.(60) "2-hit" DeRaedt ef a/.(61) "2-hit" "2-hit" in Cultures* 1  Kluwe ef a/.(62)f  1  1  Serraef a/.(63) t  17  60  15  PCR  5  6  Serra ef a/. (64) t  12  27  19  PCR  5  16  Maertens ef a/.(65)  9  38  29  PCR  4  2  186  459  121 (26%)  54  59  21 (36%)  16  16  3(19%)  5  5  1 (20%)  Total L O H Total Clonality  f Author classified neurofibromas as benign, no distinction between dermal or plexiform t A u t h o rf o u n d L O H i n S c h w a n n c e l l s b u t n o ti nf i b r o b l a s t c u l t u r e s f r o m p a t i e n t n e u r o f i b r o m a s a m p l e s  Plexiform neurofibromas occur in about halfof NF1 patients and may be either diffuse or nodular. Although these two types of plexiform tumours are clinicallydistinct, reported s t u d i e s o f L O H a n d c l o n a l i t y d o n o t d i s t i n g u i s hb e t w e e n t h e m . T h e m a j o r i t y o f p l e x i f o r m n e u r o f i b r o m a sh a v e n o ts h o w n e v i d e n c eo fc l o n a l i t yo r L O H i nt h eN F 1 r e g i o n( T a b l e 1.2). However, Schwann cell cultures derived from 4 of 6 plexiform neurofibromas showed karyotypic abnormalities, while cultures of dermal neurofibromas did not display any chromosomal abnormalities (66). Karyotypic heterogeneity was observed in the cultures from 3 of these plexiform tumours. The observation of karyotypic abnormalities in plexiform neurofibromas may indicate that such changes are frequent in these tumours or at least in those that require surgical removal. Alternatively, chromosomal abnormalities may not have been present in the tumours but may have arisen inculture.  T h e l o w p e r c e n t a g e o f o b s e r v e d N F 1 L O H i n b e n i g nn e u r o f i b r o m a s i s o f t e n a t t r i b u t e d t o technical limitations of the assays, such as their inabilityto identify point mutations or e p i g e n e t i cm e c h a n i s m s( e . g . p r o m o t e rm e t h y l a t i o n )t h a tm a y i n a c t i v a t et h en o r m allele.To date, there is no evidence that methylation plays a role in inactivating the r e m a i n i n g n o r m a l N F 1 a l l e l e ( 5 7 ,6 7 , 6 8 ) . M o r e o v e r , i ti s t h o u g h t t h a tL O H a n d , h e n c e , c l o n a l i t yi n S c h w a n n c e l l s o f t e n r e m a i n u n d e t e c t e d b e c a u s e o f t h e a b u n d a n c e o f n o n neoplastic stromal cells present in these heterogeneous tumours. A third possibility that is rarelyconsidered is that LOH is not observed because some neurofibromas do not develop as a result of "two hits" but rather as non-neoplastic proliferations of the involved tissue.  E v i d e n c e o f L O H o r s e c o n d s o m a t i c m u t a t i o n s d o e s n o t p r e c l u d e t h e p o s s i b i l i t yt h a t m u t a n tn e u r o f i b r o m i np r o t e i ne x p r e s s e df r o mt h eN F 1 g e n e c o u l d h a v er e s i d u a l f u n c t i o n . T h e r e a r e s e v e r a le x a m p l e s o f e x p r e s s i o n o f N F 1 t r a n s c r i p t st h a ta r e n o tf u l l length neurofibromin but appear to be stable.The N-isoform of neurofibromin lacks the GAP-related domain and is expressed in normal brain and brain tumours, but itis not k n o w n w h a t f u n c t i o n o r r o l et h i s i s o f o r m h a s ( 6 9 ) . N F 1 m R N A i ss u b j e c tt o m R N A e d i t i n g , a n d o n e s u c h e d i tp r o d u c e s a s t o p c o d o n i n t h e 5 ' p o r t i o n o f t h e N F 1 G A P related domain, which can resultin an incomplete transcript(70). Moreover, an NF1 patient has been reported who had a nonsense mutation that was expressed at the s a m e l e v e l a s t h e n o r m a lN F 1 a l l e l e , s u g g e s t i n g t h a t t h e m u t a t e d t r a n s c r i p tw a s s t a b l e  17  ( 7 0 ). T h e f u n c t i o n o f t h e t r a n s c r i p t i s n o t k n o w n , b u t i f i t i s n o t d e g r a d e d b y n o n - s e n s e m e d i a t e d d e c a y i tm a y f u n c t i o n t o s o m e e x t e n t ( 7 0 ) . Mast Cells within Neurofibromas Local trauma may play a role in the development of some types of neurofibromas (71), especially cutaneous neurofibromas, as the skin is prone to bumps and bruising. Local trauma results in an inflammatory response, a process that involves mast cells. Several studies have noted the presence of mast cells within neurofibromas (72, 73), but itis not clearwhat type of neurofibromas were analysed or how many of them were from individuals with NF1. Mast cells have long been thought to play a role in the development of neurofibromas, as neurofibromas often itchor burn as they develop, and individualswith NF1 have described instances in which forcefultrauma to the skin was followed by intense itching and the subsequent development of a neurofibroma  ( 7 1 ). I n a d d i t i o n , t h e c o n d i t i o n a l N f 1 k n o c k - o u t m o u s e m o d e l h a s s h o of the mast cells in the neurofibroma microenvironment. Mast cell infiltrationin the t h i c k e n e dn e r v e sa n d n e u r o f i b r o m a so ft h ed o r s a lr o o ti sm o r e s u b s t a n t i a lw h e n t h e N n u l lS c h w a n n c e l l sp r o l i f e r a t ei na n N f 1 h e t e r o z y g o u sb a c k g r o u n dt h a n i nt h ew i l dt y p e background (46),and cutaneous mast cellsoutside of the tumours are also moderately i n c r e a s e da n dd e m o n s t r a t ee n h a n c e dg r o w t h compared to wild type mice (74). Mast cells synthesize and store several factors that are involved in angiogenesis and cellularproliferation.When stimulated to degranulate, mast cells can secrete allof these products or a selection of them. Mast cells synthesize and express c-Kit, a tyrosine k i n a s e r e c e p t o r t h a t i s t h e p r o d u c t o f t h e c-Kit p r o t o - o n c o g e n e . T h e l i g a n d f o r c - K which is called SCF or KitL,promotes proliferation,migration and survival of mast cells. KitL is also expressed from normal Schwann cellsand MPNST celllinesderived from individualswith NF1, while c-Kit is not expressed in Schwann cells from normal individuals(75). The expression of KitL by Schwann cells could recruitmast cellsto the area, and, ifstimulated to degranulate by local trauma or other means, could promote the formation or subsequent growth of neurofibromas. The expression of c-Kit by abnormal Schwann cells raises the possibilityof autocrine-stimulated growth of Schwann cells.  18  Ifthey are involved in the pathogenesis of neurofibromas, itis not known whether mast cells act during tumour initiationor progression or iftheir involvement differs among differenttypes of neurofibromas. Malignant Peripheral Nerve Sheath Tumours  People with NF1 have an average decrease in lifeexpectancy of 15 years (25). MPNSTs are one of the most frequent causes of death among people with NF1 (25). MPNSTs h a v e a p o o r p r o g n o s i s b e c a u s e m e t a s t a s e s t o t h e l u n g , l i v e r ,b r a i n , s o f t tissue,bone, regional lymph nodes, skin or retroperitoneum are common (76). The lifetimeriskfor an MPNST i s 8t o 1 3 % i n N F 1 p a t i e n t s ( 7 7 ) . T h e r i s k i s e v e n h i g h e r ( 1 6 2 5 % ) f o r i n d i v i d u a l s w i t h N F 1 w h o s e p a t h o g e n i c m u t a t i o n i s a d e l e t i o n o f t h e w h o l e NF1 gene (78). MPNSTs are true neoplasms, resulting from the clonal expansion of acell population t h a t h a s s u s t a i n e d m u t a t i o n s o f b o t h NF1 a l l e l e s a n d l i k e l y a d d i t i o n a l m u t a t i o n ( s ) a t other locito allow for malignant progression. Studies performed on both fresh MPNST samples and celllinesderived from MPNSTs have demonstrated clonalityand loss of N F 1 e x p r e s s i o n , a s w e l l a s a n i n c r e a s e i n r a s a c t i v i t y ( 7 9 - 8 1 ) . M o s t , i fn o t a l l , M P N S T s in NF1 patients appear to develop from pre-existing plexiform neurofibromas (82, 83). In contrast, cutaneous neurofibromas rarely, ifever, progress to malignancy. Given the factthat many NF1 patientsdevelop hundreds or thousands of times more cutaneous neurofibromas than plexiform neurofibromas, this difference in malignant potential is striking.The number, size, and type of benign neurofibromas vary greatlyamong NF1 patients, but itis not known ifan individual'srisk for developing an MPNST bears any relationship to the burden of benign neurofibromas or ifhaving one particulartype of neurofibroma increases this risk. Research Objectives  1. Determine  i fa n i n d i v i d u a l ' sr i s k f o r a n M P N S T  is dependent  on tumour burden  or  b u r d e n o fo n e p a r t i c u l a rt u m o u rt y p e . . 2. Determine the proportion of Schwann diffuseand nodular neurofibromas.  cells and mast cells inhistologically-defined  19  3 . Determine the presence or absence of the neurofibromin protein in Schwann cells and non-Schwann cells within histologically-defined diffuse and nodular neurofibromas. 4. Determine if Schwann cells expressing or not expressing neurofibromin and nonSchwann cells expressing neurofibromin are clonal in histologically-defined diffuse and nodular neurofibromas.  Hypotheses: Risk of developing MPNSTs A s the number of neurofibromas that an individual has increases, the probability that one of them may become malignant may also increase. Since subcutaneous, nodular and diffuse plexiform neurofibromas can progress to malignancy, I hypothesize that individuals who have these types of neurofibromas have a higher risk of developing M P N S T s compared to individuals without these types of neurofibromas. Mast cell distribution in neurofibromas Individuals with NF1 complain of intense "itchiness" in areas before cutaneous neurofibromas develop and the improper tissue formation and invasiveness of diffuse plexiform neurofibromas are likely to trigger mast cell infiltration. Therefore, I hypothesize that histologically-defined diffuse neurofibromas will have a high proportion of mast cells present. The observation that mice null for Nf1 exhibit enlarged peripheral nerves and nodular plexiform neurofibromas with a higher density of mast cells on an Nf1 heterozygous background compared to a wild type background suggests that mast cells are involved in this type of neurofibroma development. Therefore, I hypothesize that the number of mast cells will be high in nodular neurofibromas because they will be drawn into the tumour by the NFT  A  Schwann cells.  Histologically-defined diffuse neurofibromas I hypothesize that histologically-defined diffuse neurofibromas develop as a result of excessive proliferation of haploinsufficient NF1 progenitor cells (Figure 1.6). Therefore, neurofibromin expression should be observed in all cell types, including Schwann cells, in these tumours, and all cells should exhibit a polyclonal phenotype. The hypothesis that histologically-defined diffuse neurofibromas develop from non-neoplastic cellular proliferation is supported by knockout mouse studies in which Nf1 ~Schwann +/  20  cells  s h o w i n c r e a s e d i n v a s i v e n e s s a n d a n g i o g e n e s i s c o m p a r e d t o S c h w a n n c e l l sf r o m w i l d t y p e l i t t e r - m a t e s ( 8 4 ) a n d s k i n f i b r o b l a s t s p r o l i f e r a t eb e y o n d t h e n o r m a l t i m e o f maturation in wound healing (85). Histologically-defineddiffuse neurofibromas include both clinically-defined cutaneous and most diffuseplexiform neurofibromas. The anecdotal evidence cited above that local trauma may be a factor in the development of cutaneous neurofibromas is consistent with their being proliferativerather than neoplastic lesions. In addition, fibroblastsobtained from scars of normal.humans display an increase in neurofibromin signal intensityby immunohistochemistry, suggesting that increased neurofibromin activitymay play a role in the normal response of fibroblasts to tissue injury (86) and that thisresponse may be abnormal in people with NF1. Clinically-defined diffuse plexiform neurofibromas almost always develop early in life. At least some diffuse plexiform neurofibromas are present before the 16 week of gestation because the hair and pigmentation patterns in skin overlying the tumours is altered in a manner that cannot occur later in fetal development (87). Itis possible that h a p l o i n s u f f i c i e n c y f o rN F 1 c o u l d d i s r u p t t h e c h e c k sa n d b a l a n c e s t h a t n o r m a l l y r e g u the growth of fetaltissues. Normal fetaldevelopment entails sequential events dependent on the proper regulation of cellularproliferation,migration, differentiation and death. In most cases these processes require coordinated regulation of several factors within and between cell types. Ifthe level of one or more of these factors is not optimal, a particular pathway may be disrupted. t h  21  Figure 1.6 Haploinsufficient model of tumour development in histologicallydefined diffuse neurofibromas in NF1.  Schwann cells are represented by circlesand allother cells are represented by s q u a r e s . T h e b l u e c o l o u r r e p r e s e n t s a l l e l e A a t t h e AR l o c u s a n d r e d r e p r e s e n t s a l l e l e B . A . A n o r m a l t i s s u e i n a n o n - N F 1 i n d i v i d u a l h a s t w o f u n c t i o n a l c o p i e s o f t h e NF1 g e n e (+/+) in all cells. When there is astimulus to proliferate the cells are kept in check by the t w o f u n c t i o n a l c o p i e s o f NF1. B . I n i n d i v i d u a l s w i t h N F 1 , a l l c e l l s a r e h e t e r o z y g o u s f o r N F 1 ( + / - ) . W h e n t h e r e i s as t i m u l u s t o p r o l i f e r a t e , a l lc e l l sw i l l e x p a n d i n n u m b e r because half the amount of neurofibromin is not sufficientto keep the cells in check, resulting in atumour from this abnormal proliferation.The tumour will be polyclonal b e c a u s e t h e r e i s a m i x t u r e o f a l l e l e A a n d a l l e l e B a t t h e AR l o c u s i n t h e c e l l s t h a t proliferated. Stimulus to proliferation  A  Normal tissue  Tumour  B  NF1  + /  -tissue  J Non S c h w a n n cell e.g. fibroblasts, mast cells, lymphocytes (~^) Schwann cell  Allele A  Allele B  Histologically-defined nodular neurofibromas  I hypothesize that histologically-defined nodular neurofibromas develop by a "second h i t " m u t a t i o n o f t h e NF1 g e n e i n S c h w a n n c e l l s ( F i g u r e 1 . 7 ) . T h e r e f o r e , n e u r o f i b r o m i n expression should not be observed in the majority of Schwann cells but willbe observed in allother cells in this tumour. Some Schwann cells may demonstrate neurofibromin expression ifthey are drawn into the tumour, along with other cell types, by the n e o p l a s t i c S c h w a n n c e l l s .S c h w a n n c e l l st h a t d o n o t e x p r e s s n e u r o f i b r o m i n s h o u l d 22  exhibit aclonal phenotype, cell types should  while Schwann  exhibit apolyclonal  cells expressing  neurofibromin  and all other  phenotype.  Figure 1.7 " S e c o n d hit" model of development for histologically-defined nodular neurofibromas in NF1.  Schwann cells are represented by circlesand allother cells are represented by s q u a r e s . A l l e l e s Aa n d Ba r e r e p r e s e n t e d b y b l u e a n d r e d , r e s p e c t i v e l y . I n a n N F 1 i n d i v i d u a l , a l l c e l l s a r e h e t e r o z y g o u s f o r NF1 ( + / - ) . I n t h i s m o d e l t h e r e i s a m i x t u r e o f allele Aand allele Bin Schwann cells and all other cell types. Asecond somatic m u t a t i o n a t t h e NF1 l o c u s o c c u r s i n o n e S c h w a n n c e l l ( - / - ) t h a t p r o v i d e s a g r o w t h a d v a n t a g e t o t h a t c e l l , w h i c h a l l o w s i tt o e x p a n d c l o n a l l y t o f o r m a t u m o u r . A s a l l N F 1 ~ Schwann cells were allderived from the one progenitor, these cells willdisplay a clonal p h e n o t y p e , w h i l e NF1 ~ S c h w a n n c e l l s a n d a l l o t h e r c e l l s w i l l b e p o l y c l o n a l b e c a u s there was a mixture of both allele Aand allele Bin the tissue to begin with. +I  C e l l s Polyclonal NF1 +/  2 n d " h i t " a t NF1 locus in Schwann cells  S c h w a n nc e l l su clonal proliferation to form a tumour NF1-'-  J Non S c h w a n n cell e.g. fibroblasts, mast cells, lymphocytes ( ^ ) Schwann cell  Allele A  Allele B  Relevance  There is currently no way of predicting which individualswith NF1 will neurofibromas or MPNSTs. MPNSTs are one of the leading causes of people with NF1, and thisis often because the MPNST has already me by the time itis detected. Itwould be helpful to be able to predict which at high riskfor developing MPNST in order to monitor them closely for malignancy, astrategy that may improve the chances of survival.  develop death amo tastasized individuals early signs  ng widely are of  Since most MPNSTs develop from pre-existingneurofibromas,understanding how differenttypes of neurofibromas develop could help treatthem and prevent some from p r o g r e s s i n g t o m a l i g n a n c y . T h e NF1 g e n e i s k n o w n t o h a v e a h i g h m u t a t i o n r a t e . L O H 23  a n d s u b t l e" s e c o n dh i t "m u t a t i o n sh a v e b e e n o b s e r v e di nt h e N F 1 g e n e , b u tt h i sa l o n e does not prove that a neurofibromin-like protein is not expressed. Looking at n e u r o f i b r o m i n e x p r e s s i o n r a t h e r t h a n L O H w i l lp r o v i d e i n f o r m a t i o n o n t h e p r o p o r t i o n o f S c h w a n n c e l l s t h a t e x p r e s s n e u r o f i b r o m i n , a n d a s s e s s i n g c l o n a l i t yw i l ls h o w h o w t h e cells proliferatedto form the tumour. This information could help in the development of treatments, since treating a tumour that has Schwann cells that do not express n e u r o f i b r o m i n m a y i n v o l v e d i f f e r e n ta p p r o a c h e s t h a n i ft h e S c h w a n n c e l l s a r e haploinsufficient,retainingsome neurofibromin expression. Most studies treat all neurofibromas as a single group, failing to take into consideration h i s t o l o g i c a la n d c l i n i c a l d i f f e r e n c e s t h a t m a y b e i n d i c a t i v e o f d i f f e r e n t p a t h o g e n i c mechanisms. 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M e t h y l a t i o n a n a l y s neurofibromatosis type 1 (NF1) promoter in peripheral nerve sheath tumours. Eur J Cancer 2004;40:2820-8. 68. Horan MP, Cooper DN, Upadhyaya M. Hypermethylation of the neurofibromatosis type 1 (NF1) gene promoter is not a common event in the inactivationof the NF1 gene in NF1-specifictumours. Hum Genet 2000;107:33-9. 6 9 . T a k a h a s h i K , S u z u k i H ,K a y a m a T , e ta l . M u l t i p l e t r a n s c r i neurofibromatosis type 1 gene in human brain and in brain tumours. Clin Sci (Lond) 1994;87:481-5. 70. Skuse GR, Cappione AJ. RNA processing and clinicalvariabilityin neurofibromatosis type I (NF1). Hum Mol Genet 1997;6:1707-12. 71. Riccardi VM. Cutaneous manifestation of neurofibromatosis: cellularinteraction, pigmentation, and mast cells.BirthDefects Orig Artie Ser 1981;17:129-47. 72. Isaacson P. Mast cells in benign nerve sheath tumours. J Pathol 1976;119:193-6. 73. Johnson MD, Kamso-Pratt J, Federspiel CF, Whetsell WO, Jr.Mast cell and lymphoreticular infiltratesin neurofibromas. Comparison with nerve sheath tumors. Arch Pathol Lab Med 1989;113:1263-70. 7 4 . Y a n gF C ,I n g r a m D A ,C h e nS , e fa l . N e u r o f i b r o m i n - d e f i c i e n t S c h w a n secrete a potent migratory stimulus for Nf 1 +/- mast cells. J Clin Invest 2003;112:1851-61. 7 5 . R y a n J J , K l e i n K A , N e u b e r g e rT J , e ta l . R o l ef o rt h es t e m c e l lf a c i n S c h w a n n c e l l n e o p l a s i a a n d m a s t c e l l p r o l i f e r a t i o na s s o c i a t e d w i t h neurofibromatosis. J Neurosci Res 1994;37:415-32. ;  29  76.  Ducatman BS, Scheithauer BW, Piepgras DG, Reiman HM, llstrupDM. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer 1986;57:2006-21. 7 7 . E v a n s D G , B a s e r M E , M c G a u g h r a n J , e ta l . M a l i g n a n t p e r tumours in neurofibromatosis 1. J Med Genet 2002;39:311-4. 7 8 . D e R a e d tT , B r e m sH , W o l k e n s t e i n P , e f a l . E l e v a t e d r i s kf o rM P N S T i n N F 1 microdeletion patients.Am J Hum Genet 2003;72:1288-92. 7 9 . B a s u T N , G u t m a n n D H , F l e t c h e rJ A ,e f a l . A b e r r a n t r e g u l a t i o n o f r a sp r o t e i malignant tumour cells from type 1 neurofibromatosis patients. Nature 1992;356:713-5. 80. DeClue JE PA, FletcherJA, Diehl SR, Ratner N, Vass WC, Lowy DR. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell 1992;69:265-73. 8 1 . G u h aA ,L a u N ,H u v a rI , e ta l . R a s - G T P l e v e l sa r ee l e v a t e d i n h u m peripheralnerve tumors. Oncogene 1996;12:507-13. 82. Korf BR. Malignancy in neurofibromatosis type 1. Oncologist 2000;5:477-85. 83. Woodruff JM. Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis. Am J Med Genet 1999;89:23-30. 84. K i m H A , L i n g B , R a t n e r N . N f 1 - d e f i c i e n tm o u s e S c h w a n n c e l l s a r e a n g i o g e n i c and invasive and can be induced to hyperproliferate:reversion of some p h e n o t y p e s b y a n i n h i b i t o ro f f a r n e s y l p r o t e i n t r a n s f e r a s e . M o l C e l l B i o l 1997;17:862-72. 85. AtitRP, Crowe MJ, Greenhalgh DG, Wenstrup RJ, Ratner N. The Nf1 tumor s u p p r e s s o r r e g u l a t e s m o u s e s k i n w o u n d h e a l i n g , f i b r o b l a s tp r o l i f e r a t i o n ,a n d collagen deposited by fibroblasts. J Invest Dermatol 1999;112:835-42. 8 6 . Y l a - O u t i n e n H , A a l t o n e n V ,B j o r k s t r a n d A S , e t a l . U p r e suppressor protein neurofibromin in normal human wound healing and in vitro e v i d e n c e f o r p l a t e l e td e r i v e d g r o w t h f a c t o r ( P D G F ) a n d t r a n s f o r m i n g g r o w t h f a c t o r - b e t a l ( T G F - b e t a 1 ) e l i c i t e di n c r e a s e i n n e u r o f i b r o m i n m R N A steady-state levels in dermal fibroblasts. J Invest Dermatol 1998;110:232-7. 87. Riccardi VM. Histogenesis control genes and neurofibromatosis 1. Eur J Pediatr 2000;159:475-6.  30  2. A S S O C I A T I O N B E T W E E N B E N I G N A N D M A L I G N A N T N E R V E S H E A T H T U M O U R S IN N F 1  PERIPHERAL  1  INTRODUCTION  Neurofibromatosis 1(NF1) is an autosomal dominant disease affecting 1in 4000 people. NF1 is characterized by multipledermal neurofibromas, plexi neurofibromas, malignant peripheralnerve sheath tumours (MPNSTs), and o t h e r C N S g l i o m a s , a s w e l l a s b y c a f e - a u - l a i ts p o t s a n d a b n o r m a l i t i e s o f t h cardiovascularand centralnervous systems. The NF1 gene is located on 17q11.2, and its protein, neurofibromin, functions as atumour suppressor.  3000 to 1 in form optic and eskeletal, chromosome  P e o p l e w i t h N F 1 h a v e ad e c r e a s e i n l i f ee x p e c t a n c y o f 1 5 y e a r s , w i t h M P N S T s as a leading cause of death in young adults (1, 2). MPNSTs have apoor prognosis because m e t a s t a s e s t o t h e l u n g , l i v e r ,b r a i n , s o f t t i s s u e , b o n e , r e g i o n a l l y m p h n o d e s , s k i n o r retroperitoneum are common (3).The frequency of MPNSTs in the general population is 0.001%, compared to 2-5% in people with NF1 (4).The lifetimeriskfor MPNST is 8 to 13% in NF1 patients (5).Most studies show that the peak incidence of MPNSTs is in the 7th decade of lifein the general population but in the 3rd or 4th decade in people with NF1 (5),although these tumours may occur at amuch younger age in either population (3). Some patientswith NF1 a example, people with NF1 gene are thought to have There are several reports another tumour, as might mutation of the NF1 tumou Most, ifnot all neurofibromas among NF1 pa MPNST bears 1  p p e a r a t g r e a t e r r i s kt h a n o t h e r s o f d e v e l o p i n g M P N S T s . For whose pathogenic mutation is adeletion of the whole NF1 alifetimeriskof 16 to 25% for developing an MPNST (6). of MPNSTs developing in the fieldafter radiation treatment for be expected for individualswho carry one constitutional r suppressor gene (3, 7).  ,MPNSTs in NF1 pa (8, 9). The number, tients, but itis not k any relationshipto  tients appear to develop from pre-existing plexiform size, and type of benign neurofibromas vary greatly nown whether an individual'sriskfor developing an the burden of benign neurofibromas.  A v e r s i o n o f t h i s c h a p t e r h a s b e e n p u b l i s h e d . T u c k e r T, W o l k e n s t e i n P, R e v u z J , Z e l l e r J , F r i e d m a n J M  A s s o c i a t i o n b e t w e e n b e n i g n a n d m a l i g n a n t p e r i p h e r a l n e r v e s h e a t h t u m o r s in N F 1 . N e u r o l o g y . 2 0 0 5 J u l 26;65(2):205-ll.  31  One study found that the greatest riskfactors associated with MPNSTs in NF1 patients w e r e p a i n r e l a t e d t o am a s s a n d t h e p r e s e n c e o f s u b c u t a n e o u s a n d cutaneous neurofibromas (10). In this study, the median ages of the NF1 patients with MPNSTs w a s 2 6 . 4 y e a r s a n d w i t h o u t M P N S T s w a s 1 4 . 7 y e a r s , b u t t h i s d i f f e r e n c ew a s n o t considered in the analysis.The presence of both subcutaneous and cutaneous neurofibromas is dependent on age, with the majority of people above the age of 16 having at least one of these tumours (11).Therefore, many people in the group of patientswith NF1 without MPNSTs would not be old enough to exhibitsubcutaneous or c u t a n e o u s n e u r o f i b r o m a s , w h e r e a s a l m o s t a l lo f t h o s e w i t h M P N S T s w o u l d b e e x p e c t e d to have dermal tumours just on the basis of their age. W e u s e d l o g i s t i c r e g r e s s i o n t o d e t e r m i n e i fa s s o c i a t i o n s e x i s t b e t w e e n M P N S T and cutaneous, subcutaneous, superficialplexiform or internalplexiform neurofibromas among patientswith NF1. METHODS Subjects and Data Description  All patients included in this analysis were diagnosed with NF1 according to es c l i n i c a lc r i t e r i a( 1 2 , 1 3 ) . D a t a w e r e o b t a i n e d o n a l l N F 1 p a t i e n t s s e e n i n t h e N F t h e H e n r i - M o n d o r H o s p i t a l( R e s e a u N F - M o n d o r ) b e t w e e n J u n e 1 9 8 8 a n d M a y the time of the analysis, thisdatabase had extensive demographic and clinica 476 NF1 probands, 31 of theiraffected parents and 16 affected children.Only were included in the present analysis.The study protocol was approved by the Ethics Committee.  tablished clinic at 2004. At ldata on probands hospital  We analyzed cross-sectionaldata on cutaneous neurofibromas, subcutaneous n e u r o f i b r o m a s , a n d i n t e r n a l a n d s u p e r f i c i a lp l e x i f o r m n e u r o f i b r o m a s r e c o r d e d a t t h e m o s t r e c e n t c o m p l e t e c l i n i c a l a s s e s s m e n t . A l l c l i n i c a l l y - a p p a r e n tn e u r o f i b r o m a s w e r e included,regardless of size. Discrete dermal tumours seen on physical exam were c o n s i d e r e d t o b e c u t a n e o u s n e u r o f i b r o m a s i ft h e y m o v e d w i t h t h e s k i n a n d s u b c u t a n e o u s n e u r o f i b r o m a s i ft h e s k i n m o v e d o v e r t h e l e s i o n ( 1 4 ) . S u p e r f i c i a l plexiform neurofibromas are more diffusethan the discrete tumours and involve the skin or subcutaneous tissue, which is thickened or raised, and the overlying skin is often hyperpigmented or hypertrichotic(14). Internalplexiform neurofibromas were not observed on physical examination but were identifiedby imaging studies. 32  Data were obtained during routine clinicalassessment of these patients in the NF Clinic. T h e s t a n d a r d p r o t o c o l w a s t o p e r f o r m ap h y s i c a l e x a m i n a t i o n a n d c l i n i c a lh i s t o r y o n each patient yearly. Between 1988 and 1995, patients also received a screening o p h t h a l m o l o g i c e x a m u s i n g as l i tl a m p , c h e s t x - r a y , a b d o m i n a l u l t r a s o u n d exam, cerebral CT or MRI, and 24-hour urinarycatecholamine analysis every fiveyears to look for common problems associated with NF1. Additionalstudies were done to investigate a n y s y m p t o m s o f i n t e r n a ln e u r o f i b r o m a s o r M P N S T t h a t o c c u r r e d . A f t e r 1 9 9 5 , t h e s c r e e n i n g w a s d i s c o n t i n u e d a n d i m a g i n g s t u d i e s w e r e o n l y d o n e t o i n v e s t i g a t ep a t i e n t symptoms. All informationwas recorded using the standard format and consistent d e f i n i t i o n s o f c l i n i c a lf e a t u r e s d e v e l o p e d f o r t h e N a t i o n a l N e u r o f i b r o m a t o s i s Foundation InternationalDatabase (15). T h e n u m b e r o f c u t a n e o u s n e u r o f i b r o m a s p r e s e n t i n ap a t i e n t w a s c o u n t e d o r e s t i m a t e d on physical exam and recorded semi-quantitativelyas none, 1-9, 10-100 or greater than 100. Subcutaneous and superficialplexiformneurofibromas were recorded as present o r a b s e n t . T h e p r e s e n c e o r a b s e n c e o f i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s w a s determined by routineabdominal ultrasoundand chest x-ray examinations. MRI or CT examinations were performed when evidence of an internalplexiformneurofibroma was seen on the ultrasound exam or chest radiograph or when the patienthad symptoms s u c h a s p a i n o r n e u r o l o g i c a l d e f i c i t ss u g g e s t i v e o f a n i n t e r n a l p l e x i f o r m n e u r o f i b r o m a . Patients coded as 'unknown' for a particular feature were not considered in models involving that feature. MPNSTs were suspected by clinicalexamination or by imaging studies and were c o n f i r m e d b y b i o p s y . P r o b a n d s w i t h M P N S T s w e r e f o l l o w e d l o n g i t u d i n a l l ye v e r y t h r e e months. Ifa patient did not attend the follow-up appointment, the status of the patient was determined by a telephone call to the individual or family. Statistical Analysis  Non-parametric Kruskal-Wallis and Mann-Whitney U tests were calculated using SPSS (SPSS, Inc.,Chicago, Illinois, 1998) to assess the relationship of age to each type of neurofibroma. A p-value less than or equal to 0.05 was considered to be significant. L o g i s t i cr e g r e s s i o n m o d e l s w e r e d e v e l o p e d u s i n g S P S S s o f t w a r e ( S P S S , I n c . , C h i c a g o , Illinois, 1998). Many NF1 features have a higher prevalence in older patients, so age 33  ( c o d e d t o t h e n e a r e s t y e a r ) a t e x a m w a s t r e a t e d a s ac o v a r i a t e i n a l l a n a l y s e s . T h e f i r s t s e t o f m o d e l s h a d t h e p r e s e n c e o f i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s a s t h e r e s p o n s e v a r i a b l e a n d e i t h e r c u t a n e o u s , s u b c u t a n e o u s o r s u p e r f i c i a lp l e x i f o r m n e u r o f i b r o m a s a s the explanatory variable.The second set of models had the presence of an MPNST as the response variable and cutaneous, subcutaneous, superficialplexiform or internal plexiform neurofibromas as the explanatory variable.Associations with explanatory v a r i a b l e s w e r e c o n s i d e r e d t o b e s i g n i f i c a n ti ft h e 9 5 % C I o f t h e o d d s r a t i o e x c l u d e d 1 . 0 . Survival rates from the time of MPNST diagnosis were calculated for allprobands with M P N S T s w h o h a d d i e d o r w e r e s t i l l l i v i n ga t t h e t i m e o f t h e a n a l y s i s ( M a y 2 0 0 4 ) . F i v e y e a r s u r v i v a lb y g e n d e r w a s d e t e r m i n e d u s i n g K a p l a n - M e i e r c u r v e s , a n d t h e s i g n i f i c a n c e o f g e n d e r o n s u r v i v a l w a s d e t e r m i n e d u s i n g al o g r a n k t e s t ( S P S S , I n c . , Chicago, Illinois, 1998). RESULTS Prevalence of Neurofibromas  W e s t u d i e d 4 7 6 p r o b a n d s . 9 3 % o f t h e s e N F 1 p a t i e n t sa r e f r o m F r a n c e . S u b j e c t a g e a t examination ranged from 1 to 77 years, with an average of 33 years (median = 31 years). S e m i - q u a n t i t a t i v ed a t a o n c u t a n e o u s n e u r o f i b r o m a s w e r e a v a i l a b l e f o r 4 4 3 N F 1 probands. Cutaneous neurofibromas were recorded as absent in 66 (15%) of the individualsand present in the rest.Sixty-eight (15%) of the patients had 1-9 cutaneous neurofibromas,170 (38%) had 10-100 cutaneous neurofibromas and 139 (31%) had more than 100 cutaneous neurofibromas: The average age (± 1 standard deviation) for i n d i v i d u a l s w i t h o u t c u t a n e o u s n e u r o f i b r o m a s w a s 2 1 ±1 0 y e a r s , f o r i n d i v i d u a l s w i t h 1 - 9 neurofibromas was 27 ±11 years, for individualswith 10-100 neurofibromas was 33 ± 12 years and for individualswith more than 100 cutaneous neurofibromas was 42 + 12 y e a r s . T h e s e v a l u e s w e r e s i g n i f i c a n t l yd i f f e r e n tf r o m e a c h o t h e r ( p < 0 . 0 0 1 ) . O f t h e 4 4 3 p r o b a n d s w i t h i n f o r m a t i o no n c u t a n e o u s n e u r o f i b r o m a s , 2 4 8 ( 5 6 % ) w e r e f e m a l e . 8 8 % o f the females had one or more cutaneous neurofibromas compared to 67% of males. The average age of females for each of the categories of cutaneous neurofibromas was higher than that of the males, but the differences are not significant.  34  Subcutaneous neurofibromas were present in 224 (48%) of the 468 individuals on whom information was available.The mean age of patients with (34 ± 14 years) and without (33 + 13 years) subcutaneous neurofibromas was not significantlydifferent. Superficialplexiform neurofibromas were present in 194 (41%) of the 471 individuals on w h o m i n f o r m a t i o nw a s a v a i l a b l e . T h e m e a n a g e o f p a t i e n t s w i t h a n d w i t h o u t s u p e r f i c i a l plexiform neurofibromas was the same (33 ± 14 years). There were 145 NF1 probands who received at least a routine abdominal ultrasound e x a m a n d c h e s t r a d i o g r a p h t o i d e n t i f yi n t e r n a l p l e x i f o r m n e u r o f i b r o m a s . E i g h t y - s i x o f these individuals underwent imaging studies as part of a routine screening protocol, and the other 59 people had imaging studies to investigatesymptoms suggestive of an i n t e r n a lt u m o u r . T h e r e w e r e 5 4 i n t e r n a l p l e x i f o r m n e u r o f i b r o m a s i d e n t i f i e d i n 4 9 ( 3 4 % ) probands. Twenty-eight plexiform neurofibromas presented with pain, and 26 were a s y m p t o m a t i c . F o r t y - f o u ro f t h e 4 9 p e o p l e w h o w e r e f o u n d t o h a v e i n t e r n a lp l e x i f o r m n e u r o f i b r o m a su n d e r w e n t C T o r M R I e x a m s . T h e C T o r M R I w a s d o n e b e c a u s e o f symptoms of an internaltumour in 30 patients and for confirmation after an abnormal abdominal ultrasound or chest x-ray exam in the other 14 individuals. The average age of patients with internalplexifor compared to 34 ± 14 years for individuals without (p=0.48). Of the internalplexiform neurofibromas the spinal nerve roots or ganglia, nine (17%) were and two (4%) were non-spinal in the chest.  m neurofibromas was 36 ± 14 years, internal plexiform neurofibromas that were identified,43 (80%) were of non-spinal in the abdomen or pelvis  There were 19 spinal neurofibromas that did not present with symptoms to the 18 i n d i v i d u a l s a f f e c t e d . T w e n t y - f o u rs p i n a l t u m o u r s w e r es y m p t o m a t i c i n2 1 i eight complained of pain, nine had neurological symptoms of spinal cord compression, five had both pain and symptoms of spinal cord compression, one complained of pain a n d h a d b o n e d e s t r u c t i o n ,a n d a n o t h e r c o m p l a i n e d o f s y m p t o m s o f c o m p r e s s i o n o f t h e duodenum. One individualwho had compression of the spinal cord experienced paralysis involvingthe region below the area of compression, and two other individuals had respiratory distress and subsequently died as a consequence of theirspinal cord compression.  35  Two individualshad non-spinal chest neurofibromas; both were asymptomatic. Nine non-spinal tumours of the abdomen or pelvic region were identifiedin eight individuals, five of whom were asymptomatic. One patient who had a neurofibroma in the region of t h e p a n c r e a s d e v e l o p e d c h o l e s t a s i s , a n o t h e r i n d i v i d u a lw i t h i n t r a - a b d o m i n a l neurofibromas had gastrointestinalbleeding,and another had abdominal pain and was found to have a neurofibroma of the pelvis. O f t h e 4 6 9 i n d i v i d u a l s w i t h i n f o r m a t i o n o n M P N S T s t a t u s ,2 5 ( 5 % ) h a d MPNSTs. Twenty-four of these 25 patients were symptomatic, with the majority of individuals complaining of pain in the area and enlargement of a previously-recognized tumour ( T a b l e 2 . 1 ) . T h e i n d i v i d u a lw h o d i d n o t p r e s e n t w i t h s y m p t o m s h a d a n M P N S T o f a lower limb that was discovered during an operation for pseudarthrosis [Patient24]. The average age of those with MPNSTs (31 ± 12 years) did not differ(p=0.35) from the a v e r a g e a g e o f i n d i v i d u a l sw i t h o u t M P N S T s ( 3 3 ± 1 2 y e a r s ) .A l l2 5 o f t h e s e MPNSTs w e r e s h o w n p a t h o l o g i c a l l yt o c o n t a i n e l e m e n t s o f b e n i g n p l e x i f o r m n e u r o f i b r o m a a n d , t h e r e f o r e , a r e l i k e l yt o h a v e a r i s e n f r o m a p r e - e x i s t i n g b e n i g n t u m o u r . T h e MPNST arose in one of the internalbenign plexiform tumours that had previously been imaged in eight of the 20 cases in which imaging studies has been performed. Seven more of these 20 MPNSTs m a y h a v e a r i s e n i n r e l a t i o n s h i pt o a t u m o u r t h a t w a s i m a g e d b u t cannot be confirmed to have done so. The remaining fiveMPNSTs arose in tumours that were apparently unrelated to the ones identifiedby imaging. The detailsof each are given in Table 2.1 and summarized below.  36  }  Table 2.1 Patient Number  Summary of Patient Data for those with M P N S T s . Sex  A g e at MPNST Diagnosis  Location of MPNST  Internal Plexiform Neurofibromas Seen on Imaging Studies  Survival (Months)  Status  1  M  22  Face  N D  30  D e a d  2  M  29  Face  N o n e  84  Alive  3  M  32  Face  L3-S1  3  D e a d  4 5  F F  39 4 9  Cervical C 2  N D C 2 - C 7  108 15  Alive D e a d  6  F  4 9  C 2 - C 3  C 2 - C 6  60  Alive  7  F  17  T r u n k .  N D  12  D e a d  8  18 27  Trunk  9  F M  Thoracic T1-T3  12 4  D e a d Alive  10  M  30  N D  29  D e a d  11  F  20  N D  29  Alive  12  M  24  Abdominal L u m b a r Spine L2  L1-L2  0  13 14  25  S a c r u m  15  F M F  31 31  48 60 19  16  F  33  L5-S2  23  D e a d  17  M  34  L4-L5 Pelvis Sciatic Nerve L2-L3  T5-S1 L4 Lumbar &  D A A D  L2-L3  14  D e a d  18  F  37  Upper  Limb  Cervical  36  Alive  19  M  53  Upper  Limb  S 1 - S 2  12  D e a d  20  M  56  Upper  Limb  N o n e  84  Alive  21  F  15  Lower  Limb  L5-S1  52  D e a d  22  F  19  Lower  Limb  3  D e a d  23  F  21  Lower  Limb  55  Alive  24  M  21  Lower  Limb  108  Alive  16  D e a d  T1  L 2 - L 3  Sacral  & T 9 - T 1 0 N o n e  Lumbar  &  L u m b a r Lower Limb M 36 25 ND =Imaging studies for internal plexiform neurofibromas not M = Male; F= Female  37  Sacral done  e a d live live e a d  Three individuals[Patients 1, 2, and 3] had MPNSTs of the face that developed from previously-identified plexiform neurofibromas. P a t i e n t 3a l s o h a d as p i n a l neurofibroma a t L 3 - S 1 . P a t i e n t 2r e c e i v e d ab o d y C T s c a n o n l y a f t e r h i s r e f e r r a l t o t h e H e n r i Mondor H o s p i t a l f o r s u s p i c i o n o f a n M P N S T , a n d n o i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s w e r e found. Patient 1 did not have imaging studies performed to look for internal plexiform neurofibromas. Eight internal MPNSTs were confirmed pathologically to originate from a previouslyidentifiedinternal plexiform neurofibroma; allwere tumours of the spinal nerves [Patients 5, 6, 9, 12, 13, 14, 16 and 17]. A pelvic MPNST was identified in an individual who had internalplexiform neurofibromas of the lumbar and sacral spine [Patient 15]. A n o t h e r i n d i v i d u a lw a s f o u n d t o h a v e a n i n t e r n a lp l e x i f o r m n e u r o f i b r o m a o f t h e t h o r a c i c spine and developed an MPNST of the trunk [Patient8].Both of these malignancies w e r e s u s p e c t e d t o h a v e o r i g i n a t e d f r o m p r e v i o u s l y - u n i d e n t i f i e di n t e r n a l p l e x i f o r m neurofibromas, and pathology reportsdemonstrated benign components in the MPNST. Three MPNSTs o f t h e u p p e r l i m b [ P a t i e n t s 1 8 , 1 9 a n d 2 0 ] a n d 5o f t h e l o w e r l i m b [ 2 1 , 22, 23, 24, and 25] developed from previously-identifiedplexiform tumours outside of the area imaged. Six of these individuals[Patients 18, 19, 21, 22, 24 and 25] also had internalplexiform neurofibromas of the spine. Both individualswho did not have internal p l e x i f o r m n e u r o f i b r o m a s [ P a t i e n t s2 0 a n d 2 3 ] h a d n o r m a l a b d o m i n a l u l t r a s o u n d a n d chest x-ray exam three years before diagnosis of the MPNST. MPNSTs of individuals[ internalplex components  the cervicalspine, lumbar spine, abdomen and trunk were identifiedin four Patients 4, 7, 10 and 11] who had no previous imaging performed to identify iform neurofibromas. Pathology reports confirmed that there were benign in each of these malignant tumours. Logistic Regression  T h e l o g i s t i cr e g r e s s i o n a n a l y s i s o f c u t a n e o u s a n d i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s w a s l i m i t e dt o 1 3 9 N F 1 p r o b a n d s w h o h a d i n f o r m a t i o n o n t h e p r e s e n c e o f i n t e r n a lp l e x i f o r m neurofibromas as well as semi-quantitativedata on cutaneous neurofibromas. There w a s n o s i g n i f i c a n t a s s o c i a t i o n b e t w e e n c u t a n e o u s a n d i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s (Table 2.2).  38  Table 2.2 Summary of odds ratios and 95% confidence intervals for logistic regression models.  Response  Variable  Explanatory Variable*  Cutaneous Neurofibromas 1-9 10-100 > 100 Internal Plexiform Age (per year) Neurofibromas Subcutaneous Neurofibromas Age (per year) Superficial Plexiform Neurofibromas Age (per year) Cutaneous Neurofibromas 1-9 10-100 >100 Age (per year) Superficial Plexiform M P N S T Neurofibromas Age (per year) Subcutaneous Neurofibromas Age (per year) Internal Plexiform Neurofibromas Age (per year) Internal Plexiform Neurofibroma Subcutaneous M P N S T Neurofibroma Age (per year) *C o m p a r i s o n s w e r em a d e w i t h f e a t u r e s t h  39  Odds Ratio (95% Confidence Interval) 1.70 0.55 0.88 1.00 3.58  (0.49, (0.14, (0.36, (0.97, (1.68,  5.9 2.1 2.1 1.0 7.6  1 4 3 3 4  ) ) ) ) )  1.00 (0.98, 1.03) 0.74 (0.36,1.54) 1.00 (0.98, 1.00) 0.79 1.20 1.56 0.98 0.90  ( ( ( ( (  0 0 0 0 0  . . . . .  1 2 5 9 6  5, 8, 1, 4, 2,  4.08 5.13 4.80 1.02 1.32  ) ) ) ) )  1.02 (1.01-1.04) 2.81 (1.14, 6.94) 0.99 (0.95, 1.01) 20.52 (5.3, 79.5) 0.94 (0.90, 0.99) 18.06 (4.55, 73.4) 2.08 (0.60, 7.24) 0.94 (0.89, 0.98) a t w e r ea b s e n t i na n i n d i v i d u a l .  T i w a q  he associationbetween cutaneous neurofib n d i v i d u a l sw h o h a d s e m i - q u a n t i t a t i v ed a t a o n ell as informationon the presence or absence ssociation between the number of cutaneous uantitativescale and the presence of MPNSTs  r o m a sa n d M P N S T s w a s t e s t e d o n 4 3 9 numbers of cutaneous neurofibromas as of MPNSTs. There was nosignificant neurofibromas measured on a semi(Table 2.2).  The logisticregression analysis of subcutaneous neurofibromas and internal plexiform n e u r o f i b r o m a s w a s l i m i t e dt o 1 4 4 i n d i v i d u a l sw h o h a d i n f o r m a t i o n o n t h e p r e s e n c e o r absence of both internalplexiform and subcutaneous neurofibromas. Seventy-six of these patientshad subcutaneous neurofibromas, 35 (46%) of whom also had internal plexiform neurofibromas. In contrast,of the 68 patients who did not have subcutaneous neurofibromas, only 13 (20%) had internalplexiform neurofibromas. There was a significantassociation between the presence of subcutaneous and internal plexiform neurofibromas, with individualshaving subcutaneous neurofibromas being 3.58 times more likelyto have internal plexiform neurofibromas than individuals without subcutaneous neurofibromas (Table 2.2). Th su su NF as 2. to  e b b 1 s  r e w e r e 4 6 4 i n d i v i d u a l s w h o h a d i n f o r m a t i o no n p r e s e n c e o r a b s e n c e o f b o t h cutaneous neurofibromas and MPNSTs. Seventeen (8%) of the 222 patientswith cutaneous neurofibromas also had MPNSTs, compared to seven (3%) of the 242 individuals without subcutaneous neurofibromas. There was a significant o c i a t i o nb e t w e e n t h e p r e s e n c e o f s u b c u t a n e o u s n e u r o f i b r o m a s a n d M P N S T s ( T a b l e 2). Individualswith subcutaneous neurofibromas were almost three times more likely h a v e a n M P N S T a s i n d i v i d u a l sw h o d i d n o t h a v e s u b c u t a n e o u s n e u r o f i b r o m a s .  The association between 143 individualswho had neurofibromas. There wa superficialand internalp  superficialand interna informationon presence s n o s i g n i f i c a n ta s s o c i a lexiform neurofibromas (  lpl or tio Tab  exiform neurofibromas was tested on absence of both kinds of plexiform n between the presence of le 2.2).  T h e r e w e r e 4 6 5 i n d i v i d u a l s w h o h a d i n f o r m a t i o no n p r e s e n c e o r a b s e n c e o f b o t h s u p e r f i c i a lp l e x i f o r m n e u r o f i b r o m a s a n d M P N S T s . T h e r e w a s n o s i g n i f i c a n ta s s o c i a t i o n between superficialplexiformneurofibromas and MPNSTs (Table 2.2). The analysis of internalplexiformneurofibromas and MPNSTs was limitedto 141 NF1 p r o b a n d s w h o h a d i n f o r m a t i o n o n t h e p r e s e n c e o r a b s e n c e o f b o t h i n t e r n a lp l e x i f o r m 40  neurofibromas and MPNSTs. Forty-nine individualshad internalplexiform neurofibromas, 17 (35%) of whom also had MPNSTs. In contrast,only three (3%) of the 9 2 i n d i v i d u a l sw i t h o u t i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s h a d M P N S T s . T h e a s s o c i a t i o n between the presence of internalplexiformneurofibromas and MPNSTs was very s t r o n g , w i t h i n d i v i d u a l sw h o h a v e i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s b e i n g o v e r 2 0 t i m e s m o r e l i k e l yt o h a v e a n M P N S T t h a n i n d i v i d u a l sw i t h o u t i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s . Age was also negatively associated with the development of MPNSTs in thisanalysis, the rate decreasing about 6% per year (Table 2.2). W h e n l o g i s t i cr e g r e s s i o n w a s d o n e w i t h b o t h s u b c u t a n e o u s a n d i n t e r n a lp l e x i f o r m neurofibromas as explanatory variables,the association of subcutaneous neurofibromas with MPNSTs was no longer significant,but the association with internalplexiform neurofibromas remained very strong (Table 2.2).After adjustment for age and the presence of subcutaneous neurofibromas, NF1 probands with internalplexiform n e u r o f i b r o m a s w e r e o v e r 1 8 t i m e s m o r e l i k e l yt o h a v e M P N S T s a s p r o b a n d s w i t h o u t internalplexiform neurofibromas. Survival Analysis  S u r v i v a l a n a l y s i s i n N F 1 p a t i e n t s w i t h M P N S T s w a s l i m i t e dt o t h MPNSTs. Fourteen individualsdied as a resultof theirMPNSTs. after diagnosis for individuals who died was 1.4 ± 1.1 years. The follow-up for individuals stillalive at the time of analysis was 5.3 y e a r s u r v i v a lw a s 4 1 % ( 9 5 % C l = 2 1 % - 6 2 % ) ( F i g u r e 2 . 1 ) f o r t h e M P N S T s a n d w a s s i m i l a rf o r m a l e s a n d f e m a l e s ( p = 0 . 7 9 ) .  41  e 25 subjects who had The mean survival average length of ± 2.5 years. The five25 patientswith  Figure 2.1 Kaplan Meier Survival of 25 NF1 patients with M P N S T s . 1.0  0.9  0.8  f  0.7  J?  0G  C  0.4  'E  1  0.3'  02  0.1 0.0  Follow-up (Months)  DISCUSSION  W e s o u g h t t o i d e n t i f yf e a t u r e s o f N F 1 t h a t c h a r a c t e r i z e p a t i e n t s w h o a r e a t t h e r i s k f o r d e v e l o p i n g M P N S T s . W e f o u n d t h a t i n d i v i d u a l sw i t h i n t e r n a l p l e x i f o r m n e u r o f i b r o m a s w e r e 1 8 t i m e s m o r e l i k e l yt o d e v e l o p M P N S T s t h a n p a t i e n t s w i internalplexiform neurofibromas, afteradjustment for age and the occurrence subcutaneous neurofibromas. In addition, individualswith subcutaneous neur w e r e m o r e t h a n t h r e e t i m e s a s l i k e l yt o h a v e i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s patientswithout subcutaneous neurofibromas.  highest thout of ofibromas as  The associationobserved between subcutaneous and internalplexiform neurofibromas m a y r e f l e c ts h a r e d p a t h o g e n i c m e c h a n i s m s a n d i s c o n s i s t e n t w i t h t h e h i s t o p a t h o l o g i c a l similaritiesamong cutaneous, subcutaneous and plexiform neurofibromas. Inaddition, loss of the normal NF1 allelehas been observed in at least some cases in all three types of neurofibromas (16-23). Itis somewhat surprising, given the histopathological similaritiesbetween these three types of benign tumours, that cutaneous neurofibromas are not associated with internalplexiform neurofibromas. However, cutaneous tumours h a v e ad i f f e r e n t n a t u r a l h i s t o r y t h a n p l e x i f o r m n e u r o f i b r o m a s b e c a u s e p l e x i f o r m t u m o u r s m a y u n d e r g o m a l i g n a n t t r a n s f o r m a t i o nw h i l e c u t a n e o u s n e u r o f i b r o m a s a l m o s t n e v e r d o . This difference in clinical behavior may reflect adifference in pathogenesis (24). A weak associationbetween cutaneous, subcutaneous and plexiformneurofibromas has been observed in one other study (25). However, the majority of plexiform tumours examined i n t h a t s t u d y w e r e s u p e r f i c i a lp l e x i f o r m n e u r o f i b r o m a s ( 2 5 ) . W e d i d n o t o b s e r v e a n y 42  association of superficial plexiform neurofibromas with either internal plexiform neurofibromas or MPNSTs in the present study, which included a subset of the data analyzed in the previous study (25). MPNSTs in people with NF1 usually arise within a preexisting plexiform neurofibroma (26). Our finding that all25 of the MPNSTs included in this study contained elements of benign plexiform neurofibroma on histopathological examination is consistent with this i n t e r p r e t a t i o n . W e f o u n d i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s i n 3 4 % o f t h e p a t i e n t s w h o h a d u n d e r g o n e i m a g i n g s t u d i e s .W e o b s e r v e d i n t e r n a l p l e x i f o r m n e u r o f i b r o m a s m o s t frequently in the spinal nerves, followed by other locations in the abdomen/pelvis, and then by other locations in the chest. Only five individuals had two clearly distinctinternal plexiform neurofibromas, which is a lower proportion of multiplicity than found in studies of plexiform neurofibromas that were apparent on clinicalexam (12.5-21%) (27, 28). The prevalence of various clinicalfeatures among the NF1 patients in this dataset may n o t r e f l e c t t h e p r e v a l e n c e a m o n g i n d i v i d u a l sw i t h N F 1 i n g e n e r a l b e c a u s e o u r d a t a w e r e o b t a i n e d f r o m p a t i e n t s s e e n a t a s p e c i a l i z e d c l i n i c ,a n d t h e a n a l y s i s w a s r e s t r i c t e d t o p r o b a n d s . T h e p a t i e n t s i n c l u d e d m a y , t h e r e f o r e ,b e m o r e s e v e r e l y a f f e c t e d t h a n t h e NF1 population in general. Itis unlikely that patient selection for imaging studies biases the results,as an equal proportion of individualswith neurofibromas was identified among those selected by routineexamination (33%) compared to those identifiedby symptoms (35%). Moreover, the prevalence and distributionof internal plexiform neurofibromas observed in this study is similar to that found in a study that performed CTs on 91 NF1 patientsover 16 years old (mean age = 35 years).Among these 91 NF1 patients,20% had internalneurofibromas on chest CT, and 40% had internal neurofibromas on abdominal/pelvic CT. Forty paraspinal tumours were identified, 17 on chest and 23 on abdominal/pelvic scan, constituting72% of allabnormal scans (29). This reportdid not state how many tumours each patienthad or how many of the patients had a single tumour that was seen on both the abdominal and chest scan. A study that performed spinal MRIs on 54 NF1 patients aged 5 to 56 years observed spinal neurofibromas in 64% of allindividualsexamined, with more than five tumour m a s s e s i n 7 9 % . A m o n g 2 4 i n d i v i d u a l sw h o c o m p l a i n e d o f s e n s o r y i m p a i r m e n t o r p a r a l y s i s , s p i n a l t u m o u r s w e r e i d e n t i f i e di n 2 3 ( 9 3 % ) . S p i n a l t u m o u r s w e r e a l s o identifiedin 12 (40%) of the 30 individuals who did not have symptoms of such a 43  tumour. Spinal tumours were seen in almost half of the asymptomatic individuals over t h e a g e o f 1 5 b u t i n o n l y 2 5 % o f a s y m p t o m a t i c i n d i v i d u a l su n d e r a g e 1 5 ( 3 0 ) . With the exception of the information on survivalafterdiagnosis of MPNST, the data a n a l y z e d f r o m t h e H e n r i M o n d o r D a t a b a s e a r e c r o s s - s e c t i o n a la n d , t h e r e f o r e ,c a n n o t b e used to assess temporal relationships.This means that the associations we observed cannot be used to predict the riskof an individual NF1 patient to develop an MPNST. A large-scale longitudinal study of the natural history of internal plexiform neurofibromas is necessary to develop predictive models. A recent study examined MRI from 50 NF1 patients who had previously-identified plexiformneurofibromas. Ten patientshad inhomogeneous-appearing tumours on MRI, and seven of these presented with pain or swelling.All 10 were diagnosed p a t h o l o g i c a l l ya s M P N S T s . T h e a g e s o f t h e s e 1 0 p a t i e n t sr a n g e d f r o m 1 3 t o 5 6 y e a r s , with a mean age of 28 years. A high internal tumour burden was observed in five of the p a t i e n t s ,t w o h a d a p a r e n t w i t h N F 1 w h o d i e d f r o m a n M P N S T a n d t w o o t h e r s h a d h a d a previous MPNST (31). MPNSTs have a poor prognosis, and in most studies the pr in NF1 patients than in those who do not have NF1 (3, 32). a five-year survival of only 21% in people with NF1 who had current study, the five-year survival was 41% in both males agreement with the suggestion that the prognosis of MPNST females with NF1 (5, 7).  ognosis appears to be worse A recent investigation found an MPNST (5).In the and females. This is not in isworse in males than  A study using the Neurofibromatosis Institute Database to identify clinical features that may be associated with mortality in patients with NF1 looked at 703 patients with a mean follow-up time of 2.4 years. Forty individualsdied during the follow-up, 27 of w h o m w h e r e a d u l t s .O f t h e 4 0 i n d i v i d u a l s w h o d i e d , 1 6 ( 4 0 % ) h a d a n M P N S T . U s i n g a g e - a d j u s t e d l o g i s t i cr e g r e s s i o n , t h e s t u d y f o u n d t h a t s u b c u t a n e o u s neurofibromas (OR= 3.6,95% Cl= 1.2,11.3) and male gender (OR=5.6, 95% Cl= 1.5,20.9) were independent predictors of mortality in adults with NF1 (33). In the present study, the f i n d i n g t h a t i n d i v i d u a l sw i t h N F 1 w h o h a v e s u b c u t a n e o u s n e u r o f i b r o m a s a r e a t increased riskof developing an MPNST, one of the leading causes of death among NF1 patients (34), supports the previous finding that subcutaneous neurofibromas are associated with mortality (33). 44  The 1997 consensus guidelines for management of NF1 patientsstate that imaging studies should not be done unless the clinicalexam identifiesproblems that require such studies (13).However, patientswith NF1 who have symptoms of an underlying tumour are not the only ones at riskfor developing internalplexiform neurofibromas, and identifying an MPNST after itbecomes symptomatic is often too late to provide a s u r g i c a l c u r e . W e f o u n d t h a t N F 1 p a t i e n t s w i t h i n t e r n a lp l e x i f o r m n e u r o f i b r o m a s w e r e m u c h m o r e l i k e l y t o d e v e l o p M P N S T s t h a n N F 1 p a t i e n t s w i t h o u t i n t e r n a lp l e x i f o r m neurofibromas, and over half of patients with internalplexiform neurofibromas were not symptomatic. This raises the question of whether adults with NF1 should have routine imaging for internal plexiform neurofibromas to identifythose at high risk of developing M P N S T s . T h e a s s o c i a t i o nb e t w e e n i n t e r n a lp l e x i f o r mn e u r o f i b r o m a s a n d s u b c u t a n e o u s neurofibromas is not strong enough to be clinicallyuseful to identifyindividuals who have a greater risk of internal neurofibromas. Eleven of the 25 individualswith MPNSTs in this study [Patients 1,5,7,8,14,15,17,20,21,22 and 24] have been tested for a constitutional NF1 microdeletion by either marker analysis,specificbreak-point PCR, semi-quantitative P C R o r F I S H ( 6 ) .T h r e e o f t h e e l e v e nw e r e f o u n dt o c a r r yd e l e t i o n so f t h e w h o l e N F 1 g e n e [ P a t i e n t s 8 , 2 1 , a n d 2 2 ] . I n t h e c u r r e n t s t u d y , i tw a s n o t p o s s i b l e t o d e t e r m i n e whether individualswith microdeletions and internalplexiform neurofibromas are at an even greater riskof developing MPNSTs compared to those with other pathogenic mutations and internalplexiform neurofibromas. Ifroutine imaging studies of adults with NF1 were advocated to look for internal plexiform neurofibromas or MPNSTs, itis not clear how such screening should be done. We do not know which type of imaging study would be most appropriateforroutine screening, how often such screening should be performed, or whether allNF1 patients o r o n l y a s u b s e t d e f i n e d b y a g e o r o t h e r c l i n i c a lf e a t u r e s s h o u l d b e s c r e e n e d . S e v e r a l imaging methods are available,including ultrasound examination, X-ray examination, CT, and MRI, each of which was used on at leastsome of the patients in the present study. Each of these techniques has advantages and disadvantages. Ultrasound examination is a safe and relatively inexpensive imaging procedure, but ithas both low spatial resolutionand poor softtissue contrast compared to other imaging techniques. 45  Conventional radiographic examination is also inexpensive and under optimal conditions has good spatial resolution,but soft tissue contrast and thus detection of internalplexiform neurofibromas is very poor. CT provides better visualizationof soft tissues and better spatial resolution but is more expensive. In addition,CT involves greater exposure to ionizing radiation, which is a particular concern in patients who carry a constitutional mutation of a tumour suppressor gene. MRI provides high spatial resolution and excellent soft tissue contrast without exposure to ionizing radiation but is expensive and more time consuming for the patient. However, itis probably the best approach to routine screening ifthe cost-benefit ratio can be shown to justify it. There is an urgent need to address the excess mortalityand morbidity associated with NF1 more effectively,and rigorous prospective studies with detailed phenotypic analysis and i m a g i n g s t u d i e s a r e n e c e s s a r y t o d e t e r m i n e t h e c l i n i c a lu t i l i t yo f r o u t i n e b o d y M R I i n affected adults.  46  REFERENCES  1.  Rasmussen SA, Yang Q, Friedman JM. Mortalityin neurofibromatosis 1: an analysis using U.S. death certificates.Am J Hum Genet 2001;68:1110-8. 2. Zoller M, Rembeck B, Akesson HO, Angervall L. Lifeexpectancy, mortality and prognostic factors in neurofibromatosis type 1. A twelve-year follow-up of an epidemiological study in Goteborg, Sweden. Acta Derm Venereol 1995;75:13640. 3. Ducatman BS, Scheithauer BW, Piepgras DG, Reiman HM, llstrup DM. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer 1986;57:2006-21. 4. Ferner RE, Gutmann DH. Internationalconsensus statement on malignant peripheral nerve sheath tumors in neurofibromatosis. Cancer Res 2002;62:15737. 5 . E v a n s D G , B a s e rM E , M c G a u g h r a n J , e ta l . M a l i g n a n tp tumours in neurofibromatosis 1. J Med Genet 2002;39:311-4. 6 . D e R a e d t T , B r e m s H ,W o l k e n s t e i n P ,e t a l . E l e v a t e d r i s k f o r M microdeletion patients.Am J Hum Genet 2003;72:1288-92. 7 . L o r e eT R , N o r t h J H J r ,W e r n e s s B A , e ta l . M a l i g n a n tp e r i tumors of the head and neck: analysis of prognostic factors.Otolaryngol Head Neck Surg 2000;122:667-72. 8. Woodruff JM. Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis. Am J Med Genet 1999;89:23-30. 9. Korf BR. Malignancy in neurofibromatosis type 1. Oncologist 2000;5:477-85. 10. K i n g A A , D e b a u n M R , R i c c a r d iV M , G u t m a n n D H . M a l i g n a n t p e r i p h e r a l n e r v e sheath tumors in neurofibromatosis 1. Am J Med Genet 2000;93:388-92. 11. H u s o n S M . N e u r o f i b r o m a t o s i s 1 : a c l i n i c a la n d g e n e t i c o v e r v i e w . I n : H u s o n SM, Hughes RAC, editors.The neurofibromatoses: a pathogenetic and clinical overview. 1sted. London: Chapman and Hall Medical; 1994. p. 160-203. 12. National Institutesof Health Consensus Development Conference Statement: neurofibromatosis. Bethesda, Md., USA, July 13-15, 1987. Neurofibromatosis 1988;1:172-8. 1 3 . G u t m a n n D H ,A y l s w o r t hA , C a r e yJ C , e ta l . T h ed i a g n o multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2. J A M A1 9 9 7 ; 2 7 8 : 5 1 - 7 . 14. Friedman JM. Epidemiology of neurofibromatosis type 1. Am J Med Genet 1999;89:1-6. 15. Friedman JM, Birch P, Greene C. National Neurofibromatosis Foundation InternationalDatabase. Am J Med Genet 1993;45:88-91. 1 6 . D a s c h n e rK , A s s u m G , E i s e n b a r t hI ,e fa l .C l o n a lo r i g i no ft u m o rc e l l si n a plexiform neurofibroma with LOH in NF1 intron38 and in dermal neurofibromas withoutLOH of the NF1 gene. Biochem Biophys Res Commun 1997;234:346-50. 47  e r P N p h  s t  17. 18. 19. 2 0 . 2 1 . 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.  Colman SD, Williams CA, Wallace MR. Benign neurofibromas in type 1 neurofibromatosis(NF1) show somatic deletionsof the NF1 gene. Nat Genet 1995;11:90-2. Kluwe L, Friedrich R, Mautner VF. Loss of NF1 allele in Schwann cells but not in fibroblastsderived from an NF1-associated neurofibroma. Genes Chromosomes Cancer 1999;24:283-5. K l u w e L , F r i e d r i c h R E , M a u t n e r V F . A l l e l i cl o s s o f t h e N F 1 g e n e i n N F 1 associated plexiformneurofibromas. Cancer Genet Cytogenet 1999;113:65-9. R a s m u s s e n S A , O v e r m a n J , T h o m s o n S A , etal. C h r o m o s o m e 1 7 l o s s heterozygosity studies in benign and malignant tumors in neurofibromatosis type 1. Genes Chromosomes Cancer 2000;28:425-31. S e r r a E ,P u i g S , O t e r o D , e ta l . C o n f i r m a t i o n o fa d o u b l e - h i tm o d e lf o gene in benign neurofibromas. Am J Hum Genet 1997;61:512-9. Serra E, Ars E, Ravella A, et al.Somatic NF1 mutational spectrum in benign n e u r o f i b r o m a s : m R N A s p l i c ed e f e c t s a r e c o m m o n a m o n g p o i n t m u t a t i o n s . H u m Genet 2001;108:416-29. Skuse GR, Kosciolek BA, Rowley PT. The neurofibroma in von Recklinghausen neurofibromatosis has a unicellular origin.Am J Hum Genet 1991;49:600-7. Tucker T, Friedman JM. Pathogenesis of hereditary tumors: beyond the "two-hit" hypothesis. Clin Genet 2002;62:345-57. S z u d e k J , B i r c h P , R i c c a r d i V M , E v a n s D G , F r i e d m a n J M . A s s o c i a t i o n s o fc l i n i c a l features in neurofibromatosis 1 (NF1). Genet Epidemiol 2000;19:429-39. McGaughran JM, Harris Dl, Donnai D, et al. A clinical study of type 1 neurofibromatosis in north west England. J Med Genet 1999;36:197-203. Huson SM, Harper PS, Compston DA. Von Recklinghausen neurofibromatosis. A clinicaland population study in south-east Wales. Brain 1988; 111:1355-81. Friedman JM, Birch PH. Type 1 neurofibromatosis: a descriptive analysis of the disorder in 1,728 patients. Am J Med Genet 1997;70:138-43. Tonsgard JH, Kwak SM, Short MP, Dachman AH. CT imaging in adults with neurofibromatosis-!" frequent asymptomatic plexiform lesions. Neurology 1998;50:1788-60. Thakkar SD, Feigen U, Mautner VF. Spinal tumours in neurofibromatosis type 1: a n M R I s t u d y o f f r e q u e n c y , m u l t i p l i c i t ya n d v a r i e t y .N e u r o r a d i o l o g y 1 9 9 9 ; 4 1 : 6 2 5 9. Mautner VF, Friedrich RE, von Deimling A, et al.Malignant peripheral nerve sheath tumours in neurofibromatosis type 1: MRI supports the diagnosis of malignant plexiform neurofibroma. Neuroradiology 2003;45:618-25. Loree TR, North JH Jr,Werness BA, et al.Malignant peripheral nerve sheath tumors of the head and neck: analysis of prognostic factors.Otolaryngol Head Neck Surg 2000;122:667-72.  48  33. 34.  Khosrotehrani K, Bastuji-Garin S, R neurofibromas are associated with study of 703 patients.Am J Med Ge Rasmussen SA, Friedman JM. NF1 2000;151:33-40.  iccardi VM, et al. Subcutaneous mortality in neurofibromatosis 1: A cohort net 2005;132:49-53. gene and neurofibromatosis 1. A J Epidemiol  49  3. H E T E R O G E N E T I T Y O F M A S T C E L L D I S T R I B U T I O N IN NF1 NEUROFIBROMAS  2  INTRODUCTION  Neurofibromatosis 1(NF1) is an autosomal dominant disorder with an incidence of 1:3500 (1).Benign neurofibromas are the hallmark feature of NF1. All neurofibromas contain Schwann cells (the suspected progenitor celltype), but fibroblasts,mast cells, lymphocytes, perineurial cells,endoneurial fibroblasts and endothelial cells also occur in varying numbers (2). Neurofibromas also vary in terms of their age of appearance, natural history,and malignant potential.Consequently, several differentclassifications of neurofibromas have been proposed (Table 3.1). Table 3.1 Examples of different classifications for NF1-associated neurofibromas. Source Clinical  Crowe,  Schull & Neel  Riccardi Huson  Types of Neurofibromas  (4) & Hughes  Korf & Rubenstein Histopathological  Harkin and Reed Masson Woodruff  2  (3)  (5) (6) (7)  (8) (9)  Cutaneous Subcutaneous Plexiform C u t a n e o u s Subcutaneous Nodular plexiform Diffuse plexiform Dermal Nodular Plexiform Dermal Plexiform Plexiform Non-plexiform Encapsulated Diffuse Localized cutaneous Localized subcutaneous Plexiform Diffuse (skin or subcutaneous Massive soft tissue  layer)  A version of this c h a p t e r h a s been s u b m i t t e d for publication. T u c k e r T, Riccardi V M , Sutcliff M , Vielkind J,  W e c h s l e r J , W o l k e n s t e i n P, F r i e d m a n J M . H e t e r o g e n e i t y o f m a s t c e l l d i s t r i b u t i o n i n N F l n e u r o f i b r o m a s .  50  N o n e o f t h e s e c l a s s i f i c a t i o n sh a s a c h i e v e d u n i v e r s a l a c c e p t a n c e , a n d t h el i t e r a t u r e contains many examples of inconsistentuse and confusion of various clinical and pathological descriptive terms. Most studies of neurofibroma pathogenesis treat all n e u r o f i b r o m a s a s as i n g l e c l a s s d e s p i t e t h e i r c l i n i c a l a n d p a t h o g e n i c h e t e r o g e n e i t y . T h i s heterogeneity is important because some kinds of neurofibromas have little,if any, malignant potential,while other kinds may progress to malignancy. The resulting neoplasms, malignant peripheralnerve sheath tumours (MPNSTs), are one of the leading causes of death in NF1 patients (10). There is currently no effective treatment known for benign neurofibromas or MPNSTs except surgical removal in favourable cases. Although the Schwann cell isthe prominent celltype in neurofibromas, mast cells may play an important role in neurofibroma development (11), and mast cell infiltrationisa prominent feature of some neurofibromas (12).In the present study, we used histologicaland immunohistochemical techniques to study the occurrence and d i s t r i b u t i o no f m a s t c e l l s i n n e u r o f i b r o m a s f r o m i n d i v i d u a l s w i t h N F 1 . Neurofibromas were classifiedhistopathologically as diffuse or nodular (single or plexiform). Nodular neurofibromas are circumscribed by the perineurium while diffuse neurofibromas are not. We found that distributionof w i t h i n at u m o u r not seen in the a s s o c i a t e d M  diffuse neurofib mast cellsthan region showed surrounding no P N S T s .  r o m a s h a d ah i g h e r p r o p o r t i o n a n d nodular neurofibromas. Mast cells no obvious clustering around bloo rmal tissue. Mast cellswere rarely  more un that were d vessels seen in  iform present and were NF1-  MATERIALS & METHODS Sample Collection  We collectedformalin fixed-paraffinembedded samples France), The Neurofibromatosis Institute(La Crescenta, H o s p i t a l ( V a n c o u v e r , C a n a d a ) a n d C h i l d r e n ' sa n d W o m e n ' C a n a d a ) . A l l i n d i v i d u a l s h a d ac o n f i r m e d d i a g n o s i s o f N F 1 c r i t e r i a( 1 3 ) . I n t o t a l w e s t u d i e d 4 9 b e n i g n n e u r o f i b r o m a s MPNSTs from 4individuals.The study protocol was appr B r i t i s hC o l u m b i a R e s e a r c h E t h i c s C o m m i t t e e . 51  f r o m C r e t e i lH o s p i t a l( P a r i s , California),Vancouver General s Hospital (Vancouver, according to established from 37 individuals and 4 oved by the University of  Histopathological Classification of Neurofibromas  E a c h n e u r o f i b r o m a w a s c l a s s i f i e d h i s t o p a t h o l o g i c a l l yo n t h e b a s i s o f i t sa p p e a r a n c e i n hemotoxylin and eosin (H&E) stained sections as diffuseor nodular according to the d e f i n i t i o n s u s e d b y o n e o f t h e a u t h o r s ( J . W . ) .T h e d i f f u s e n e u r o f i b r o m a s w e r e predominantly located in the dermis and involved the thin terminal branches of peripheral nerves. Diffuse neurofibromas were characterized by proliferationof cells that are not limitedby the perineurium and can encircle normal structures (exocrine glands, hairfolliclesand vessels) without destroying them. The proliferatingcells were irregularly dispersed within a fibrous and/or myxoid background. Myelin fibrilswere rare or absent. N o d u l a r n e u r o f i b r o m a s u s u a l l y i n v o l v e d d e e p e r t i s s u e s , a n d t h e c e l l u l a rp r o l i f e r a t i o ni s i n t r a n e u r a l ,e n c l o s e d w i t h i n l a r g e h y p e r t r o p h i c n e r v e s c i r c u m s c r i b e d b y t h e p e r i n e u r i u m . The nodular tumours included both single and plexiformtypes. Single nodular tumours i n v o l v e j u s t o n e f a s c i c l e ,w h i l e n o d u l a r p l e x i f o r m t u m o u r s m a y i n v o l v e s e v e r a l f a s c i c l e s , nerve branches or a nerve plexus. Dispersed or fascicular myelin fibrilsare usually found in the central area of the nodules. On transverse section, the central area is loose w i t h am y x o i d b a c k g r o u n d s u b s t a n c e , w h i l e t h e p e r i p h e r a l a r e a i s u s u a l l y m o r e c e l l u l a r . Some neurofibromas contain both usually found in the deep dermis a surrounded by diffuse extraneural normal pre-existing structures. The histopathology reports.  d i f f u s ea n d n o d u l a r c o m p o n e n t s ; s u c h t u m o u r s a r e nd subcutaneous tissue.The nodular component is c e l l p r o l i f e r a t i o n ,i n f i l t r a t i n g t h e f a t t i s s u e a n d o t h e r diagnosis of all4MPNSTs was obtained from the  Identification of Granulated Mast Cells  Serialsections were cut from each neurofibroma. One section was stained with H&E, a n d as e c o n d s e c t i o n w a s s t a i n e d w i t h 0 . 1 % t o l u i d i n e b l u e , am e t a c h r o m a t i c d y e t h a t u n i q u e l y i d e n t i f i e sn o r m a l g r a n u l a t e d m a s t c e l l s .S e c t i o n s w e r e d e p a r a f f i n i z e d i n x y l e n e , hydrated through graded alcohols (100%, 95%, and 70%) and stained withtoluidine blue for 30 seconds and rinsed inwater. The slides were then dehydrated through graded alcohol (70%, 95%, 100%) and cleared in xylene.  52  Identification of Mast Cells and Schwann Cells  Immunohistochemistry u s i n g ac - K i t a n t i b o d y w a s p e r f o r m e d t o i d e n t i f y a l l m a s t c e l l s , regardless of whether they contain toluidineblue-positivegranules. Serial sections were c u t f r o m 9b e n i g n n e u r o f i b r o m a s ( 5 d i f f u s e a n d 4n o d u l a r ) a n d f r o m 3M P N S T s . One s e c t i o n o f e a c h f o r c - K i t ,a n d o n e w a s d u a l l a b e l e d f o r c - K i t a n d S 1 0 0 ( a S c h w a n n c e l l marker). After deparaffinization,heat antigen retrievalwas performed at 70°C in EDTA b u f f e rp H 6 . 0 . E n d o g e n o u s p e r o x i d a s e a c t i v i t yw a s b l o c k e d w i t h 3 % h y d r o g e n peroxidase solution.The tissue sections were incubated with 1:400 mouse anti-human c - K i ta n t i b o d y ( Z y m e d , S a n F r a n c i s c o , C A ) o v e r - n i g h ta t r o o m t e m p e r a t u r e . T h e s e c t i o n s w e r e i n c u b a t e d w i t h ab i o t i n - l a b e l e d g o a t a n t i - m o u s e I g G s e c o n d a r y antibody a n d v i s u a l i z e d u s i n g A B C E l i t ea n d N o v a R e d ( V e c t o r L a b o r a t o r i e s ,C a n a d a I n c , Burlington,ON). Nuclei were identifiedusing hemotoxylin. Dual immunohistochemisty was performed as above with the additionof 1:400 rabbitanti-human S100 antibody ( D a k o C y t o m a t i o n , M i s s i s s a u g a , O N ) a n d m o u s e a n t i - h u m a n c - K i ta n t i b o d y . S e c o n d a r y a n t i b o d i e s w e r e c o n j u g a t e d w i t h A l e x a 4 8 8 o r A l e x a 5 6 8 ( I n v i t r o g e nC a n a d a I n c . , Burlington, ON) Tumour Characteristics  H&E and toluidineblue-stainedtumours were independently reviewed by two of the a u t h o r s ( M . S . a n d T . T . ) . E a c h t u m o u r w a s g r a d e d a s a w h o l e o n a3 - p o i n t L i k e r t s c a l e for cellularityand vascularity and on a 4-point Likert scale for mast cell density and l y m p h o c y t i c i n f i l t r a t i o n . F o r c e l l u l a r i t ya n d v a s c u l a r i t y ,s c a l i n g w a s d e t e r m i n e d a t 2 0 0 x m a g n i f i c a t i o no n t h e H & E s e c t i o n . M a s t c e l ld e n s i t y w a s d e t e r m i n e d a t 4 0 0 x magnification on toluidineblue-stained slides and rated as 0, 1-5, 6-10 and >10 mast cells per field.Lymphocytic infiltrationwas determined at 400x magnification as a percentage of the totalcells.Five to eight fieldschosen at random were taken to represent the tumour as a whole. In addition, the total number of mast cells per section w a s d e t e r m i n e d b y m a n u a l c o u n t i n g o f o n e s e c t i o n o n b o t h t h e t o l u i d i n eb l u e a n d c - K i t s t a i n e d s e c t i o n s . T h e s a m p l e s w e r e s c a n n e d t o d e t e r m i n e i ft h e r e w a s a n y c l u s t e r i n g o f m a s t c e l l s i n p a r t i c u l a r r e g i o n s o r a r o u n d p a r t i c u l a rt u m o u r f e a t u r e s , a n d t h e a d j a c e n t normal tissue,when present, was compared to the tumour tissue to determine if there were differences in cellularity,vascularity or mast cell density.  53  Statistical Analysis  K e n d a l l ' s c o r r e l a t i o nw a s c a l c u determine the relationshipbetw Mann-Whitney U rank test was 2001) to determine ifthere was the two types of neurofibromas. be significant.  lated using SPSS (SPSS een vascularity,cellul performed using SPSS a significant difference A p-value less than or  , Inc.Chicago, a r i t ya n d m a s t c (SPSS, Inc. Chi in the mast cell equal to 0.05 wa  Illinois,2001) to elldensity. The cago, Illinois, density between s considered to  RESULTS  Mast cells are present in many neurofibromas, but itis not known ifthe quantity or d i s t r i b u t i o n o f m a s t c e l l s d i f f e r sb e t w e e n d i f f e r e n tt y p e s o f n e u r o f i b r o m a s . W e s t u d i e d t h e c e l l u l a r i t y , v a s c u l a r i t y , a n d d e n s i t y a n d d i s t r i b u t i o no f m a s t c e l l s i n 4 9 b e n i g n n e u r o f i b r o m a s f r o m 3 7 N F 1 p a t i e n t s a n d i n 4M P N S T s from 4NF1 patients. Each of the b e n i g n t u m o u r s w a s c l a s s i f i e d h i s t o l o p a t h o l o g i c a l l y a s ad i f f u s e o r n o d u l a r neurofibroma. 32 of the neurofibromas we studied (from 27 individualswith NF1) were classified histopathologically as diffuse neurofibromas. 15 of the neurofibromas (from 9 individualswith NF1) were classifiedas nodular tumours. Two neurofibromas (from 2 i n d i v i d u a l s ) w e r e am i x o f b o t h d i f f u s e a n d n o d u l a r c o m p o n e n t s a n d w e r e n o t i n c l u d e d in the statisticalanalysis due to the small number. Figure 3.1 cellularit distributi staining is  shows examples of diffuse and nodular neurofibromas with typicalpatterns of y , v a s c u l a r i t y ,a n d l y m p h o c y t i c i n f i l t r a t i o n o n H & E s t a i n i n g .T h e m a s t c e l l onin each of these same neurofibromas as determined by toluidine blue also shown.  54  Figure 3.1 Images of typical neurofibromas in the current study that represent the majority of the tumour. A. H&E section of a diffuse neurofibroma at 100x magnification. B. Serial section to (A) stained with toluidine blue to identifymast cells (*)at 100x magnification taken at the periphery of the tumour. C. H&E section of a nodular neurofibroma at 100x magnification. D. Serial section to (C) stained with toluidine blue to identifymast cells at 100x magnification.Note the higher cellularityand abundance of smaller blood vessels (arrow) in A compared to C and the lymphocytic infiltration (L) in C, some of which is indicated. Also note the presence of the reddish-purple mast cells (*)in the diffuse neurofibroma (B) and the absence of mast cells in the nodular neurofibroma (D).  Cellularity  N o d u l a r n e u r o f i b r o m a s t e n d t o b e l e s s c e l l u l a rt h a n d i f f u s e n e u r o f i b r o m a s ( F i g u r e 3 . 1 ) , with almost 80% of nodular tumours falling into category 1 for cellularity(Figure 3.2). T h e s e t u m o u r s d i s p l a y e d m o r e d e n s e f i b r o t i ct i s s u e a n d a r e a s o f d e g e n e r a t i o n i n t h e central portion of the neurofibroma but not at the periphery. In contrast, the diffuse t u m o u r s h a d h i g h e r c e l l u l a r i t yw i t h am o r e u n i f o r m d i s t r i b u t i o n o f c e l l s t h r o u g h o u t t h e t u m o u r . T h e r e w a s a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n c e l l u l a r i t ya n d h i s t o p a t h o l o g i c a l tumour type (Kendall's tau = -0.384, p=0.007).  55  Figure 3.2 Distribution of pathological features of neurofibromas classified histopathologically.  A. Cellularity estimated on a3-point Likert scale with 3being the highest cellularity; B. V a s c u l a r i t y e s t i m a t e d o n a3 - p o i n t L i k e r t s c a l e w i t h 3b e i n g t h e h i g h e s t v a s c u l a r i t y ; a n d C. Mast cell density estimated on a4-point Likert scale with 4being the highest mast cell density. • D i f f u s en e u r o f i b r o m a s • N o d u l a rn e u r o f i b r o m a s  Cellularity  B 1.0000  £: 0.2000 0.0000 2 Vascularity  T w w w  here was ithin diff ithinthe hen they  o f t e n ah i g h e r p r o use neurofibromas. nodular neurofibro were observed in  portion of lymphocytes within nodular neurofibromas than Lymphocytic infiltrationwas generally distributed evenly mas (Figure 3.1),while lymphocytes were more localized diffuse tumours.  56  Vascularity  Nodular neurofibromas typicallyhad fewer large vessels than diffusetumours, which h a d m o r e s m a l l e r v e s s e l s ( F i g u r e 3 . 1 ) . V e s s e l s w e r e e v e n l y d i s t r i b u t e dt h r o u g h o u t t h e tumour in diffuse and nodular neurofibromas. Overall, vascularity was notsignificantly correlated with tumour type (Kendall's tau = -0.243, p=0.085). However, vascularity was s i g n i f i c a n t l yc o r r e l a t e d w i t h c e l l u l a r i t y ( K e n d a l l ' s t a u = 0 . 5 1 0 , p < 0 . 0 0 1 , ) , a s m o r e c e l l u l a r n e u r o f i b r o m a s h a d ah i g h e r n u m b e r o f b l o o d v e s s e l s . Mast Cell Distribution Within Neurofibromas and MPNSTs  The majority of mast cells were located within the tumour component, with only a few located in the surrounding normal tissue. Nodular neurofibromas had significantly fewer mast cells than diffuse neurofibromas (p=0.021) (Figures 3.1 & 3.2). The few mast cells seen within nodular neurofibromas were located at the periphery of the tumours (Figure 3.3A &3.3B). The mast cellswere evenly distributedthroughout diffuse neurofibromas, with no obvious mast cellclusters forming around particularstructures (Figure 3.1). A r e a s o f n o d u l a r n e u r o f i b r o m a s s e e n w i t h i n am i x e d n o d u l a r a n d d i f f u s e t u m o u r c o n t a i n e d v e r y f e w m a s t c e l l s ,a l t h o u g h t h e s u r r o u n d i n g d i f f u s e t u m o u r t i s s u e c o n t a i n e d many mast cells (Figure 3.3C & 3.3D).  57  Figure 3.3 Mast cell distribution within neurofibromas. A. H&E staining of a nodular neurofibroma and adjacent perineurium at 200x magnification. B. Toluidine blue staining of a serial section of the same nodular neurofibroma at 200x magnification showing clustering of mast cells (*)in the perineurium at the periphery of the neurofibroma. C. H&E staining at 50x magnification of a neurofibroma that has both diffuse (D) and nodular components (N). D. Toluidine blue staining of a serial section of the same neurofibroma in (D) showing mast cells (*) withinthe diffuse component (D), but few are observed within the nodular component (N).  Mast cells did not appear to cluster around blood vessels or within areas of lymphocytic infiltration,but mast celldensity was correlated with overall tumour vascularity (Kendall's tau =0.513, p<0.001) and cellularity(Kendall's tau =0.456, p=0.001). Four MPNSTs  were also stained with toluidineblue.Two had no observable mast cells,  a n d a n o t h e r h a d v e r y f e w m a s t c e l l s .T h e f o u r t h M P N S T component  included a  benign-appearing  from which the malignancy may have arisen. In thissample, mast cells were  frequent in the benign region but absent from the malignant portion (Figure 3.4). 58  Figure 3.4 Mast cell distribution in MPNSTs. A. H&E section of a histologically benign neurofibroma component adjacent to a MPNST at 50x magnification. B. Serial section of a histologically benign neurofibroma a d j a c e n t t o a n M P N S T s t a i n e d f o r t o l u i d i n eb l u e s h o w i n g m a s t c e l l s ( * )a t 5 0 x m a g n i f i c a t i o n .C . H & E s e c t i o n o f a m a l i g n a n t c o m p o n e n t o f t h e s a m e M P N S T showing high cellularityand mitoses at 100x magnification. D. Serial section of the malignant component stained for toluidineblue showing the absence of mast cellsat 100x magnification.  Distribution of Granulated and Non-granulated Mast Cells  To ensure that the differences we found in mast celldensity were not biased by observing only the granulated mast cellsshown with toluidineblue, we used c-Kit immunostaining to identifyallmast cells,both granulated and degranulated. The number of c-Kitpositive cellswas higher than the number of toluidineblue positive cells in each of the tumours studied. To ensure that this increase was not due to neoplastic Schwann cellsas well as mast cellsexpressing c-Kit,we dual immunolabeled an adjacent section of each tumour with antibodies to c-Kitand S100. No colocalization of t h e c - K i ta n d S 1 0 0 s i g n a l s w a s s e e n w i t h c o n f o c a l i m a g i n g ( F i g u r e 3 . 5 ) .W e c o n c l u d e that the greater frequency of c-Kit-positivethan toluidine blue-positive cells in these 59  tumours probably indicates that some  mast celldegranulation occurs in both types of  neurofibromas. Figure 3.5 Confocal image of a diffuse neurofibroma stained with S100 and c-Kit.  Confocal neurofib showing Schwann cells.Th c-Kit.  image of a diffuse roma at 400x magnification staining for both S100+ (red) cellsand c-Kit+(green) mast e Schwann cellsdo not express  There was no relationship between the increase in the number of c-Kit positive-cellsin comparison to the number of toluidineblue-positivecells and neurofibroma type; however, there was astrong correlation between toludine blue counts and c-Kit staining (Kendall's tau =0.894, p<0.001). As expected from the toluidine blue results,the total n u m b e r o f c - K i t - p o s i t i v e c e l l s w a s s i g n i f i c a n t l yl o w e r i n n o d u l a r n e u r o f i b r o m a s t h a n i n diffuse neurofibromas (p=0.014). DISCUSSION  Mast cells are aconsistent finding in neurofibromas in people with NF1. However, the role of the mast cell in the development of neurofibromas in NF1 individuals is poorly u n d e r s t o o d . T h e p u r p o s e o f t h i s s t u d y w a s t o d e t e r m i n e i ft h e d e n s i t y a n d d i s t r i b u t i o n o f mast cells differsbetween neurofibromas classifiedby their histopathological appearance and ifmast cell infiltrationis related to other histological characteristics of these tumours, which may indicatedifferentpathogenesis for each type of n e u r o f i b r o m a . W e f o u n d t h a t n o d u l a r n e u r o f i b r o m a s h a v e s i g n i f i c a n t l yf e w e r m a s t c e l l s than diffuse neurofibromas and that MPNSTs show littleor no mast cellinfiltration. Mast cellsare formed in the bone marrow. They are released intothe blood  as  u n d i f f e r e n t i a t e d p r e c u r s o r s a n d d o n o t d i f f e r e n t i a t e u n t i lt h e y l e a v e t h e v a s c u l a t u r e 60  and  enter the target tissue. Mast cells can be found in endoneurial and perineurialspaces of peripheral nerves (14), particularlywhen the nerve is damaged and in need of repair. Previous studies that have investigated mast cellswithin neurofibromas found results t h a t a r e g e n e r a l l y s i m i l a r t o t h o s e o f o u r s t u d y . S o m e , b u t n o t a l l ,n e u r o f i b r o m a s were found to have a high density of mast cells (12, 15, 16), but itwas not clear what types of neurofibromas were examined inthose studies or how many of the tumours came from individuals with NF1. Isaacson (12) described 4 neurofibromas that were densely hyalinised with an appearance similarto some of the dense fibrous nodular neurofibromas in the current study. Fewer than 5 mast cells per 400x field were observed in these tumours; this is similar to our findings in the current study. In addition, Isaacson noted that an entrapped thickened nerve within what was called a "plexiform neurofibroma" displayed fewer mast cellsthan the tumour tissue;thiswas also observed in the present study in 2 neurofibromas that were a mix of both nodular and diffuse tissue (Figure 3.3). Johnson and coworkers noted the abundance and even distributiono throughout a large number of neurofibromas but did not observe mast m a j o r i t yo f M P N S T s e x a m i n e d ( 1 5 ) .O n e n e u r o f i b r o m a w a s c l a s s i f i e and ithad a greater concentration of mast cells at the periphery of the observed in some of the nodular neurofibromas in the present study.  f mast cells cells in the das subcutaneous, tumour (15), as  Patients with NF1 neuropathy also have enlarged nerves, but sural nerve biopsies from such individuals showed a disorganized perineurium and hypercellular epineurium with mast cellsprimarilylocated close to blood vessels within the epineurium and endoneurium (17).The location of mast cellswithin these thickened nerves contrasts to the location observed in nodular neurofibromas in the present study, where mast cells w e r e f o u n d p r i m a r i l ya t t h e p e r i n e u r i u m a n d / o r e p i n e u r i u m b o r d e r . T h e r e s t r i c t i o no f mast cellsto the endoneurium or perineurium may distinguishneuropathy and frank neurofibroma development. Z h u Usin i n v m a i thes  e t a l u s e d a u n i q u es y s t g a Cre transgene under e s t i g a t o r sp r o d u c e da n t a i n i n ga n N f 1 h e t e r o e mice are enlarged, and  e m t o s t u d yn e u r o f i b r o m a d e v e l o p m e n t i n a m o u s e m o d e l . the control of the Schwann cell Krox20 promoter, these h o m o z y g o u sk n o c k o u to f N f 1 i nS c h w a n n c e l l sw h i l e z y g o u ss t a t ei n a l lo t h e rc e l l s( 1 1 ) . T h e p e r i p h e r a ln e r v e so f some of the animals develop dorsal root neurofibromas 61  that resemble nodular plexiform neurofibromas in NF1 patients. Interestingly, formation o f n e u r o f i b r o m a s o c c u r s o n l y i fo t h e r c e l l s i n t h e s e m i c e a r eh e t e r o z y g o u sf N f 1 h a p l o i n s u f f i c i e n tt u m o u re n v i r o n m e n ta p p e a r s n e c e s s a r yf o rt h ep a t h o g e these histologically-mixedtumours (11).The thickened nerves in these mice show i n f i l t r a t i o n o f m a s t c e l l s , a n d t h i s i n f i l t r a t i o n i s m o r e s u b s t a n t i a l w h e n t h e N f 1n u l l S c h w a n n c e l l sp r o l i f e r a t ei na n N f 1 h e t e r o z y g o u sb a c k g r o u n dt h a ni nt h ew i l dt y p e background. However, itis not clear where the mast cells were found within the thickened nerves. Mast cells are normally found in close proximity to the Schwann cells that myelinate these nerves (7).Moreover, cutaneous mast cells outside of these tumours are also moderately increased and demonstrate enhanced growth in vitro in N f 1 h e t e r o z y g o u s m i c ec o m p a r e dt ow i l dt y p e m i c e( 1 8 ) . T h e s eo b s e r v a t i o n ss u g g e that mast cells are involved in neurofibroma formation in this mouse model, but the p a t h o g e n i c p r o c e s s m a y d i f f e rs o m e w h a t f r o m t h a t w h i c h o c c u r s n a t u r a l l yi n h u m a n s with NF1. Riccardi (4) has distinguished 4 types of neurofibromas clinicallythat differwith respect to theirage of appearance, natural history,and malignant potential (Table 3.1). This c l i n i c a lc l a s s i f i c a t i o n d o e s n o t c o r r e l a t e w i t h t h e p a t h o l o g i c a l c l a s s i f i c a t i o n u s e d i n t h e present study. Riccardi's "cutaneous neurofibromas" occur in the skin surface and develop from the terminal branches of peripheral nerves. "Subcutaneous neurofibromas" are described as firm and lyingdeeper in the subcutaneous layer of the skin. They have discrete margins but may extend along a nerve. "Nodular plexiform n e u r o f i b r o m a s " ( w h i c h m a y o r m a y n o t e x h i b i tt h e p a t h o l o g i c a lf e a t u r e s u s e d t o m a k e this diagnosis in the present study) arise within major peripheral nerves and may lie just beneath the dermis or much deeper in the body. They are often found in the spinal nerves, nerve roots or ganglia. Riccardi also describes "diffuseplexiform neurofibromas" t h a t d o n o t c o r r e s p o n d t o t h e p a t h o l o g i c a l c l a s s i f i c a t i o nu s e d i n t h e p r e s e n t s t u d y . Riccardi's "diffuse plexiform neurofibromas" usually are apparent at birthor within the f i r s tf e w y e a r s o f l i f e . T h e s e n e u r o f i b r o m a s c a n b e s u p e r f i c i a l ,i n v o l v i n g t h e s k i n a n d subcutaneous tissue,or involve deeper tissue.The tumours do not have discrete borders but invade adjacent normal tissues. Most of the tumours in our study were not c h a r a c t e r i z e d u s i n g t h i s c l i n i c a lc l a s s i f i c a t i o n ,a n d t h e h i s t o l o g i c a l a n d c l i n i c a l approaches do not classifyindividualtumours intothe same groups. Both cutaneous and "diffuseplexiform" tumours usually are diffuse neurofibromas histologically, 62  although some congenital or early onset tumours are mixed n e u r o f i b r o m a s . T u m o u r s t h a t a r e c l a s s i f i e dc l i n i c a l l ya s s u b plexiform neurofibromas are usually nodular neurofibromas However, the only way to determine the histological type of examine it histologically.  diffuseand nodular cutaneous or nodular on histopathological exam. a neurofibroma is to  In the present study, the number of mast cellswas lower in histologically-defined nodular neurofibromas than in histologically-defineddiffuse neurofibromas. Nodular n e u r o f i b r o m a s d i f f e rf r o m d i f f u s e n e u r o f i b r o m a s i n t h a t n o d u l a r t u m o u r s a r e s u r r o u n by an intactepineurium and perineurium, although the organization of the endoneurim disrupted with abnormal accumulation of cells (8). Itis tempting to speculate that the epineurium and/or perineurium acts as an anatomical barrierthat excludes mast cell from nodular neurofibromas. In contrast, mast cells might have directaccess to the tumour tissue in diffuse neurofibromas, which develop distalto the end of the epineurium and perineurium or in which the epineurium and perineurium are broken d o w n ( 8 ) . T h e s t r i k i n gd i f f e r e n c e b e t w e e n t h e n u m b e r o f m a s t c e l l s i n n e u r o f i b r o m a s d i f f e r e n tt y p e s m a y i n d i c a t e t h a t s o m e w h a t d i f f e r e n tm e c h a n i s m s u n d e r l i e t h e development of these differentkinds of neurofibromas. However, we cannot exclude p o s s i b i l i t yt h a t t h e d i f f e r e n c e i n m a s t c e l l n u m b e r a n d d i s t r i b u t i o nw e o b s e r v e d a r e largely a result of the differences in overall tumour cell number and distribution seen nodular and diffuse neurofibromas.  ded is s  of the in  Mast cells have been observed in a number of malignant neoplasms including breast cancer (19),squamous epithelialcarcinoma (20), colorectal cancer (21), and uterine cancer (22). In melanoma, mast cellsare located at the margin of tumour and normal tissue (23), and mast cellcounts within the tumour correlate strongly with microvascular density and shorter patient survival(24). In our study we found an abundance of mast cells in the histopathologically-defined diffuse neurofibromas, which include clinicallydefined cutaneous neurofibromas that almost never progress to malignancy, and we found fewer mast cellsin MPNSTs and histologically-definednodular neurofibromas, w h i c h c a n p r o g r e s s t o m a l i g n a n c y . H o w e v e r , w e c a n n o t r u l e o u t t h e p o s s i b i l i t yt h a t t h e nodular neurofibromas we examined might subsequently have progressed to malignancy or that the MPNSTs developed from neurofibromas that were infiltratedby mast cells at an earlier point in the tumorigenic process. In fact, the conjunction of mast 63  c e l l - i n f i l t r a t e d h i s t o l o g i c a l l y - b e n i g n n e u r o f i b r o m a t i s s u e a n d m a s t c e l l - f r e eM P N S T one NF1 patient (Figure 3.4) is compatible with the malignancy developing from a benign neurofibroma that is heavily infiltratedwith mast cells.  in  We found mast cellsto be evenly distributedin the tumour component of diffuse neurofibromas but not in the surrounding normal tissue. While there was no clustering of m a s t c e l l s a r o u n d b l o o d v e s s e l s , t h e r e w a s as i g n i f i c a n t c o r r e l a t i o n b e t w e e n t h e d e n s i t y o f m a s t c e l l s a n d v a s c u l a r i t y w i t h i n at u m o u r ( p < 0 . 0 0 1 ) . T h i s i s c o n s i s t e n t w i t h m a s t cellspromoting angiogenesis by releasing factors such as vascular endothelial growth factor(VEGF), matrix metalloproteinases(MMP2, MMP9) and chymase. Similarly,there w a s ac o r r e l a t i o n b e t w e e n m a s t c e l l d e n s i t y a n d c e l l u l a r i t y , s u g g e s t i n g t h a t m a s t c e l l s release factors that either directly or indirectly(e.g., by promoting angiogenesis) lead to increased cellular proliferation.  Also of interestis the abundance of lymphocytes we observed in nodular neurofibromas compared to diffuseneurofibromas. Johnson and coworkers studied lymphocytic i n f i l t r a t i o n i n av a r i e t y o f S c h w a n n c e l l t u m o u r s a n d f o u n d t h a t t h e n u m b e r o f B lymphocytes varied greatly within neurofibromas (15). 18/32 neurofibromas studied by J o h n s o n e ta l . s h o w e df e w e rt h a n 5 0 B - l y m p h o c y t e sp e rs e c t i o n , a n had more than 150 B-lymphocytes. Only two of the nine MPNSTs studied showed Blymphocytic infiltration.Itis not clear what types of neurofibromas were examined in the J o h n s o n s t u d y o r i ft h e M P N S T s w e r e f r o m i n d i v i d u a l sw i t h N F 1 . T h e a b u n d a n c e o f lymphocytic infiltrationin the nodular neurofibromas we studied suggests that these tumours may be triggeringan immune response. Mast cells synthesize and express c-Kit,a tyrosine kinase receptor that is the product t h e c-Kit p r o t o - o n c o g e n e . T h e l i g a n d f o r c - K i t , w h i c h i s c a l l e d S C F o r K proliferation,migration and survival of mast cells.Neurofibromin and c-Kitare involv i n o v e r l a p p i n g c e l l s i g n a l i n g p a t h w a y s . N e u r o f i b r o m i n a c t s a s an e g a t i v e r e g u l a t o r o f Ras pathway, converting active Ras-GTP to inactiveRas-GDP, while c-Kitactivation KitL induces the phosphorylation of Akt via the PI-3K pathway (25). In culture, it has been shown that Schwann cellsexpress membrane-bound KitL (26) and secrete KitL (27), both of which would attractmast cells.  of i t L ,p r o ed the by  C u l t u r e d S c h w a n n c e l l s f r o m Nf1' m i c e s e c r e t e f i v e t o s i x t i m e s m o r e w i l dt y p eS c h w a n nc e l l s .T h i si n c r e a s e d K i t Ls e c r e t i o n i n c r e a s e sm i g r a t i A  64  m a s tc e l l st o w a r d s N f 1 S c h w a n nc e l l s ( 2 7 ) . I na d d i t i o n , N f 1 m a s t c e l l s e x h i b i t e f o l d i n c r e a s e i n m i g r a t i o n a n d a2 - f o l d i n c r e a s e i n p r o l i f e r a t i o n c o m p a r e d with wild type c e l l s i n r e s p o n s e t o a g i v e n d o s e o f K i t L ( 2 7 ) . T h e m i g r a t i o n o f t h e Nf1 ' m a s t c e l l w a s r e d u c e d t o w i l d t y p e l e v e l s w h e n Nf1 ~ m a s t c e l l s w e r e t r a n s d u c e d w i t h a r e t r o v i r u s c a r r y i n g N f 1 , b u t l i t t l e e f f e c tw a s o b s e r v e d w h e n aN f 1 t r a n s g e n e h a r b o u r i n g a pathogenic mutation was introduced (27).Greater accumulation of mast cells occurred a t t h e s i t e o f K i t L c u t a n e o u s i n f u s i o n i n Nf1 ~ m i c e t h a n i n w i l d t y p e m i c e ( 2 8 ) . +/  +/  +/  Normal adult Schwann cellsdo not express c-Kit(25),but Badache and coworkers demonstrated ac o r r e l a t i o n b e t w e e n a n i n c r e a s e i n t h e l e v e l o f c - K i t s y n t h e s i s a n d a d e c r e a s e i n n e u r o f i b r o m i n s y n t h e s i s i n S c h w a n n c e l l l i n e s d e r i v e d f r o m 3M P N S T s from NF1 individuals(29). In the present study, we did not observe c-Kitstaining of Schwann cellsin any of the three MPNSTs examined, and we only found mast cellsin low numbers in one MPNST. None of these MPNSTs expressed neurofibromin(data not shown). The difference in resultscould be because we assessed c-Kitexpression in primary tumours while Badache and coworkers used celllinesderived from NF1 M P N S T s . A d d i t i o n a l l y , w e a s s e s s e d c - K i te x p r e s s i o n u s i n g i m m u n o h i s t o c h e m i s t y w h i l e B a d a c h e u s e d ad i f f e r e n t m e t h o d o f d e t e c t i o n , W e s t e r n b l o t t i n g . T h e b e n i g n neurofibromas we studied did not show any evidence of S100+ Schwann cells expressing c-Kitwhen slides were dual labeled with antibodies for both S100 and c-Kit antigens (Figure 3.5). The samples used in thisstudy were archivalmaterial from surgical specimens that had b e e n s u b m i t t e d f o r c l i n i c a l p a t h o l o g y . T h e r e f o r e , i tw a s a s s u m e d t h a t t h e s a m p l e w a s representative of the whole tumour and that the sections used to study tumour characteristics and mast cell density and distributionrepresent the tumour as a whole, although some variabilityamong differentportionswould be expected because of the heterogeneous nature of neurofibromas. I n s u m m a r y , w e f o u n d t h a t m a s t c e l l s a r e s i g n i f i c a n t l yl e s s f r e q u e n t i n h i s t o l o g i c a l l y nodular neurofibromas than in diffuse neurofibromas. This difference is consistent with c l i n i c a lo b s e r v a t i o n s t h a t d i f f e r e n t k i n d s o f n e u r o f i b r o m a s h a v e d i f f e r e n t n a t u r a l h i s t o r i e s a n d d i f f e r e n t r i s k s o f m a l i g n a n t d e g e n e r a t i o n . T h e d i f f e r e n c e i n m a s t c e l l i n f i l t r a t i o na n d d i s t r i b u t i o ns u g g e s t s t h a t m a s t c e l l s m a y p l a y d i f f e r e n t r o l e s i n t h e p a t h o g e n e s i s o f differentkinds of neurofibromas. 65  REFERENCES  1. 2.  3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.  Friedman JM. Epidemiology of neurofibromatosis type 1. Am J Med Genet 1999;89:1-6. F r i e d m a n J M , R i c c a r d i V M . C l i n i c a la n d e p i d e m i o l o g i c a l f e a t u r e s . I n : G u t m a n n D H , M a c C o l l i n M , R i c c a r d i V M , e d i t o r s .N e u r o f i b r o m a t o s i s : p h e n o t y p e , n a t u r a l h i s t o r y ,a n d p a t h o g e n e s i s . 3 r d e d . B a l t i m o r e : J o h n H o p k i n s U n i v e r s i t y P r e s s ; 1999. p.26-86. Crowe F, Schull W, Neel J. Multiple Neurofibromatosis. Springfield: Charles C Thomas; 1956. RiccardiVM. An Overview of NF-1: Dysplasia and Neoplasia. In:Riccardi VM, editors.Neurofibromatosis: Phenotype, Natural History and Pathogenesis. 2nd ed. Baltimore: The Johns Hopkins UniversityPress; 1992. p. 18-39. Wiesteler O, Radner H. Pathology of neurofibromatosis 1 and 2. In:S. Huson, R. Hughes, editors. The neurofibromatoses: A pathogenetic and clinical overview, ed. London: Chapman and Hall Medical; 1994. p.135-59. Korf B, Rubenstein A. Neurofibromatosis: A handbook for patients, families and h e a l t h c a r e p r o f e s s i o n a l s . 2 n d e d i t i o n ,N e w Y o r k : T h i e m e M e d i c a l ; 2 0 0 5 . Harkin J, Reed R. Atlas of tumor pathology. Washington: Armed Forces Institute of Pathology; 1968. M a s s o n P . H u m a n T u m o r s : H i s t o l o g y ,D i a g n o s i s a n d T e c h n i q u e . 2 n d e d i t i o n , Detroit:Wayne State UniversityPress; 1970. Woodruff JM. Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis. Am J Med Genet 1999;89:23-30. 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P A T H O G E N E T I C  H E T E R O G E N E I T Y O F NF1  NEUROFIBROMAS  ASSOCIATED  3  INTRODUCTION  Neurofibromatosis 1 ( N F 1 ) i s ac o m m o n dominantly inherited autosomal disease with a n i n c i d e n c e o f 1 i n 3 5 0 0 ( 1 ) . N F 1 i s c a u s e d b y m u t a t i o n s i n t h e NF1 g e n e t h a t e n c o d e s n e u r o f i b r o m i n , a u b i q u i t o u s l y e x p r e s s e d p r o t e i n . M o s t c o n s t i t u t i o n a l m u t a t i o n s i n NF1 r e s u l t i n a l o s s o f f u n c t i o n o f t h e p r o t e i n . L o s s o f t h e n o r m a l NF1 a l l e l e ( L o s s o f Heterozygosity or LOH) has been observed in pheochromocytomas (2),juvenile myeloid leukemia (3) and malignant peripheral nerve sheath tumours (MPNSTs) (4) in N F 1 p a t i e n t s , c o n f i r m i n g NF1 a s a t u m o u r s u p p r e s s o r g e n e . T h e h a l l m a r k f e a t u r e o f N F 1 i s t h e n e u r o f i b r o m a , ab e n i g n t u m o u r c o m p o s e d of S c h w a n n c e l l s ,f i b r o b l a s t s ,p e r i n e u r a l c e l l s ,m a s t c e l l s a n d e n d o t h e l i a l c e l l s . S c h w a n n c e l l s c o n s t i t u t e al a r g e p r o p o r t i o n ( 4 0 - 8 0 % ) o f n e u r o f i b r o m a s a n d a r e t h o u g h t t o b e t h e progenitor celltype because culturesderived from some NF1 patient neurofibromas d i s p l a y l o s s o f t h e n o r m a l NF1 a l l e l e i n S c h w a n n c e l l s b u t n o t i n f i b r o b l a s t s ( 5 - 7 ) . N e u r o f i b r o m a s a r e t h o u g h t t o a r i s e a c c o r d i n g t o t h e " t w o - h i t "h y p o t h e s i s , w i t h a n i n a c t i v a t i n g s o m a t i c m u t a t i o n i n t h e n o r m a l NF1 a l l e l e a s t h e r a t e - l i m i t i n g s t e p . L O H s e c o n d s o m a t i c m u t a t i o n s o f t h e NF1 l o c u s h a v e b e e n o b s e r v e d i n 2 6 % o f d e r m a l neurofibromas and 36% of plexiformneurofibromas studied (8-24) .The low percentage o f o b s e r v e d L O H i s u s u a l l y a t t r i b u t e d t o t e c h n i c a l l i m i t a t i o n so f t h e a s s a y o r t o "contamination" of the neoplastic Schwann cellclone by an abundance of nonneoplastic stromal cells present in these heterogeneous tumours. T a p p t " 3  here is also evidence that Schwann cellscomprising an NF1 -associated neurofibroma re themselves heterogeneous. Immunohistochemical analyses of some dermal and lexiformneurofibromas have shown both neurofibromin-negativeand neurofibrominositiveSchwann cells (25).The former are usually interpretedas the neoplastic clone h a t h a s l o s t b o t h f u n c t i o n a l c o p i e s o f t h e NF1 g e n e , a n d t h e l a t t e r a s n o r m a l contaminants" of the tumour that have proliferatedin response to the neoplastic A version of this c h a p t e r has been s u b m i t t e d for publication. Tracy Tucker, C a r o l y n B r o w n , John Fee,  V i n c e n t M. R i c c a r d i , M a r g a r e t S u t c l i f f e , J a n i n e W e c h s l e r , P i e r r e W o l k e n s t e i n J a n M. F r i e d m a n . P a t h o g e n e t i c h e t e r o g e n e i t y of N F l - a s s o c i a t e d  neurofibromas  68  process. A similar interpretation has been made ( p r e s u m a b l y S c h w a n n c e l l )l i n e s g r o w n f r o m N F contain a mixture of neurofibromin-positive and m i x t u r eo f c e l l sw i t ha n d w i t h o u tN F 1 L O H The "two readily ex example, i n v o k  h i t "h y p o t h e s i s e x p l a i n s t h e d e v e l o p m plain all of the clinicaldata regarding the following observations in NF1 pa i n g a dh o cm o d i f i c a t i o n  of observations that S100-positive 1-associated neurofibromas may neurofibromin-negative cells or a ( 6 ,2 5 ,2 6 )  ent o neuro tients so ft  f some fibroma are not h e" t  neurofibromas, but does not development. For easily explained without w oh i t "h y p o t h e s i s :  •  T h e a p p a r e n ta s s o c i a t i o no ft r a u m aw i t ht h es u b s e q u e n td e v e l o p m e n to f a neurofibroma at the same site (27).  •  E r u p t i v en e u r o f i b r o m a t o s i s ,i nw h i c hi n n u m e r a b l ed e r m a ln develop over a period of a few months in a person with NF1 (28).  •  R a p i d i n c r e a s ei nt h ed e v e l o p m e n to fn e u r o f i b r o m a sd u r i n g p r e g n a subsequent regression of some tumours afterdelivery (29).  •  T h e f a i l u r et of i n d e v i d e n c e o f L O H o ra " s e c o n d h i t " N F 1 m u t a t i o n i nm a n neurofibromas, despite extensive molecular genetics studies (8-24).  We have proposed an alternativeinterpretationof these data: that NF1-associated neurofibromas, which are clinicallyheterogeneous in appearance, natural history, and propensityto progress to MPNSTs, (30) are also pathogenicallyheterogenous (31). We s u g g e s t t h a t i n p e o p l e w i t h N F 1 s o m e n e u r o f i b r o m a s a r i s e t h r o u g h i n a c t i v a t i o no f t h e second (normal)NF1 alleleina Schwann celland expansionof the resultingneoplastic c l o n e ,w h i l eo t h e rn e u r o f i b r o m a sa r i s ea s p o l y c l o n a lp r o l i f e r a t i o n so f N F 1 haploinsufficient cells. We used immunohistochemistry to determine the expression of neurofibromin (the p r o t e i n p r o d u c to f t h e N F 1 l o c u s ) i nS c h w a n n c e l l sa n d n o n - S c h w a n nc e l l so f N F 1 associated neurofibromas classifiedhistopathologicallyas diffuseor nodular. We observed a significantly lower proportion of S100+/Nf+ cells and a significantly higher proportion of S100-/Nf- in nodular neurofibromas than in diffuse neurofibromas. W e t h e n u s e d l a s e r m i c r o d i s s e c t i o nt o i s o l a t eS 1 0 0 + / N f + , S 1 0 0 + / N f - a n d S 1 0 0 - / N f + c e l l sf r o m t u m o u r s t h a t h a d b e e n r e m o v e d f r o m f e m a l e s w i t h N F 1 . W e t e s t e d f o r 69  c l o n a l i t y i n e a c h c e l l t y p e u s i n g X - l i n k e d AR p o l y m o r p h i s m s . W e f o u n d c l e a r e v i d e n c l o n a l i t ya m o n g t h e S 1 0 0 + / N f - c e l l s i n s o m e t u m o u r s , a s w o u l d b e e x p e c t e d i f t h e neurofibromas arose through clonal expansion of neoplastic Schwann cells after a "second hit"eliminated all neurofibromin expression. In contrast, other tumours showed no evidence of clonalityin any of the celltypes examined, as would be expected if these n e u r o f i b r o m a sa r o s ea s p o l y c l o n a lp r o l i f e r a t i o n so fN F 1 h a p l o i n s u f f i c i e n tc e l METHODS Sample Collection  W e c o l l e c t e df o r m a l i n f i x e d - p a r a f f i ne m b e d d e d s a m p l e s f r o m C r e t e i lH o s p i t a l( P a r i s , France), The Neurofibromatosis Institute(La Crescenta, California),Vancouver General H o s p i t a l ( V a n c o u v e r , C a n a d a ) , a n d C h i l d r e n ' sa n d W o m e n ' s H o s p i t a l ( V a n c o u v e r , C a n a d a ) . W e s t u d i e d at o t a l o f 3 8 b e n i g n - n e u r o f i b r o m a s f r o m 2 9 p e o p l e w h o a l l h a d a confirmed diagnosis of NF1 according to established criteria(32). The tumours used in t h i s s t u d y a r e as u b s e t o f t h o s e u s e d i n C h a p t e r 3 . T h e s t u d y p r o t o c o l w a s a p p r o v e d b y t h e U n i v e r s i t y o f B r i t i s hC o l u m b i a R e s e a r c h E t h i c s C o m m i t t e e . Histopathological Classification of Neurofibromas  Each neurofibroma was classified histopathologicallyon the basis of itsappearance in hemotoxylin- and eosin- (H&E) stained sections as diffuse or nodular according to definitionsused by one of the authors (J.W.).Diffuse neurofibromas were predominantly located in the dermis. They involved the thin terminal branches of peripheral nerves and were characterized by proliferationof cellsthat were not limitedby the perineurium. The proliferating cells were irregularly dispersed within a fibrous and/or myxoid background and sometimes encircled normal structures (exocrine glands, hairfolliclesand vessels) without destroying them. Myelin fibrilswere rare or absent. N o d u l a r n e u r o f i b r o m a s u s u a l l y i n v o l v e d d e e p e r t i s s u e s . T h e c e l l u l a rp r o l i f e r a t i o ni n these tumours was intraneural,enclosed within large hypertrophic nerves circumscribed b y t h e p e r i n e u r i u m . S i n g l e n o d u l a r t u m o u r s i n v o l v e d j u s t o n e f a s c i c l e ,w h i l e n o d u l a r plexiform tumours involved several fascicles, nerve branches or a nerve plexus. On transverse section, the central area of nodular tumours was loose, with a myxoid background substance, usually containing dispersed or fascicular myelin fibrils.The peripheralarea of these tumours was usually more cellular. 70  S o m e n e u r o f i b r o m a s c o n t a i n e d b o t h d i f f u s ea n d n o d u l a r c o m p o n e n t s ; s u c h t u m o u r s were usuallyfound in the deep dermis and subcutaneous tissue.The nodular component was surrounded by a diffuse extraneural cellular proliferation thatinfiltrated the fattissue and other normal structures. Tumour Characteristics  H&E sections of each neurofibroma were independently reviewed by two of us (M.S. a n d T . T . ) E a c h t u m o u r w a s g r a d e d a t 2 0 0 x m a g n i f i c a t i o n o n a3 - p o i n t L i k e r t s c a l e f o r cellularityand vascularity.Lymphocytic infiltrationwas determined at 400x magnification a s ap e r c e n t a g e o f t h e t o t a l c e l l s . O n l y n e u r o f i b r o m a s w i t h < 3 0 % l y m p h o c y t i c i n f i l t r a t i o n w e r e s e l e c t e d f o r t h i s s t u d y . L y m p h o c y t i c i n f i l t r a t i o n w a s t h e n r a t e d o n a3 - p o i n t L i k e r t scale of <5%, 6-20%, 21-30%. Five to eight fieldschosen at random were taken to represent the tumour as a whole. The section was then scanned to identifyregions that r e p r e s e n t e d t h e f u l lr a n g e o f c e l l u l a r i t yw i t h i n t h e s p e c i m e n . T h e s e r e g i o n s w e r e marked for subsequent imaging and analysis afterstainingfor S100 (a Schwann cells marker) and neurofibromin. Immunofluorescent Staining for S100 and Neurofibromin  F o u r s e r i a l5 p m s e c t i o n s w e r e c u t a f t e rt h e H & E s e c t i o n f r o m e a c h o f t h e n e u r o f i b r o m a s and stained respectively for S100 only, neurofibromin (Nf) only, S100 and neurofibromin t o g e t h e r o r w i t h n o p r i m a r y a n t i b o d y a s an e g a t i v e c o n t r o l . S e c t i o n s o f a t r a u m a t i c n e u r o m a f r o m ap e r s o n w h o d o e s n o t h a v e N F 1 w e r e u s e d a s ap o s i t i v e c o n t r o l , a n d sections of an MPNST f r o m ap e r s o n w i t h N F 1 w e r e u s e d a s an e g a t i v e c o n t r o l f o r t h e specificityof neurofibromin staining.Traumatic neuromas are composed of similar cell types as neurofibromas, mainly Schwann cellsand fibroblasts,both of which express neurofibromin.An average of 92% of cellsinthe traumatic neuroma were neurofibromin positive.There was no evidence of S100 or neurofibromin expression in the MPNST, although Schwann cellsin an adjacent histologicallybenign component were S100 and neurofibromin positive. All sections were deparaf 95%, and 70%) and equi was performed in 10mM treated with fresh 1mg/ml  finized in xylene, hyd libratedin phosphatesodium citratepH 6. sodium borohydride 71  rated through graded alcohols (100%, buffered saline. Heat antigenretrieval 0 at 70°C for 10 minutes. Samples were for 20 minutes at room temperature to  reduce autofluorescence and then blocked with 5% normal goat serum for 30 minutes. P r i m a r y a n t i b o d y t o S 1 0 0 ( p o l y c l o n a lr a b b i ta n t i - c o w ; D A K O C a n a d a I n c . ; M i s s i s s a u g a O N , C a n a d a ) w a s a d d e d a t ac o n c e n t r a t i o n o f 1 : 4 0 0 a n d n e u r o f i b r o m i n ( m o n o c l o n a l mouse anti-human; McNFN27; Novus Biologicals Inc.;Littleton,CO) at a concentration of 1:100. The sections were incubated in antibody solution overnight at room temperature. Goat anti-mouse and anti-rabbitIgG antibodies labeled with Alexa 488 and 568 fluorochromes, respectively,were then added, and the sections were incubated for 90 minutes at room temperature. Nuclei were identifiedby stainingwith DAPI. Slides were cover-slippedwith VECTASHIELD© m o u n t i n g m e d i u m ( V e c t o rL a b o r a t o r i e s C a n a d a I n c . ;B u r l i n g t o n ,O N , C a n a d a ) . Image Acquisition  Slides stained for S100 only or for neurofibromin only were scanned to ensure that the staining worked. Blood vessels within the tumour served as an internalpositive control for neurofibromin staining.On the section that had been dual labeled for both S100 and neurofibromin,two images were taken using each of three filters(DAPI, FITC, RHOD) withineach region that had been selected forexamination on the H&E stainedslide. Areas that had few blood vessels were chosen to avoid includingS100-/Nf+ endothelial c e l l s i n t h e c o u n t s . A d d i t i o n a l i m a g e s w e r e t a k e n i n a r e a s w h e r e t h e r e w a s ac h a n g e i n the staining patterns for S100 and neurofibromin. All samples were imaged on a Zeiss A x i o P l a n 2m i c r o s c o p e e q u i p p e d w i t h a1 4 b i t A x i o C a m H R m c a m e r a a t 4 0 0 x magnification. Image resolution was 1388 X 1040 pixels, arid images were saved in TIF format. Image Analysis  Cellomics (Cellomics Inc, Pittsburg PA) bioimaging software was used to quantify the totalnumber of cellswithin an image and the number of cellsthat were S100+/Nf+, S 1 0 0 + / N f - , S 1 0 0 - / N f + a n d S 1 0 0 - / N f - .T h e b a c k g r o u n d p i x e li n t e n s i t yw a s measured from five random areas for each of the images on each fluorescent channel. A cell was c o n s i d e r e d t o b e p o s i t i v e w h e n i t sp i x e l i n t e n s i t yw a s t w o s t a n d a r d d e v i a t i o n s a b o v e t h e mean background pixelintensity.This analysis was performed on each of the images, and averages were calculatedforeach tumour.  72  Statistical Analysis  Kendall's correlationwas calculated using SPSS (SPSS, Inc. Chicago, Illinois,2001) to determine the relationship between vascularity, cellularity,lymphocyticinfiltration, tumour type and staining patterns. The Mann-Whitney U test was performed using S P S S ( S P S S , I n c . C h i c a g o , I l l i n o i s , 2 0 0 1 ) t o d e t e r m i n e i ft h e r e w e r e s i g n i f i c a n t differencesbetween the S100+/Nf+, S100+/Nf-, S100-/Nf+, and S100-/Nf- cells as percentages of the total number of cells within nodular and diffuse neurofibromas. A pvalue less than or equal to 0.05 was considered to be significant. Method Development for Immunofluorescently Labeled-formalin Fixed Samples for Laser Microdissection  Metal cassettes and normal glass slides were found to be incompatible with the staining procedure described above. Glass slideswith polyethylene naphtholate (PEN) membrane mounted on top were, therefore,used for this study. L a s e r m i c r o d i s s e c t i o n r e q u i r e s ad r y s a m p l e , b o t h f o r c u t t i n g a n d c a p t u r i n g . F o r m a l i n fixed samples have high overall levels of tissue autofluorescence, which is further h e i g h t e n e d w h e n t h e s a m p l e s a r e v i s u a l i z e dd r y . X y l e n e , 1 0 0 % e t h a n o l , a n d 9 5 % e t h a n o l w e r e t e s t e d t o d e t e r m i n e i ft h e y c o u l d b e u s e d t o w e t t h e s a m p l e f o r visualizationbut would dry quickly enough to allow microdissection of the desired cells. Xylene provided the best visual picture of the tissue and evaporated quickly. However, x y l e n e c o u l d o n l y b e a p p l i e d t o t h e t i s s u e t h r e e t i m e s b e c a u s e i td e g r a d e d t h e P E N membrane to which the tissuewas attached. After applying xylene to the tissue, tiledimages of 120-140 fieldsat 600x were taken. Selected cells were microdissected on a Veritas microdissector using the ultraviolet (UV) laser and collected using the infrared(IR) laser.There are several parameters withinthe UV cuttingthat can be adjusted. These include the strength of the laser, the n u m b e r o f t i m e s t h e s e l e c t i o n i s c u t , a n d t h e " t a b s " ,w h i c h a r e s p a c e s o n t h e s p e c i m e n t h a t a r e l e f tu n c u t t o h e l p p r e v e n t t i s s u e c u r l i n g . M a n y e x p e r i m e n t s w e r e r e q u i r e d t o develop conditions that provided satisfactoryrecovery of these formalin-fixed tissues with single cell microdissection when hundreds of cells were being selected in a small a r e a . I tw a s d e t e r m i n e d t h a t t h e b e s t o p t i o n w a s t o e l i m i n a t e t h e " t a b s " f o r c u t t i n g , w h i c h p e r m i t t e d g o o d c o l l e c t i o n w i t h as i n g l e c u t . 73  T g c A  he last parameter tested was iven tiledimage. This varied ollectedfrom tissue that was ssessment of the tissue was,  the number of selections that could be collected within a between samples. A greater number of samples could be m o r e c e l l u l a rt h a n f r o m t i s s u e t h a t w a s m o r e f i b r o u s . therefore,required before beginning the microdissection.  Tissue Preparation for Laser Microdissection  S e r i a l s e c t i o n s w e r e c u t f r o m 3d i f f u s e n e u r o f i b r o m a s from 3 females with NF1 and 3 nodular neurofibromas from 1 female with NF1. The firstsection was cut at 5pm, s t a i n e d w i t h H & E , a n d u s e d a s ar e f e r e n c e s l i d e . A l l o t h e r s e c t i o n s w e r e c u t a t 8 p m and m o u n t e d o n P E N g l a s s m e m b r a n e s l i d e s( M o l e c u l a rD e v i c e s , S u n n y v a l e , C A ) . O n e section was immunofluorescently stained (as described above) with rabbit anti-human s m o o t h m u s c l e a c t i n ( S M A ) ( A n a S p e c , I n c . , S a n J o s e , C a l i f o r n i a ) a t ac o n c e n t r a t i o n o f 1:400 to identifyblood vessels withinthe neurofibromas. The DNA purifiedfrom the b l o o d v e s s e l s w i t h i n t h e n e u r o f i b r o m a s s e r v e d a s ac o n t r o l f o r m o l e c u l a r a n a l y s i s b e c a u s e p e r i p h e r a l b l o o d w a s n o t a v a i l a b l e .S u b s e q u e n t s e c t i o n s w e r e s t a i n e d f o r b o t h neurofibrominand S100 as described above, with the exception thatAlexa 594 was used instead of Alexa 568 as the labelon the secondary antibody. Sample Collection by Laser Microdissection  Tiled images were taken using two filters(FITC, RHOD) on the Veritasmicrodissector. T h e i m a g e s w e r e c o m p a r e d t o i d e n t i f yc e l l st h a t w e r e S 1 0 0 + / N f + , S 1 0 0 + / N f - a n d S 1 0 0 /Nf+. Cells of each class were selectively cut with aUV laser and attached to a c o l l e c t i o nc a p u s i n g a n i n f r a r e d l a s e r . B a s e d o n t h e i n i t i a l D N A a s s e s s m e n t o f t h e w h o l e tumour, enough cellswere microdissected to yield20ng of DNA for each celltype. DNA Purification  DNA was extracted from microdissected samples by overnight incubationat 42°C in 50pl of DNA extractionbufferper microdissectioncap. The buffercontained 10mM TrisH C I p H 8 . 0 ,1 m M E D T A , 1 % T w e e n 2 0 a n d 0 . 1 % P r o t e i n a s eK .P h e n o l / C h l o r o f o r m extractionwas performed followed by ethanol precipitationwith 4pg/ul of glycogen a d d e d t o e a c h 5 0 u l d i g e s t i o n a s ac a r r i e r . E t h a n o l p r e c i p i t a t i o n w a s c a r r i e d o u t a t - 2 0 ° C for a minimum of 2 hours in 0.1 M sodium acetate and 95% ethanol pH 5.2 solution. Samples were resuspended in 10ul of water.  74  XCI Clonality Assay  S k e w i n g o f X - c h r o m o s o m e - i n a c t i v a t i o n ( X C I ) w a s u s e d t o a s s e s s c l o n a l i t yi n e a c h o f the microdissected samples by means of a DNA methylation-sensitiye assay using p o l y m o r p h i c v a r i a n t s o f t h e a n d r o g e n r e c e p t o r ( A R ) g e n e . 5 u lo f p u r i f i e d g e n o m w a s d i g e s t e d w i t h Hpall a n d Rsal, a n d a n u n d i g e s t e d c o n t r o l w i t h 5 u w a s d i g e s t e d w i t h Rsal a l o n e . B o t h d i g e s t i o n s w e r e c a r r i e d o u t i n a t o t a l v o l u m e o f 1 0 u l . N e s t e d P C R w a s p e r f o r m e d o n t h e D N A d i g e s t s w i t h t h e f o l l o w i n gp r i m e r s f o r 3 5 c y c l e s i ne a c h r o u n d : F i r s t r o u n d - A R N 1 F ( 5 ' C T G C A G C G A C T A C C G C A T C 3 ' ) , A R N 2 R GCTCTGGGACGCAACCTC 3');second round:ARF (5' T C C A G A A T C T G T T C C A G A G C G T G C 3 ' ) ,A R R ( 5 ' G C T G T G A A G G T T G C T G T T C C T C A T 3 ' ) .T h e s e c o n d r o u n d o f P C R w a s p e r f o r m e d w i t h af o r w a r d p r i m e r l a b e l e d w i t h t h e H E X A B I P r i s m D y e , a n d p r o d u c t s w e r e s i z e d u s i n g c a p i l l a r y e l e c t r o p h o r e s i so n a n A B I Prism 310 genetic analyzer (Applied Biosystems, Foster City,CA). Fluorescence was detected by ABI Prism data-collectionsoftware and analysed with GeneScan software. T h e p e a k a r e a f o r e a c h a l l e l ew a s u s e d t o d e t e r m i n e X C I s k e w i n g . T o a c c o u n t f o r p r e f e r e n t i a la m p l i f i c a t i o n , t h e a r e a s o f t h e p e a k s o f t h e d i g e s t e d s a m p l e w e r e normalized in relationto measurements from the undigested sample. T r r >  he degre epresent epresent 90% ske  e of skewing was the peak areas of the peak areas of wing were conside  cal the the red  culatedas (d1/u1)/(d1/u1+d2/u2),where d1 and d2 t w o a l l e l e sf r o m t h e d i g e s t e d s a m p l e s , a n d u 1 a n d u 2 t w o a l l e l e sf r o m t h e u n d i g e s t e d s a m p l e s . S a m p l e s w i t h to be clonal.  RESULTS  W e s t u d i e d t h e e x p r e s s i o n o f n e u r o f i b r o m i n a n d S 1 0 0 , aS c h w a n n c e l l m a r k e r , i n 3 8 b e n i g n n e u r o f i b r o m a s f r o m 2 9 N F 1 p a t i e n t s .2 8 o f t h e t u m o u r s ( f r o m 2 3 p a t i e n t s ) w e r e classified histopathologically as diffuse neurofibromas, 8 tumours (from 5 individuals) were classified as nodular neurofibromas, and two tumours (from 2individuals) had a mix of both diffuseand nodular components. The mixed tumours were not included in the statisticalanalysis. Figure 4.1 shows examples of each type of neurofibroma with characteristic patterns of cellularity,vascularity, and lymphocytic infiltration.  75  Figure 4.1 Images of typical neurofibromas in the current study that represent the majority of the tumour.  A. H&E section of anodular neurofibroma at 400x magnification. B. Serial section to (A) stained for neurofibromin and counterstained with DAPI at 400x magnification. C. Serial section to (A) stained for S100 and counterstained with DAPI at400x. D. Merged image of (B) and (C). E. H&E section of adiffuse neurofibroma at 400x magnification. F. Serial section to (E) stained for neurofibromin and counterstained with DAPI at 400x magnification. G. Serial section to (E) stained for S100 and counterstained with DAPI at 4 0 0 x . H . M e r g e d i m a g e o f ( F ) a n d ( G ) . N o t e t h e h i g h e r c e l l u l a r i t y i n Ec o m p a r e d to A and the lymphocytic infiltration(L) in A. Also note the presence of blood vessels marked with an arrow and stained positive in (B) and (F) for neurofibromin.  76  77  Cellularity  N o d u l a r n e u r o f i b r o m a s t e n d e d t o b e l e s s c e l l u l a rt h a n d i f f u s e n e u r o f i b r o m a s ( F i g u r e 4.1, Figure 4.2), with almost 75% of nodular tumours fallinginto the lowest cellularity category. In comparison to diffusetumours, nodular neurofibromas displayed more dense fibrotictissue and areas of degeneration in the central portion, but not at the periphery. In contrast, the diffuse tumours had higher overall cellularitywith a more u n i f o r m d i s t r i b u t i o n o f c e l l s t h r o u g h o u t t h e t u m o u r . T h e r e w a s as i g n i f i c a n t c o r r e l a t i o n b e t w e e n c e l l u l a r i t ya n d h i s t o p a t h o l o g i c a l t u m o u r t y p e ( K e n d a l l ' s t a u = - 0 . 4 7 4 , p = 0 . 0 0 3 ) (Table 4.1). Lymphocytic Infiltration  Only neurofibromas with less than 30% lymphocytic infiltrationwere chosen for this study, as lymphocytes would contributeto the overallcellularityas determined by DAPI staining and cause an over-estimation of the percentage of cells within a neurofibroma that were neurofibromin positiveand an under-estimation of those that were S100p o s i t i v e .A l l n e u r o f i b r o m a s t h a t w e r e e x c l u d e d f r o m t h i s s t u d y w e r e n o d u l a r neurofibromas. For the 38 tumours included in the study, the average lymphocytic infiltrationfor nodular neurofibromas was 13% compared to 9% in diffuse neurofibromas. The correlationof lymphocytic infiltrationand tumour type could not be tested statisticallybecause the most extreme cases were eliminated in the tumour selection process. Lymphocytic infiltrationwas generally distributedevenly w neurofibromas, while lymphocytes were more localizedin s o b s e r v e d i n d i f f u s e n e u r o f i b r o m a s . T h e r e w a s as i g n i f i c a n t lymphocytic infiltrationwith both cellularity (Kendall's tau = vascularity (Kendall's tau = -0.510, p=0.001) (Table 4.1).  ithin the nodular mall clusterswhen they were inverse correlation between -0.353, p=0.022) and  Vascularity  Nodular n had more throughou correlated  eurofibromas typicallyhad fewer large vessels than smaller vessels (Figure 4.1, Figure 4.2).Vessels we t both diffuseand nodular neurofibromas. Vasculari with tumour type (Kendall's tau = -0.187, p=0.254)  78  diffusetumours, which re evenly distributed ty was notsignificantly but was significantly  correlated with cellularity (Kendall's tau =0.479, p=0.002), as more cellular neurofibromas had a higher number of blood vessels (Table 4.1). Figure 4.2 Distribution of pathological features of neurofibromas classified histopathologically.  A. Cellularity estimated on a 3-point Likert scale with 3 being the highest cellularity; B. Vascularity estimated on a 3-point Likert scale with 3 being the highest vascularity; and C. Lymphocytic infiltration estimated on a 3-point Likert scale with 3 being the highest lymphocytic infiltration. I Diffuse neurofibromas •  Nodular neurofibromas  79  Table 4.1 Summary of Kendall's tau correlations for pathological features in histologically-defined nodular and diffuse neurofibromas from patients with NF1.  T u m o u r T u m o u r T y p e Coefficient Significanc Cellularity Coefficient Significanc Vascularity Coefficient Significanc Lymphocytes Coefficient Significanc  T y p e  Cellularity  Vascularity  e  1.0  e  -0.474 P=0.003  1.0  e  -0.187 P=0.254  0.479 P=0.002  1.0  e  0.204 P=0.209  -0.353 P=0.022  -0.510 P=0.001  Lymphocytes  1.0  Neurofibromin & S100 Staining within Benign Neurofibromas  Fi hi we Th ne  gure 4.1 shows stologically-d re scattered t ere were area rve fibers.  examples of typicalstaining patterns for S100 and neurofibro efineddiffuse and nodular neurofibromas. S100-positive (Schwa hroughout allneurofibromas, often separated by S100-negative s t h a t s h o w e d c o m p a c t S 1 0 0 - p o s i t i v e c e l l s , i n d i c a t i n gt r a p p e d  min in nn) cells cells. axons or  W h e n s l i d e s w e r e d u a l l a b e l e d f o r S 1 0 0 a n d n e u r o f i b r o m i n ,2 5 . 4 ± 9 . 6 % ( m e a n ± 1 S D ) o f cellswithin nodular neurofibromas and 41.9±14.2% of cellswithin diffuse neurofibromas s t a i n e d p o s i t i v e l yf o r b o t h S 1 0 0 a n d n e u r o f i b r o m i n ( t h e p h e n o t y p e e x p e c t e d f o r n o r m a l Schwann cells)(Figure 4.3). This difference was highly statisticallysignificant (p=0.005). The proportion of S100-positive cellsthat were also neurofibromin-positive was somewhat lower in nodular neurofibromas (61.1±20.0%) than in diffuse neurofibromas (74.4±12.7%), but this difference did not reach statisticalsignificance (p=0.071). Fifteen of the 38 tumours showed neurofibromin expression in more than 75% of S100-positive c e l l s .T h r e e t u m o u r s , o n e n o d u l a r a n d t w o d i f f u s e ,s h o w e d n e u r o f i b r o m i n s t a i n i n g i n more than 90% of S100-positive cells.In one of these diffuse neurofibromas, 100% of S100-positive cells also expressed neurofibromin.  80  Figure 4 . 3 Boxplot summarizing S 1 0 0 and neurofibromin staining in nodular and diffuse neurofibromas.  The boxes represent the interquartilerange, with the top of the box representing t 75th percentile and the bottom the 25th percentilefor the data. The linewithin the represents the median. The whiskers on the boxes display the spread of the data. c i r c l e s r e p r e s e n t o u t l i e r s t h a t a r e b e t w e e n 1 . 5 a n d 3b o x l e n g t h s f r o m t h e u p p e r o r l o w e r e d g e o f t h e b o x . T h e s t a r s r e p r e s e n t o u t l i e r s t h a t a r e g r e a t e r t h a n 3b o x l e n g from the upper edge of the box. [ |D i f f u s e n e u r o f i b r o m a s I N o d u l a rn e u r o f i b r o m a s  S100+M+  S100+M-  S100-M+  he box The ths  S100-/Nf-  Overall, there did not appear to be large aggregations of S100+/Nf- Schwann cells in any of the tumours. Rather, S100+/Nf- cellsand S100+/Nf+ cellswere both found t h r o u g h o u t t h e t u m o u r s . T h e r e w a s n o s i g n i f i c a n td i f f e r e n c eb e t w e e n n o d u l a r a n d diffuse neurofibromas in the mean percentage of cellsthat was S100-positive and neurofibromin-negative (the phenotype predicted for aSchwann cell neoplasm that a r i s e s b y c o m p l e t e i n a c t i v a t i o n o f t h e NF1 l o c u s ) ( F i g u r e 3 ) . C e l l s n e g a t i v e f o r b o t h S100 and neurofibromin were scatteredthroughout both types of neurofibromas. The mean percentage of these double negative cellswas significantlyhigher in nodular neurofibromas (31.3±9.9%) than in diffuse neurofibromas (15.7±10.4%) (p=0.003, Figure 4.3). Five nodular neurofibromas and two diffuseneurofibromas had more than 35% of cellsnegative for both S100 and neurofibromin. Nodular neurofibromas usually 81  h a v e c e n t r a l a r e a s o f d e g e n e r a t i o n o r n e c r o s i s t h a t t e n d t o b e n e g a t i v e f o r b o t h S100 a n d n e u r o f i b r o m i n , b u t p a r t i c u l a rc a r e w a s t a k e n t o a v o i d t h e s e a r e a s f o r i m a g i n g because they may not represent the tumour as a whole. There was no difference between  nodular and diffusetumours in the percentage of cells  that was S100-negative and neurofibromin-positive(the phenotype expected for normal s t r o m a lc e l l s ) ( F i g u r e 4 . 3 ) . T h e s e c e l l s w e r e a l s o d i s t r i b u t e d a m o n g t h e S100-positive cells. N i n e n e u r o f i b r o m a s , . 7 d i f f u s e a n d 2n o d u l a r , s h o w e d a r e a s o f n o n - u n i f o r m s t a i n i n g f o r either S100 or neurofibromin. Six of the diffuse neurofibromas showed littlevariabilityin the percentage of any of the celltypes throughout the specimen, but one diffuse tumour s h o w e d ad r o p i n p e r c e n t a g e o f a l l c e l l t y p e s w i t h o n e a r e a t h a t w a s completely negative for both S100 and neurofibromin.Of the two nodular neurofibromas that s h o w e d v a r i a t i o n i n s t a i n i n g , o n e s h o w e d ad r o p i n t h e p e r c e n t o f a l l c e l l t y p e s a n d t h e o t h e r s h o w e d ad r o p i n t h e p e r c e n t S 1 0 0 + / N f + c e l l s i n o n e p a r t i c u l a r a r e a o f t h e s l i d e . Four neurofibromas, alldiffuse, showed variations in tumour appearance according to the H&E, and more images were taken in these differingareas. Three of these neurofibromas did not show any difference in the percentage of each of the celltypes in comparison to the other areas of the tumour, while one neurofibroma showed some variation in the number of cells that were positive for both neurofibromin and S100. Correlations  T h e r e w a s as i g n i f i c a n t i n v e r s e c o r r e l a t i o n b e t w e e n t h e n u m b e r o f S 1 0 0 + / N f + c e l l s a n d the number of S100-/Nf- cells in atumour (p<0.001) (Table 4.2). S100+/Nf+ cells were also inversely correlated with S100-/Nf+ cells (p=0.018) but not with S100+/Nf- cells (Table 4.2). S100-/Nf+ and S100+/Nf- cellswere negatively correlated with each other ( p < 0 . 0 0 1 ) . T h e n u m b e r o f l y m p h o c y t e s i n an e u r o f i b r o m a w a s d i r e c t l y c o r r e l a t e d w i t h the number of S100-/Nf- cells (p=0.034) and inversely correlated with the number of S100+/Nf+ cells (p= 0.01) (Table 4.2).  82  Table 4.2 Summary of Kendall's tau correlations for S100 and neurofibromin staining in histologically-defined noduiar and diffuse neurofibromas from patients with NF1.  S100+/Nf+ S100+/Nf+ Coefficient Significanc S100+/NfCoefficient Significanc S100-/Nf+ Coefficient Significanc S100-/NfCoefficient Significanc  S100+/Nf-  S100-/Nf+  e  1.0  e  0.007 P=0.956  1.0  e  -0.285 P=0.018  -0.544 P O . 0 0 1  1.0  e  -0.595 P O . 0 0 1  -0.124 P=0.299  0.142 P=0.238  S100-/Nf-  1.0  Clonality ofS100+/Nf+, S100+/Nf- and S100-/Nf+ Cells  Ifaneurofibroma develops from an S100-positive (Schwann) cell that sustains a " s e c o n d h i t " m u t a t i o n o f t h e NF1 l o c u s , t h e n e o p l a s t i c c e l l s s h o u l d b e c l o n a l l y r e l a t e d t o each other and should not express neurofibromin. Other cellswithin the tumour that are not part of the neoplastic clone would be expected to continue to express neurofibromin a n d t o b e p o l y c l o n a l . I n c o n t r a s t , i f n e u r o f i b r o m a s a r i s e b y p r o l i f e r a t i o n o f NF1 haploinsufficient cells,allcelltypes, including Schwann cells,should be polyclonal and should continue to express neurofibromin. Six neurofibromas, 3diffuse and 3nodular, that had been removed from female NF1 patientswere selected for microdissection based on DNA quality.After sections of each t u m o u r w e r e d o u b l e s t a i n e d f o r S 1 0 0 a n d n e u r o f i b r o m i n ,a n d S 1 0 0 + / N f + c e l l s ( p u t a t i v e l yn o r m a l S c h w a n n c e l l s ) ,S 1 0 0 + / N f - c e l l s ( p u t a t i v e l y n e o p l a s t i c S c h w a n n c e l l s t h a t h a d s u s t a i n e d a " s e c o n d h i t " NF1 m u t a t i o n ) , a n d S 1 0 0 - / N f + c e l l s ( p u t a t i v e l y n o r m a l non-Schwann cells)were isolated by laser capture microdissection (Figure 4.4).  83  Figure 4.4 Images showing selective capture of single cell microdissection of S100+/Nf-negative cells from heterogeneous tissue.  A. Diffuse neurofibroma stained for neurofibromin. Red circles indicate cells that are S100-/Nf+ when compared side-by-sideto images of S100 stainingat 600x magnification. B. Same tissue in (A) stained for S100. Green circles indicate cells that are S100+/Nf- and blue circlesindicatecellsthat are S100+/Nf+ when compared sideby-side to images of neurofibromin staining at 600x magnification. C. Image of tissue after cutting allof the selected cells types. Red arrows indicate S100-/Nf+ cells cut with a UV laser, green arrows indicate S100+/Nf- cells cut with aUV laser, and the blue arrows indicate S100+/Nf+ cells cut with aUV laser at 600x magnification. After cutting, only the S100+/Nf- cellswere captured using the infrared laser onto the collection cap. D. Images of collectioncap showing specific removal of only S100+/Nf- cells from heterogeneous tissue at 400x magnification.  84  85  The X-chromosome inactivation was then measured to determine each tumour and tested to determ samples from these patients were summarized in Table 4.3.  ( X C I ) r a t i oo f D N A i s o l a t e d f r o m e a c h g r o u p o f c e l l s clonality.Blood vessels were also microdissected from ine the XCI ratioin normal tissue because blood not available(Figure 4.5).The results are  Figure 4.5 Images of Blood Vessel Microdissection.  A . T i s s u e s e c t i o n s t a i n e d w i t h s m o o t h m u s c l e a c t i n ( S M A ) t o i d e n t i f yb l o o d v e s s e l s w i t h i n a d i f f u s e n e u r o f i b r o m a a t 200x m a g n i f i c a t i o n . B . T i s s u e f o l l o w i n g t i s s u e c u t w i t h a U V l a s e r . A r r o w s i n d i c a t e v e s s e l s t h a t h a v e b e e n c u t a t 200x m a g n i f i c a t i o n . C T i s s u e f o l l o w i n g s e l e c t i v e c o l l e c t i o n o f s a m p l e s o n t o ac o l l e c t i o n c a p u s i n g a n i n f r a r e d l a s e r a t 200x m a g n i f i c a t i o n . D . I m a g e o f c o l l e c t i o n c a p s h o w i n g s p e c i f i c r e m o v a b l o o d v e s s e l s a t 200x m a g n i f i c a t i o n . A  •  86  Table 4.3 Summary of clonality results from histologically-defined nodular and diffuse neurofibromas and the proportion of each cell type within the neurofibromas.  D D D N N N  1 2 3 1 2 3  XCI Ratios  Staining Pattern (%)  T u m o u r S100+/Nf+ 38 38 57 25 21 20  S100+/Nf30 25 14 13 22 18  D = Diffuse neurofibroma N= Nodular neurofibroma + = positive staining -=n e g a t i v e s t a i n i n g Nf = neurofibromin  S100-/Nf+ 30 24 19 30 19 28  S100-/Nf3 14 10 32 38 34  Whole Tumour 54:44 45:55 66:34 56:44 46:54 55:45  Blood ssels 8:32 0:20 4:56 91:9 95:5 87:13  Ve 6 8 4  S100+ 62: 43: 53: 77: 54: 41:  / 3 5 4 2 4 5  Nf+ 8 7 7 3 6 9  S100+/Nf90:10 21:79 44:56 7:93 23:77 46:54  S100-/Nf+ 55:45 42:58 51:49 96:4 58:42 84:16  Tumour D1 is a diffuse neurofibroma with 30% S100+/Nf- cells.Normal blood vessels from this patient showed an XCI ratioclose to the expected 50:50 (56:44). In contrast, t h e S 1 0 0 + / N f - c e l l s d e m o n s t r a t e d c l o n a l i t yw i t h a n X C I r a t i oo f 9 0 : 1 0 ( T a b l e 4 . 3 ) . A s expected, the XCI ratiosof S100+/Nf+ and S100-/Nf+ cellswere similarto that of the blood vessels. The proportion of S100-/Nf- cells isthistumour was very low (3%). These findings are consistent with this neurofibroma arising through clonalproliferation o f a S c h w a n n c e l lp r e c u r s o r t h a t s u s t a i n e d a " s e c o n d h i t "N F 1 m u t a t i o n t h a t a b r o g a t e d neurofibromin expression. D 2 w a s t h e s e c o n d d i f f u s en e u r o f i b r o m a s t u d i e d .T h i s n e u r o f i b r o m a h a d 2 5 % S100+/Nf- cells.The XCI ratioof this cellpopulation was opposite (21:79) that of the blood vessels (80:20). S100+/Nf+ and S100-/Nf+ cellsboth had XCI ratiosclose to the 5 0 : 5 0 e x p e c t e d f o r r a n d o m X C I , a l t h o u g h t h i s w a s s o m e w h a t d i f f e r e n tf r o m t h e r a t i o found in the blood vessels (Table 4.3). Although the XCI ratio in the S100+/Nf- cells did not meet our definition of clonality (90:10), itseems likelythat this neurofibroma also arose through clonal proliferation of a Schwann cell precursor that sustained a "second h i t "N F 1m u t a t i o n . T h e t h i r dd i f f u s e n e u r o f i b r o m a a n did not show any evidence of clo vessels (44:56) (Table 4.3). This cells,which also had an XCI rati n e u r o f i b r o m aa r e c o n s i s t e n tw i haploinsufficient cells. The three nodular neur tumours removed from neurofibromas showed 4.3). Allthree of these 38%).  alysed (D3) had nality.The XCI tumour showed oclose to that of t ht h e t u m o u r a r  1 4 % S 1 0 0 + / N f - c e l l s ,a n d t h e s e c e l l s r a t i ow a s t h e s a m e a s i n t h e b l o o d a high proportion (57%) of S100+/Nf+ the blood vessels. The findings in this isingas a polyclonalproliferationof NF1  o f i b r o m a s ( N 1 , N 2 a n d N 3 ) e x a m i n e d f o r c l o n a l i t yw e r e t h e s a m e p a t i e n t .T h e n o r m a l b l o o d v e s s e l s i n a l lt h r e e s i m i l a rX C I s k e w i n g ( 9 1 : 9 , 9 5 : 5 a n d 8 7 : 1 3 , r e s p e c t i v e l tumours also showed high proportions of S100-/Nf- cell  Only 13% of the cells in N1 stained positivelyfor S100 but negatively The XCI ratiofor this celltype was highly skewed (7:93) to the opposi the blood vessels. S100+/Nf+ cellsand S100-/Nf+ cells both had XCI that of the normal blood vessels. The findings in this neurofibroma ar 88  all spinal of these y) (Table s (32-  for neurofibromin. te allelethan in ratiossimilar to e consistent with  clonalproliferationof a Schwann mutation.  c e l lp r e c u r s o r t h a t s u s t a i n e d a " s e c o n d h i t " N F 1  Nodular neurofibroma N2 had 22% S100+/Nf- cells.This celltype was not clonal (23:77) but the XCI ratiodifferedgreatly from that of the blood vessels, which was highly skewed in the opposite direction(95:5) (Table 4.3).The S100+/Nf+ cellsand S100-/Nf+ cells in this sample both had XCI ratiosclose to that expected for random X-inactivation a n d t h u s d i f f e r e n tf r o m t h e s k e w e d X C I r a t i oo b s e r v e d i n t h e b l o o d v e s s e l s . T h e i n t e r p r e t a t i o no f t h i s c a s e i s l e s s c l e a r , b u t t h e n e u r o f i b r o m a m a y a l s o h a v e a r i s e n through clonal proliferation of a Schwann cell precursor that sustained a "second hit" N F 1m u t a t i o n . The thirdnodu XCI ratioof th d i f f e r e n tf r o m in this tumour the S100+/Nf+ blood vessels.  lar neurofibroma tested (N3) had 20% of cellsthat w ese cells is compatible with random X-inactivation (4 that of the normal blood vessels (87:13) (Table 4.3). had an XCI ratiosimilarto that of the blood vessels, cells resembled that of the S100+/Nf- cells and not The interpretationof these data with respect to clon  ere S100+/Nf-. The 6:54) but somewhat The S100-/Nf+ cells but the XCI ratio of that of the normal alityis unclear.  DISCUSSION  NF1 neurofibromas are thought to develop according to Knudson's "two-hit"hypothesis, as a resultof inactivationof both allelesof the NF1 locus (33-35). People with neurofibromatosis1 have a constitutionalmutation of NF1 in every cellof theirbodies, and somatic mutation of the second (normal) allele in a Schwann cell is assumed to be t h e r a t e - l i m i t i n gs t e p i n n e u r o f i b r o m a f o r m a t i o n . A l t h o u g h t h e " t w o - h i t " m e c h a n i s m is widely accepted as the basis of neurofibroma formation in NF1, the development of all neurofibromas as a result of "second hit"mutations does not readily explain allof the available data.  We have suggested that some neurofibromas arisethrough inactivationof the second ( n o r m a l ) N F 1 a l l e l e i n a S c h w a n n c e l la n d s u b s e q u e n t c l o n a le x p a n s i o n t o f o r m a n e o p l a s m , b u to t h e rn e u r o f i b r o m a sa r i s ea s p o l y c l o n a l p r o l i f e r a t i o n so f N F 1 h a p l o i n s u f f i c i e n t c e l l s .T h e d e v e l o p m e n t o f s o m e n e u r o f i b r o m a s a s p o l y c l o n a l p r o l i f e r a t i o n so f N F 1 h a p l o i n s u f f i c i e n tc e l l sc o u l de x p l a i nt h ee x c e p t i o n a lo b s e r l i s t e d a b o v e a n d o t h e r s t h a t a r e n o t e a s i l y r e c o n c i l e d w i t h t h e " t w o - h i t "m o d e l o f t u m o u r development. There is increasing evidence that haploinsufficiency of other tumour 89  s u p p r e s s o rg e n e s s u c h a sT tumours without a "second hit" (36-39).  S  C  1  , TSC2,  a n d  P  5  7  c a na l s ol e a dt  The purpose of this study was to quantify the percentage of cells with immunohistological features of normal Schwann cells (S100+/Nf+ cells), neoplastic S c h w a n n c e l l st h a th a v e s u s t a i n e d a " s e c o n d h i t "N F 1 m u t a t i o n ( S 1 0 0 + / N f -c e l l s ) , a n d normal non-Schwann c e l l s ( S 1 0 0 - / N f + c e l l s ) a n d t o d e t e r m i n e i ft h e p r o p o r t i o n s o f t h e s e c e l lt y p e s d i f f e ri n h i s t o l o g i c a l l y - d e f i n e d n o d u l a r a n d d i f f u s e n e u r o f i b r o m a s . W e a l s o w a n t e d t o d e t e r m i n e i fS c h w a n n c e l l s t h a t n o l o n g e r e x p r e s s n e u r o f i b r o m i n w e r e c l o n a l , as would be expected fora neoplasm thatarose as the resultof a "second hit" NF1 m u t a t i o n . W e f o u n d a s i g n i f i c a n td i f f e r e n c e b e t w e e n t h e m e a n p e r c e n t a g e s o f S100+/Nf+ cellsand of S100-/Nf- cells in nodular and diffuse neurofibromas. The f i n d i n g s o f o u r c l o n a l i t ys t u d i e s s u p p o r t t h e i n t e r p r e t a t i o nt h a t s o m e n e u r o f i b r o m a s a r e c l o n a l S c h w a n n c e l l n e o p l a s m s w h i l e o t h e r n e u r o f i b r o m a s a r e p o l y c l o n a lp r o l i f e r a t i o n s o fN F 1 h a p l o i n s u f f i c i e n t c e l l s . H a p l o i n s u f f i c i e n c y a n d l o s so f N F 1 a r e t h o u g h t t o l e a dt o t u m o u rf o r m a t i o n b e c a u s e n e u r o f i b r o m i n , t h e p r o t e i n p r o d u c to f t h e N F 1 g e n e , f u n c t i o n s t o d o w n - r e g u l a t e R a s , a key cellcycle control protein. Neurofibromin enhances the conversion of active RasG T P t o i n a c t i v eR a s - G D P . R a s m o d u l a t e s s e v e r a l i m p o r t a n t s i g n a l t r a n s d u c t i o n pathways, one of which isthe MAP-kinase pathway. Inactivatingmutations in one or b o t hc o p i e so f N F 1 l e a dt o h i g h e rR a s c o n c e n t r a t i o n sa n d a n i n c r e a s e i n c e l l u l a r proliferation (40-42). T h e r e i s s u b s t a n t i a l e v i d e n c et h a tN F 1 f u n c t i o n s a s a t u m o u r s u p p r e s s o rg e n e , a n d L O H o r s e c o n ds o m a t i cm u t a t i o n sf o rN F 1 h a v eb e e n o b s e r v e d i ns o m en e u r o f i b r o m a in accordance with the "two-"hit"hypothesis. Overall, LOH or second somatic mutations have been observed in 26% of dermal neurofibromas and 36% of plexiform n e u r o f i b r o m a s s t u d i e d t o d a t e ( 8 - 2 4 ) .T h e l o w p e r c e n t a g e o f L O H o r s e c o n d s o m a t i c m u t a t i o n s o b s e r v e d i n N F 1 n e u r o f i b r o m a s h a s b e e n a t t r i b u t e dt o d e t e c t i o n m e t h o d s t h a t c a n n o ti d e n t i f ya l lm u t a t i o n s ,m u t a t i o n so u t s i d eo ft h e r e g i o no f t h e N F 1 g e n e t h a t w a s t e s t e d ,o rt h ef r e q u e n ti n a c t i v a t i o no ft h es e c o n d ( n o r m a l )N F 1 a l l e l eb y a ne p i g e n e t i c m e c h a n i s m .H o w e v e r ,n o e v i d e n c eo fe p i g e n e t i ci n a c t i v a t i o no ft h eN F 1 g e n e h a s b e observed (14, 43, 44).  90  To better understand NF1 neurofibroma development, Zhu and coworkers (45) used a c r e / l o xs y s t e mt o d e l e t e N f 1 i nS c h w a n n c e l l sw h i l em a i n t a i n i n g N f 1 h e t e r o z y g o s other celltypes in mice. These animals showed thickened peripheral nerves and developed both cranialand spinal nerve neurofibromas. Interestingly,these features w e r e o n l y o b s e r v e d w h e n o t h e r c e l l s w e r e h e t e r o z y g o u s f o rN f 1 ,p o i n t i n g t o t h e importance of the A/77 haploinsufficient background in neurofibroma development in m i c e . S i m i l a r i l y , e m b r y o n i c d a y 1 2 . 5 Nf1~'~ S c h w a n n c e l l s i m p l a n t e d i n t o t h n e r v e o f Nf1 ~ m i c e d e v e l o p i n t o t u m o u r s t h a t r e s e m b l e p l e x i f o r m n e u r o f i b r H u m a n NF1~'~ S c h w a n n c e l l s d e r i v e d f r o m n e u r o f i b r o m a s o f N F 1 p a t i e n t s e x h i b i t growth advantage in culture and produce tumours that resemble plexiform neurofibromas when transplanted intothe peripheralnerves of scid mice (25). The tumours produced in these experimental systems are models of nodular neurofibromas that develop in people with NF1, but the relevance of these models to other types of neurofibromas that occur as part of NF1 is unknown. Itis interesting to note that multipl cutaneous neurofibromas, the feature that gives NF1 itsname, and congenital diffuse neurofibromas do not develop in any of the NF1 mouse models described to date (4750). Moreover, there are important differencesbetween human nodular neurofibromas, which are presumed to arise as clonal outgrowths from a single mutant precursor that has sustaineda "second hit"atthe NF1 locus,and these mouse models,in which tumours develop from a large number of neurofibromin-deficient Schwann cells. +I  Muir et al (25) also stained benign neurofibromas from people with NF1 for neurofibromin and S100. They found four basic patterns of neurofibromin staining in S100-positive regions: neurofibromin-negative, predominantly neurofibromin-negative with focal areas of distinctpositive staining, predominantly neurofibromin-positive with f o c a l a r e a s o f d i s t i n c t n e g a t i v e s t a i n i n g ,a n d n e u r o f i b r o m i n - p o s i t i v e . W e a l s o o b s e r v e d t w od i f f e r e n tp o p u l a t i o n so fS c h w a n n c e l l s- t h o s et h a te x p r e s s e d n e u r o f i b t h o s et h a td i dn o t- b u tw e r a r e l ys a w l a r g e a g g r e g a t e s o f S 1 0 0 - p o s i t i v e c e l l st h a td i n o t e x p r e s s n e u r o f i b r o m i n .O u r t u m o u r s a l m o s t a l w a y s s h o w e d m i n g l i n g o f n e u r o f i b r o m i n - n e g a t i v e a n d n e u r o f i b r o m i n - p o s i t i v e S c h w a n n c e l l s .O u r f i n d i n g s a r e similar to those obtained by molecular analysis of a dermal neurofibroma in which the " s e c o n d h i t " s o m a t i c m u t a t i o n o f t h e N F 1 l o c u sw a s i d e n t i f i e d ( 1 0 ) . I n t h i s t u m o u r ,b o t h t h e m u t a n ta n d n o n - m u t a n tv e r s i o no f t h e s e c o n d N F 1 a l l e l ew e r e f o u n d i n a homogenous-appearing area of tumour cells. 91  S100-positive (presumably Schwann) cellculturesgrown from neurofibromas removed f r o m N F 1 p a t i e n t s c o n t a i n b o t h n e u r o f i b r o m i n - p o s i t i v ea n d n e u r o f i b r o m i n - n e g a t i v e c e l l populations (6, 25, 26). Itis not known how the heterogeneity of neurofibromin expression seen in these cultures relatesto the tumours from which the cultures were grown. In the present study, we found that S100+/Nf- cells (i.e.,cells with the phenotype e x p e c t e d f o r S c h w a n n c e l l s t h a t h a v e s u s t a i n e d a n i n a c t i v a t i n gm u t a t i o n o f t h e i r n o r m a l N F 1 a l l e l e ) m a d e u p o n l y1 8 % o f t h e n e u r o f i b r o m a s s t u d i e d , a n d t h e p r o p o r t i o n w a s similar in diffuse and nodular tumours. However, the proportion of S100+/Nf+ cells (the p h e n o t y p e e x p e c t e d f o r n o r m a l S c h w a n n c e l l s )w a s s i g n i f i c a n t l yh i g h e r ( 4 2 % ) i n d i f f u s e neurofibromas than in nodular neurofibromas (24%). The percentage of S100-positive (presumably Schwann) cellsthat expressed neurofibromin averaged 61% (range, 25100%) in nodular neurofibromas and 74% (range, 39-100%) in diffuse neurofibromas. These values are consistent with two other studies that estimated the proportion of S c h w a n n c e l l sw i t ha n i n a c t i v eN F 1 g e n e , i . e . ,t h e r e c i p r o c a lf r a c t i o n , a s 1 6 - 6 2 % i n c u l t u r e sd e r i v e d f r o m N F 1 n e u r o f i b r o m a s ( 8 ) a n d a s 3 0 - 6 0 % a s e s t i m a t e d f r o m L O H o n sequence analysis of a whole tumour (19). One possible explanation for the difference observed in the proportion of S100+/Nf+ cells between diffuse and nodular neurofibromas is that the diffuse neurofibromas were m u c h m o r e l i k e l yt o h a v e b e e n o b t a i n e df r o mi n d i v i d u a l sw i t h c o n s t i t u t i o n a lw h o l e g e n e d e l e t i o n s w h i l e t h e n o d u l a r n e u r o f i b r o m a s w e r e m o r e l i k e l yt o h a v e b e e n o b t a i n e d from individuals with constitutional intragenic mutations. The "second hits" in n e u r o f i b r o m a s f r o m i n d i v i d u a l sw i t h c o n s t i t u t i o n a lw h o l e g e n e d e l e t i o n s a r e u s u a l l y subtle mutations that might not completely abrogate neurofibromin expression (51), while "second hits"in neurofibromas from individuals with other constitutional mutations a r e m o r e l i k e l yt o c a u s e L O H a n d t h u s l a c k n e u r o f i b r o m i n e x p r e s s i o n ( 1 9 , 2 3 ) . T h i s explanation is unlikely to account for the difference in frequency of S100+/Nf+ cells that w e o b s e r v e d b e t w e e n n o d u l a r a n d d i f f u s en e u r o f i b r o m a s b e c a u s e w h o l e g e n e d e l e t i o n s account for only about 5% of constitutionalmutations in people with NF1 (52-54). We found that about 31% of in diffuse neurofibromas did o u t t h e p o s s i b i l i t yt h a t m a n y positive cells that did not sta  the cellsin nodular neurofibromas and about 15% of cells not express eitherS100 or neurofibromin. We cannot rule of the S100-/Nf- cellsmay actuallybe neurofibrominin for neurofibromin. However, the sensitivityof the 92  antibody should be equal in both diffuseand nodular neurofibromas, and an average of 92% of cells in traumatic neuroma, which were used as a positive control in our study, were neurofibromin-positive. We do not know what kind of cellsthe S100-/Nf- population represents, and Muir et al. (25) did not comment on this cell population in their study. One intriguingpossibility is that the S100-/Nf- cells are dedifferentiated Schwann cells or, alternatively, immature Schwann stem cells that are part of a neoplastic clone that has suffered a "second hit" N F 1 m u t a t i o n . T h i s i s c o n s i s t e n t w i t hs t u d i e s s h o w i n gt h a t i n c r e a s e d s i g n a l i n g t h r o u g the Ras/Raf/Erk pathway can drive Schwann cells to a dedifferentiated state (55). It is not clear why such cellsshould be more common in nodular than diffuse neurofibromas when S100+/Nf- cells are observed in a similar proportion in both kinds of tumour. cAMP has been shown to induce ^differentiationof Schwann cells(55) and could be present in higher levels in diffuse than nodular neurofibromas. S100-/Nf- cells are often observed in MPNSTs (56), and the higher proportion S100-/Nf- cells in nodular neurofibromas raises concern about the riskof this tumour type progressing to malignancy. Itis important to define the nature of the S100-/Nf- cells and to determine if they are more tumorigenic than other celltypes within neurofibromas. The correlationwe observed between lymphocytic infiltrationand the proportio S100-/Nf- cells might indicate that this celltype is triggering an immune response, is also possible that the prevalence of both lymphocytes and S100-/Nf- cells both some other underlying feature.In any case, we may not have completely defined r e l a t i o n s h i pb e t w e e n t h e s e t w o c e l lt y p e s b e c a u s e o u r s t u d y w a s r e s t r i c t e d t o neurofibromas in which lymphocytes constituted less than 30% of the totalcell population.  n of but it reflect the  R i c c a r d i ( 3 0 ) h a s d i s t i n g u i s h e d f o u rt y p e s o f n e u r o f i b r o m a s- c u t a n e o u s , n o d u l a rp l e x i f o r ma n d d i f f u s ep l e x i f o r m- o n t h e b a s i so f t h e i rc l i n i c a lf e a t u r e s .A m a benefit of Riccardi's classification is that tumours with different natural histories are placed into different groups, and neurofibromas with a high potential for malignant degeneration (nodular plexiform and diffuseplexiform tumours) are distinguished from those with a much lower riskof malignancy (cutaneous and subcutaneous tumours). T h e t u m o u r s i n o u r s t u d y w e r e n o t a l l c l a s s i f i e d b y t h i s c l i n i c a lm e t h o d , a n d t h e h i s t o l o g i c a la n d c l i n i c a l a p p r o a c h e s d o n o t p l a c e a l l t u m o u r s i n t o t h e s a m e g r o u p s . B o t h 93  cutaneou histolog neurofib plexifor However, examine  s and diffuseplexiform tumours usually are diffuse neurofibroma ically,but some diffuseplexiform neurofibromas are mixed diffus r o m a s . T u m o u r s t h a t a r e c l a s s i f i e dc l i n i c a l l ya s s u b c u t a n e o u s o r m neurofibromas are usually nodular neurofibromas on histopathol the only way to determine the histological type of a neurofibroma it histologically.  s eand nodular nodular ogical exam. is to  T h r e e p r e v i o u s s t u d i e s h a v e a s s e s s e d c l o n a l i t yi n N F 1 n e u r o f i b r o m a s . A l l o f t h e s e studies tested the XCI ratioof whole tumours, which are made up of several different cell populations, rather than looking at each particular cell type separately. In total, 35 neurofibromas, 30 dermal and 5 plexiform neurofibromas, were previously assessed (8, 11, 57). One study did not find clonalityin any of the 14 neurofibromas tested (57). In another study, eight dermal neurofibromas were found to have a monoclonal origin, a l t h o u g h n o n e o f t h e s et u m o u r s d i s p l a y e d L O H f o rN F 1 ( 8 ) . T h e l a s t s t u d y f o u n d clonality in 3 dermal neurofibromas and 1 plexiform neurofibroma (11). The whole tumour XCI ratios in the current study are consistent with a polyclonal phenotype. U s i n g l a s e r m i c r o d i s s e c t i o n , w e a s s e s s e d t h e X C I r a t i o i n t h r e e d i f f e r e n tc e l l u l a r populations defined by S100 and neurofibromin staining,as well as in blood vessels (as n o r m a l t i s s u e c o n t r o l s ) ,f r o m s i x N F 1 - a s s o c i a t e d n e u r o f i b r o m a s . O u r f i n d i n g s a r e compatible with a heterogenous origin of neurofibromas, with some tumours developing through clonal proliferationof S100+/Nf- cells,and other neurofibromas developing through a polyclonal process. There was evidence for both mechanisms in both histologically-defineddiffuse and histologically-definednodular neurofibromas. Clear e v i d e n c e f o r c l o n a l i t yw a s s e e n i n o n e n o d u l a r ( N 1 ) a n d o n e d i f f u s e ( D 1 ) n e u r o f i b r o m a . N1 was defined clinicallyas a nodular plexiform neurofibroma, and D1 was defined clinically as a cutaneous neurofibroma. In both of these cases, itis likely that the S100+/Nf- cells represent a neoplastic clone of Schwann cells that sustained a "secondh i t " m u t a t i o no f t h e N F 1 l o c u s .I ti s i n t e r e s t i n g t h a tt h i sp o p u l a t i o n m a d e u p o n l y 3 0 % o f the cells in diffuse neurofibroma D1 and only 13% in the cells in nodular neurofibroma N1. Cells negative for both S100 and neurofibromin made up the greatest proportion of cells in neurofibroma N1. Unfortunately, we could not isolatethese cells by m i c r o d i s s e c t i o n w i t h o u t a s p e c i f i c m a r k e r , a n d w e d o n o t k n o w i ft h e y w e r e c l o n a l o r  94  not. Itis interesting to speculate that they might be dedifferentiated (or just primitive)Schwann cell precursors that are part of the neoplastic clone.  more  Two other neurofibromas (N2 and D2), which were classified clinicallyas a nodular plexiform neurofibroma and a cutaneous neurofibroma, respectively, probably also arose through clonal proliferation of a Schwann cell (or Schwann cell precursor) that s u s t a i n e da " s e c o n d h i t " i n a c t i v a t i n g m u t a t i o na t t h e N F 1 l o c u s . T h e X C I r a t i o i n S 1 0 0 + / N f - c e l l s i n t h e s e t w o t u m o u r s d i d n o t m e e t o u r f o r m a l d e f i n i t i o no f s k e w i n g ( 9 0 : 1 0o r g r e a t e r ) , b u t b o t h s h o w e d a p r e d o m i n a n c e o f t h e N F 1 a l l e l et h a tw a s l e s s frequent among cellsin the normal blood vessels. The most parsimonious explanation isthat these tumours were clonal but the S100+/Nf- cellstested were "contaminated" by non-clonal tissue of some type. There was no evidence for clonalityamong the S100+/Nf- cellsin one diffuse n e u r o f i b r o m a ( D 3 ) . T h i s t u m o u r w a s c l a s s i f i e d c l i n i c a l l ya s a d i f f u s e p l e x i f o r m neurofibroma. All celltypes within this tumour had an XCI ratiosimilarto the blood vessels. S100+/Nf- cells made up only 14% of the totalin thistumour, and S100-/Nfc e l l so n l y 1 0 % . W e c a n n o t r u l e o u t t h e p o s s i b i l i t yt h a t t h e d o u b l e n e g a t i v e p o p u l a t i o n was clonal in this tumour, but ifitwas itrepresents an unusually small proportion of the total cell population, and itdoes not seem to have contributed substantially to the S100positive population. Interpretationof the clonalitydata in neurofibroma N3, which was classifiedclinically as a nodular plexiform neurofibroma, is less clear. A polyclonal origin may be most likely. A largerfraction(34%) of the cells in thistumour were negative for both S100 and n e u r o f i b r o m i n , a n d t h e p o s s i b i l i t yt h a t t h i s p o p u l a t i o n r e p r e s e n t s d e d i f f e r e n t i a t e d o r more primitive Schwann cell precursors that have sustained a "second-hit" inactivating mutation of the NF1 locus cannot be ruled out. There are several possible explanations for the skewing observed in blood vessels from t h e i n d i v i d u a lf r o m w h o m a l l t h r e e n o d u l a r n e u r o f i b r o m a s e x a m i n e d f o r c l o n a l i t y w e r e obtained. The firstpossibility is that the individual herself has skewed X inactivation throughout her body. 10% of women have a greater than 90% expression of one X chromosome by chance in blood (58). A second possibilityis that the mesodermal stem cellpool from which the blood vessels arose was small and that skewed X chromosome inactivation occurred in derivatives of this pool by chance as a result of sampling. A third , 9 5  possibility is that the tumour arose focally within a small mixed population of cells and the cells that produced neovasculature of the tumour allarose from a tiny initial pool within the focus of involvement. Growth of the tumour vasculature from a small population of cells could result in skewed XCI which, by chance, happened to be in the same direction in allthree tumours. Without testing other tissues from this individual, we c a n n o t d i s t i n g u i s h w h i c h o f t h e s e p o s s i b i l i t i e si s c o r r e c t . Another m a y b e normal extreme general  possibilityis that the skewing observed in norm d u e t o N F 1 h a p l o i n s u f f i c i e n c y .T h e r e f o r e tissues of females with NF1 should be explored (>90%) XCI skewing is greater than the propor population.  al tissues such as blood vessels , a s t u d ya s s e s s i n gX C I r a t i o si n t o s e e i ft h e p r o p o r t i o n o f tion (10%) observed in the  In summary, we found that a high proportion of S100-positive (presumably Schwann) cellsin NF1-associated neurofibromas express neurofibromin. Although some neurofibromas appear to develop as clonal neoplasms as a result of "second hit" m u t a t i o n so f t h e n o r m a l N F 1 a l l e l e i n a S c h w a n n c e l l , t h e o r i g i n o f s o m e o t h e r n e u r o f i b r o m a s a p p e a r s t o b e d i f f e r e n t .W e f a v o u r t h e p o s s i b i l i t yt h a t s o m e n e u r o f i b r o m a s a r i s ea s p o l y c l o n a l p r o l i f e r a t i o n s o f N F 1 h a l p l o i n s u f f i c i e n t c e l l s , b u t i ti a l s o p o s s i b l e s o m e n e u r o f i b r o m a s a r i s e f r o m m o r e p r i m i t i v e o r d e - d i f f e r e n t i a t e dc e l l s o f t h e S c h w a n n c e l l l i n e a g e , r a t h e r t h a n f r o m m a t u r e S c h w a n n c e l l s .T h e s e o b s e r v a t i o n s need to be confirmed in a larger sample set to ensure they are not unique to the few t u m o u r s e x a m i n e d . F u r t h e r s t u d i e s a r e n e e d e d t o c l a r i f yt h e n a t u r e o f t h e S 1 0 0 - / N f cells that often make up a substantial fraction of the total cell population within n e u r o f i b r o m a s a n d t o d e t e r m i n e t h e r e l a t i o n s h i po f t h e s e a n d o t h e r c e l l u l a r c o m p o n e n t s to the development of MPNST in people with NF1.  96  REFERENCES  1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.  Friedman JM. Epidemiology of neurofibromatosis type 1. Am J Med Genet 1999;89:1-6. Xu W, Mulligan LM, Ponder MA, et al.Loss of NF1 alleles in phaeochromocytomas from patients with type I neurofibromatosis. Genes Chromosomes Cancer 1992;4:337-42. Side L, Taylor B, Cayouette M, et al. Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders. N Engl J Med 1997;336:1713-20. Legius E, Marchuk DA, Collins FS, Glover TW. 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Nat Genet 1995;11:90-2. S a w a d a S , F l o r e l l S , P u r a n d a r e S M , e t a l . I d e n t i f i c a t i o no f N F 1 m u t a t i o n s i n b o t h alleles of a dermal neurofibroma. Nat Genet 1996;14:110-2. Daschner K, Assum G, Eisenbarth I,et al. Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron38 and in dermal neurofibromas w i t h o u tL O H o f t h e N F 1 g e n e . B i o c h e m B i o p h y s R e s C o m m u n 1 9 9 7 ; 2 3 4 : 3 4 6 - 5 0 . K l u w e L , F r i e d r i c h R E , M a u t n e r V F . A l l e l i cl o s s o f t h e N F 1 g e n e i n N F 1 associated plexiformneurofibromas. Cancer Genet Cytogenet 1999;113:65-9. John AM, Ruggieri M, Ferner R, Upadhyaya M. A search for evidence of somatic mutations in the NF1 gene. J Med Genet 2000;37:44-9. L u i j t e n M , R e d e k e r S , v a n N o e s e l M M , e t a l . 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Pathogenesis of hereditary tumors: beyond the "two-hit" hypothesis, Clin Genet 2002;62:345-57. National Institutesof Health Consensus Development Conference Statement: neurofibromatosis. Bethesda, Md., USA, July 13-15, 1987. Neurofibromatosis 1988;1:172-8. G o t t f r i e dO N , V i s k o c h i l D H , F u l t s D W , C o u l d w e l l W T . M o l e c u l a r , g e n e t i c , a n d c e l l u l a rp a t h o g e n e s i s o f n e u r o f i b r o m a s a n d s u r g i c a l i m p l i c a t i o n s . N e u r o s u r g e r y 2006;58:1-16. Reed N, Gutmann DH. Tumorigenesis in neurofibromatosis:new insights and p o t e n t i a lt h e r a p i e s .T r e n d s M o l M e d 2 0 0 1 ; 7 : 1 5 7 - 6 2 . Arun D, Gutmann DH. Recent advances in neurofibromatosis type 1. Curr Opin Neurol 2004;17:101-5. Henske EP, Scheithauer BW, Short MP, et al.Allelicloss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am J Hum Genet 1996;59:400-6. Niida Y, Stemmer-Rachamimov AO, Logrip M, et al.Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic m e c h a n i s m s f o r p a t h o g e n e s i s o f T S C l e s i o n s .A m J H u m G e n e t 2 0 0 1 ; 6 9 : 4 9 3 503. Ma L, Teruya-Feldstein J, Behrendt N, et al.Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in t u m o r s u p p r e s s i o n .G e n e s D e v 2 0 0 5 ; 1 9 : 1 7 7 9 - 8 6 . Trkova M, Foretova L, Kodet R, Hedvicakova P, Sedlacek Z. A Li-Fraumeni syndrome family with retained heterozygosity for a germline TP53 mutation in two tumors. Cancer Genet Cytogenet 2003;145:60-4. Bernards A. Neurofibromatosis type 1 and Ras-mediated signaling: fillingin the GAPs. Biochimica Biophysica Acta 1995;1242:43-59. Feldkamp MM, Angelov L, Guha A. Neurofibromatosis type 1 peripheral nerve tumors: aberrant activationof the Ras pathway. Surg Neurol 1999;51:211-8. HarrisinghMC, Lloyd AC. Ras/Raf/ERK signallingand NF1. Cell Cycle 2004;3:1255-8. Horan MP, Cooper DN, Upadhyaya M. Hypermethylation of the neurofibromatosis type 1 (NF1) gene promoter is not a common event in the inactivation of the NF1 gene in NF1-specifictumours. Hum Genet 2000;107:33-9. Harder A, Rosche M, Reuss DE, et al.Methylation analysis of the neurofibromatosis type 1 (NF1) promoter in peripheral nerve sheath tumours. Eur J Cancer 2004;40:2820-8. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann celloriginand role of tumor environment. Science 2002;296:920-2. 99  46. 47. 48. 49. 50. 51. 52. 53.  54. 55. 56. 57. 58.  W u M , W a l l a c e M R , M u i r D . T u m o r i g e n i c p r o p e r t i e so fn e u r o f i b r o m i n - d e f i c i e n t Schwann cells in culture and as syngrafts in Nf1 knockout mice. J Neurosci Res 2005;82:357-67. Jacks T, Shih TS, Schmitt EM, et al.Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet 1994;7:353-61. McClatchey Al, Cichowski K. Mouse models of neurofibromatosis. Biochimica et Biophysica Acta 2001;1471:M73-m80. Gutmann DH, Giovannini M. Mouse models of neurofibromatosis 1 and 2. Neoplasia 2002;4:279-90. Cichowski K ST, Schmitt E, Santiago S, Reilly K, McLaughlin ME, Bronson RT, Jacks T. Mouse models of tumor development in neurofibromatosis type 1. Science 1999;286:2172-6. Skuse GR, Cappione AJ. RNA processing and clinicalvariabilityin neurofibromatosis type I (NF1). Hum Mol Genet 1997;6:1707-12. Tonsgard JH, YelavarthiKK, Cushner S, Short MP, Lindgren V. Do NF1 gene deletions result in a characteristic phenotype? Am J Med Genet 1997;73:80-6. Upadhyaya M, Ruggieri M, Maynard J, et al.Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and c o m m o n l y a s s o c i a t e d w i t h a l e a r n i n g d i s a b i l i t y ,d y s m o r p h i c f e a t u r e s a n d developmental delay.Hum Genet 1998;102:591-7. Leppig KA, Kaplan P, Viskochil D, et al. Familial neurofibromatosis 1 m i c r o d e l e t i o n s : c o s e g r e g a t i o n w i t h d i s t i n c tf a c i a l p h e n o t y p e a n d e a r l y o n s e t o f cutaneous neurofibromata. Am J Med Genet 1997;73:197-204. Harrisingh MC, Perez-Nadales E, Parkinson DB, et al.The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation. Embo J 2004;23:306171. Frahm S, MautnerVF, Brems H, et al.Genetic and phenotypic characterization of tumor cellsderived from malignant peripheral nerve sheath tumors of neurofibromatosis type 1 patients. Neurobiol Dis 2004;16:85-91. Fialkow PJ, Sagebiel RW, Gartler SM, Rimoin DL. Multiple celloriginof hereditary neurofibromas. N Engl J Med 1971;284:298-300. Busque L, Mio R, MattioliJ, et al. Nonrandom X-inactivation patterns in normal females: lyonization ratiosvary with age. Blood 1996;88:59-65.  100  5. G E N E R A L D I S C U S S I O N  Summary Neurofibromas are the hallmark feature of NF1. Almost allindividualswith NF1 develop neurofibromas in theirlifetime,and some individualsdevelop hundreds or thousands of t h e s e t u m o u r s . C u t a n e o u s n e u r o f i b r o m a s c a n h a v e ag r e a t e f f e c t o n q u a l i t y o f l i f e , a n d other kinds of neurofibromas may cause pain, deformity, functional impairment, or bleeding because of erosion into adjacent structures.Congenital diffuse and nodular p l e x i f o r m n e u r o f i b r o m a s m a y g i v e r i s e t o M P N S T s , w h i c h a r e am a j o r c a u s e o f premature death in people with NF1 (1). Mouse models of benign neurofibromas an intotumour development, but these models situation.Cultures derived from NF1 neuro but cannot be used to study some critical environment.  d M P N S T h a v e p r o v i d e d v a l u a b l ei n s i g h t s d o n o t f u l l yr e c a p i t u l a t et h e h u m a n fibromas also provide valuable information host factors or the role of the tumour  In order to understand tumour development in people with NF1 fully,itis necessary to s t u d y h o w n e u r o f i b r o m a s a r i s e in vivo a n d t o t a k e i n t o c o n s i d e r a t features and natural histories of each type of neurofibroma. I used histology and i m m u n o h i s t o c h e m i s t r y t o d e t e r m i n e t h e c e l l u l a rc o m p o s i t i o n a n d e x p r e s s i o n o f n e u r o f i b r o m i n i n d i f f u s e a n d n o d u l a r n e u r o f i b r o m a s a n d a s s e s s e d c l o n a l i t yt o d e f i n e t h e cell populations that give rise to these tumours. I also investigated associations of b e n i g n a n d m a l i g n a n t p e r i p h e r a l n e r v e s h e a t h t u m o u r s i n al a r g e s e r i e s o f N F 1 p a t i e n t s i n o r d e r t o i d e n t i f y ag r o u p o f p a t i e n t s w h o a r e a t h i g h e s t r i s k f o r d e v e l o p i n g MPNSTs. Risk of MPNST Development  The strong association I observed between the occurrence of internal neurofibromas a n d M P N S T s i n p e o p l e w i t h N F 1 ( C h a p t e r 2 ) s u g g e s t s t h a t s u c h i n d i v i d u a l sa r e a t higher riskof developing MPNSTs than NF1 patientswithout internal neurofibromas. The large size and older age of subjects in the clinicaldatabase I used in this study are u n i q u e i n N F 1 r e s e a r c h a n d a l l o w e d m e t o a s s e s s t h i s a s s o c i a t i o n f o r t h e f i r s tt i m e . M y investigation was largely cross-sectional, but ifthe association I found with the d e v e l o p m e n t o f m a l i g n a n c y c a n b e c o n f i r m e d i n l o n g i t u d i n a ls t u d i e s o f N F 1 p a t i e n t s 101  w s d d  ith ign iag eat  internalneurofi s and symptoms nosis and more h of about 10%  bromas, such individualsmay benefit from closer monitoring for of malignant progression.Closer monitoring may permitearlier e f f e c t i v et r e a t m e n t o f M P N S T s , w h i c h c u r r e n t l ya c c o u n t f o r t h e of people with NF1 (1).  The optimal approach to clinicalmonitoring of NF1 patients for MPNST development is unknown. Screening forsymptoms isprobably inadequate because most MPNSTs are m e t a s t a t i c a t t h e t i m e o f c l i n i c a ld i a g n o s i s ( 2 ) . R o u t i n e w h o l e - b o d y i m a g i n g s t u d i e s using CT or MRI would permit recognitionof most internalneurofibromas and MPNSTs but cannot reliablydistinguish benign and malignant peripheral nerve sheath tumours. PET scanning can distinguishbenign neurofibromas from MPNST in most cases (3), but PET and MRI scans are expensive,and PET or CT imaging would requireexposing NF1 patients to ionizing radiation,which itselfmay increase their risk of developing an MPNST (2, 4). A thorough cost-benefit analysis is needed to determine the clinical value of and optimal protocol for screening selected NF1 patients for malignancy. My observation that cutaneous neurofibromas were not associated with the development of MPNST differsfrom the findings in one study that found a weak association with cutaneous neurofibromas. However, the median age ofindividuals without MPNSTs was lower than those with MPNSTs in that study (5),and the patients with MPNSTs were of the approximate age when cutaneous neurofibromas begin to appear. Therefore, the majority of individualswithout MPNSTs would be expected to h a v e f e w i fa n y c u t a n e o u s n e u r o f i b r o m a s b a s e d o n a g e a l o n e . O u r o b s e r v a t i o n i s consistent with the clinicalobservation that cutaneous neurofibromas have not been reported to progress to malignancy, even though individualswith NF1 may have thousands of these benign tumours (6). The association Iobserved between internaland subcutaneous neurofibromas could point to a common mechanism of tumour development. In contrast, the lack of a s s o c i a t i o n b e t w e e n c u t a n e o u s n e u r o f i b r o m a s a n d e i t h e r i n t e r n a lo r s u b c u t a n e o u s neurofibromas suggests that more than one mechanism of neurofibroma pathogenesis may exist in people with NF1. The results from Chapters 3 and 4 are consistent with this interpretation.  102  Benign Neurofibroma Development  T h e c l a s s i f i c a t i o n o f b e n i g n n e u r o f i b r o m a s u s e d f o r C h a p t e r s 3a n d 4d i f f e r s f r o m t h a t used in Chapter 2. Chapter 2 classified externally-visible neurofibromas according to their clinical features, while Chapters 3a n d 4 u s e d ah i s t o p a t h l o g i c a l classification. Clinically-definedcutaneous and diffuseplexiform neurofibromas are generally classifiedas diffuse neurofibromas histopathologically,and clinically-defined s u b c u t a n e o u s a n d n o d u l a r p l e x i f o r m n e u r o f i b r o m a s a r e u s u a l l y c l a s s i f i e da s n o d u l a r neurofibromas histopathologically.However, exceptions do occur, and theclinical classification of a neurofibroma cannot always be determined from its histopathological appearance or the histopathological classificationfrom the clinical appearance. The internalneurofibromas diagnosed by MRI or CT scan in Chapter 2 were not classified clinically,but most are probably nodular plexiform neurofibromas in Riccardi's clinicalclassification (7) or nodular neurofibromas in the histopathological classification u s e d i n C h a p t e r s 3a n d 4 . T h u s , t h e l a c k o f a s s o c i a t i o n b e t w e e n i n t e r n a l ( p r e d o m i n a n t l y nodular) neurofibromas and cutaneous (generally diffuse) neurofibromas found in C h a p t e r 2 i s c o n s i s t e n t w i t h t h e h i s t o l o g i c a l f i n d i n g s p r e s e n t e d i n C h a p t e r s 3a n d 4 , which are suggestive of pathogenetic differences between nodular and diffuse neurofibromas. In Chapter 4, Ifound that S100-/Nf- cells were abundant in nodular neurofibromas but much less common in diffuse neurofibromas. Ido not know what cell population the S100-/Nf- cells represent. Itis unlikely that these cells are really S100-/Nf+ cells (normal stromal cells)that did not stain for neurofibromin because the sensitivityof the antineurofibromin antibody was 92% in the controland should be equal in nodular and diffuse neurofibromas. Two interesting possibilitiesare that the S100-/Nf- cells are 1. n e o p l a s t i c S c h w a n n c e l l s t h a t h a v e s u s t a i n e d a" s e c o n d - h i t " o f t h e N F 1 l o c u s a n d l o s t S 1 0 0 e x p r e s s i o n t h r o u g h d e d i f f e r e n t i a t i o no r 2 . n e o p l a s t i c p r i m i t i v e S c h w a n n c e l l p r e c u r s o r s ( s t e m c e l l s ) t h a t s u s t a i n e da" s e c o n d h i t " N F 1 m u t a t i o n . E i t h e r p o s s i b i l i t y c o u l d p r o v i d e ap o p u l a t i o n o f n e o p l a s t i c c e l l s t h a t d r i v e s t u m o u r f o r m a t i o n a n d l e a d s t o a high risk of malignant degeneration, accounting for the association observed in Chapter 2. We were not able to assess clonalityin the S100-/Nf- population because we d i d n o t h a v e ac e l l m a r k e r o r n u c l e a r s t a i n t o i d e n t i f y t h e s e c e l l s . T h e u s e o f n u c l e a r stains lowered the qualityof DNA in the samples that were already compromised from 103  the formalin-fixationprocess. However, the ab neurofibromas suggests that these cells have this tumour type. Itis important to assess the are the neoplastic clone that gave riseto the  undance of S100- /Nf- cells in nodular an important role in the development of clonality of these cells to determine if they tumour.  Ifound fewer mast cells in histologically-defined nodular than diffuse neurofibromas (Chapter 3).The low numbers of mast cellsobserved in nodular neurofibromas could s i m p l y r e f l e c ta n a t o m i c a l d i f f e r e n c e s t h a t e x i s t b e t w e e n n o d u l a r a n d d i f f u s e t u m o u r s . Diffuse tumours are in direct contact with adjacent tissues, while nodular tumours are s u r r o u n d e d b y t h e p e r i n e u r i u m , w h i c h m a y s e r v e a s a n a n a t o m i c a l b a r r i e rt o i n v a s i o n b y m a s t c e l l s . A l t e r n a t i v e l y ,t h e r e l a t i v e a b u n d a n c e o f S 1 0 0 - / N f - c e l l s a n d t h e r e l a t i v e paucity of S100+/Nf+ cells in nodular neurofibromas may provide less stimulus to mast cell invasion than in diffuse neurofibromas. Most of the histologically-defined nodular neurofibromas that I studied were classified clinicallyas nodular plexiform tumours. The histologicaland clinicalsimilaritiesbetween these neurofibromas and subcutaneous tumours suggest that they share a common pathogenesis, but more subcutaneous neurofibromas need to be studied to assess thispossibility. Although clinically-definedcutaneous and diffuseplexiform neurofibromas have similar histologicalcharacteristics, the pathogenesis of these two kinds of tumours is likely to d i f f e r b e c a u s e t h e y a r i s e a t d i f f e r e n t t i m e s o f l i f ea n d h a v e v e r y d i f f e r e n t n a t u r a l histories. Two of the diffuse neurofibromas I studied for clonality were classified c l i n i c a l l ya s c u t a n e o u s t u m o u r s ( C h a p t e r 4 ) . O n e s h o w e d d e f i n i t ec l o n a l i t y , a n d t h e other, possible clonalityof S100+/Nf- cells.These tumours had few S100-/Nf- cells, in contrastto nodular plexiform neurofibromas. My observation that cutaneous n e u r o f i b r o m a s o f t e n s h o w a b u n d a n t m a s t c e l l i n f i l t r a t i o n m a y r e f l e c ts t r o n g a t t r a c t i o n o r s t i m u l a t i o n o f m a s t c e l l s b y S 1 0 0 + / N f - S c h w a n n c e l l s ,a s h a s b e e n o b s e r v e d i n t h e mouse model (8). Only one of the diffuse neurofibromas examined for clonalityin this study was classified clinicallyas a congenital diffuse plexiform tumour (Chapter 4). Ithad few neurofibrominnegative cells (either S100-positive or S100-negative) and demonstrated polyclonality in allcell types. This observation is consistent with the hypothesis that diffuse plexiform neurofibromas arise during fetaldevelopment through excessive proliferationof poorlyr e g u l a t e d N F 1 h a p l o i n s u f f i c i e n t c e l l s r a t h e rt h a n t h r o u g he x p a n s i o no f a n e o p l a s t i c 104  Schwann cell clone that has sustained a second-hit" that annuls neurofibromin expression. The abundance of mast cellsin thistumour might be explained by recruitment or proliferation of mast cells in response to excessive cellularproliferation. However, thisneurofibroma was located superficially,and the presence of many mast cells might simply reflectitsproximity to the skin, where many mast cells normally r e s i d e . M o r e d i f f u s e p l e x i f o r m n e u r o f i b r o m a s f r o m b o t h d e e p a n d s u p e r f i c i a ll o c a t i o n s need to be examined to confirm my findingsregarding the numbers, distributionand clonality of various cell types. How this Study fits into current NF1 research T h e f u n c t i o no f t h e N F 1 g e n e h a s n o ty e tb e e n f u l l ye l u c i d a t e d .T h e b e s tc h a r a c t e r i z e function of neurofibromin is as a negative regulator of the Ras signaling pathway (9), but N F 1 i sa l s oi n v o l v e d i no t h e rs i g n a l i n g p a t h w a y s , i n c l u d i n g c A M P ( 1 0 ) . Haploinsufficiencyor complete absence of neurofibromin has been shown to affect the proliferation of a variety of cell types (11-13), but research into the responsible mechanisms is ongoing. The ubiquitous expression of neurofibromin, itsinteractions w i t h n u m e r o u s o t h e r c e l l u l a rp a t h w a y s , a n d t h e v a r i e t y a n d v a r i a b i l i t yo f p h e n o t y p e s observed in people with NF1 show that NF1 is a complex disease. Itis,therefore, not s u r p r i s i n gt h a t m o r e t h a n o n e p a t h o g e n i c m e c h a n i s m m a y l e a d t o t h e d e v e l o p m e n t o f neurofibromas, and tryingto cut too deeply with Occam's razor may not be the best way to understand the disease. T d e s t  here is no cure for neurofibromas except surgical removal in favourable cases. Small iscretetumours can usually be removed successfully,but plexiform neurofibromas, specially diffuse plexiform tumours, usually cannot be removed in theirentirety without ubstantialneurological compromise caused by surgicaldamage to nerves that traverse he tumour. Incompletely removed neurofibromas usually grow back.  Most studies of neurofibromas either lump allof these tumours together as a single benign group or categorize them as "dermal" or "plexiform",with the "dermal" group containing both cutaneous and subcutaneous neurofibromas, as defined by Riccardi's clinicalclassification,and the "plexiform"group containing both nodular and diffuse p l e x i f o r m n e u r o f i b r o m a s . T h e r e s u l t so f m y w o r k s h o w t h a t t h e s e g r o u p i n g s a r e insufficient.At the very least,clinically-definedcutaneous neurofibromas should be analysed separately from subcutaneous neurofibromas, and clinically-definednodular 105  and diffuseplexiform neurofibromas should be analysed separately because their histologicalappearance, cellularcomposition, and natural history are different. The differences Iobserved between histologically-defined diffuse and nodular neurofibromas help clarifyhow differentkinds of neurofibromas develop and may p r o v i d e i n s i g h ti n t o w h y s o m e n e u r o f i b r o m a s p r o g r e s s t o m a l i g n a n c y , w h i l e m o s t d o not. My resultssupport the current theory that neurofibromas can develop by clonal e x p a n s i o n o f n e o p l a s t i c S c h w a n n c e l l s t h a th a v e s u s t a i n e d a " s e c o n d - h i t " N F 1 mutation. However, I also found evidence that other neurofibromas develop by a d i f f e r e n tm e c h a n i s m- p o l y c l o n a l p r o l i f e r a t i o no fN F 1 h a p l o i n s u f f i c i possible that some neurofibromas, particularlynodular neurofibromas, develop from a S c h w a n n c e l l p r e c u r s o r r a t h e r t h a n f r o m m a t u r e S c h w a n n c e l l s . F u r t h e rc l i n i c a l , pathology, cellbiology, and molecular genetic studies of neurofibromas are necessary t o d e t e r m i n e w h y i n d i v i d u a l sw i t h N F 1 a n d c e r t a i n k i n d s o f n e u r o f i b r o m a s a r e m o r e l i k e l yt o d e v e l o p M P N S T s t h a n N F 1 p a t i e n t sw i t h o u t s u c h t u m o u r s . Future Directions The technique that Ideveloped for laser microdissection of cells in formalin-fixed paraffin-embedded samples is novel and described. Fixed tissue of this kind is readily available, and the molecular genetic studies on cellsisolatedfrom such tissue on immunofluorescent features should facilitatemolecular genetic tumours without the need for additionalsample collection.This applied to other kinds of tumours associated with NF1 as well a lesions that occur in some patients with this condition.  immunofluorescently-labeled has not previously been a b i l i t yt o p e r f o r m the basis of studies of a variety of technique can also be s to bony and vascular  The association Ifound between internalneurofibromas and MPNSTs is striking, but because thisstudy was based on cross-sectionaldata, the resultscan not be used to assess individual risk.Nevertheless, my findings show the need for a longitudinal study of neurofibroma growth and MPNST development in young adults with NF1. Current clinicalstandards for care of NF1 patients recommend imaging studies for internal n e u r o f i b r o m a s o n l y i ft h e y a r e s y m p t o m a t i c . M y d a t a s h o w t h a t i n d i v i d u a l s w i t h a s y m p t o m a t i c i n t e r n a ln e u r o f i b r o m a s m a y a l s o b e a t r i s k f o r d e v e l o p i n g MPNSTs. These findings raise important questions about whether routinewhole-body imaging should be considered for allNF1 patients and whether those found to have internal 106  plexiformtumours should be followed more closelyforthe development of an MPNST. Clinicalresearch and cost-benefit studies of available imaging techniques for people with NF1 are clearly needed. M o s t i n t e r n a l n e u r o f i b r o m a s i n c l u d e d i n m y a s s o c i a t i o n s t u d y o f M P N S T r i s kw o u l d b e c l a s s i f i e d c l i n i c a l l y a s n o d u l a r p l e x i f o r m n e u r o f i b r o m a s . T h e r e l a t i v e l yh i g h p r o p o r t i o n o f S100-/Nf- cells I observed in histologically-defined nodular neurofibromas may be evidence of a previously-unappreciated dedifferentiated or precursor Schwann cell population that is of particular importance in the pathogenesis of these tumours and in theirmalignant progression. The techniques used in the current study precluded further molecular analysis of this celltype, but future studies of the origin,clonal nature, and genetic stabilityof the S100-/Nf- cells may provide criticalinsight into processes that lead to the formation and malignant progression of nodular plexiform neurofibromas. My observations suggest that cellular interactions play a key role in neurofibroma d e v e l o p m e n t a n d i m p l y t h a t m o d i f y i n g g e n e s m a y b e o f i m p o r t a n c e . I n d i v i d u a l sw i t h N F 1 w h o s e c o n s t i t u t i o n a l m u t a t i o ni s a m i c o d e l e t i o n o f t h e e n t i r e N F 1 l o c u s a n d i t s adjacent genomic region are characterised by unusual facial features, a high tumour burden and an unusually high riskof developing MPNST (14, 15). There are 17 genes contained within the 1.4Mb region that is usually deleted in these patients.The products o f t h e s e o t h e r g e n e s m a y i n t e r a c tw i t ht h e N F 1 g e n e , w i t hn e u r o f i b r o m i n , o r w i t h o t h e r factors in the signaling pathways that neurofibromin regulates. Proteins produced by genes elsewhere in the genome also appear to interactwith neurofibromin (16, 17). Studies of factors that influence neurofibromin activityand function are a major area of study at NF research laboratoriesthroughout the world. NF1 is a very complex disease, and my studies show that neurofibroma development is n o t s i m p l y a m a t t e r o f a S c h w a n n c e l l s u s t a i n i n g a " s e c o n d h i t "o f t h e N F 1 g e n e . I n addition to having different clinicalcharacteristics, different kinds of neurofibromas e x h i b i t c l e a r d i f f e r e n c e s i n c e l l u l a rc o m p o s i t i o n a n d g e n e e x p r e s s i o n . M y r e s u l t s p r o v e that the clinicaland pathological heterogeneity of neurofibromas needs to be taken into a c c o u n t b y r e s e a r c h e r s w h o a r e t r y i n gt o u n d e r s t a n d h o w t h e s e t u m o u r s d e v e l o p a n d , in some instances, progress to malignancy.  107  References 1. Rasmussen SA, Yang Q, Friedman JM. Mortalityin neurofibromatosis 1: an analysis using U.S. death certificates.Am J of Hum Genet 2001;68:1110-8. 2. Ducatman BS, Scheithauer BW, Piepgras DG, Reiman HM, llstrup DM. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer 1986;57:2006-21. 3 . F e r n e rR E , L u c a sJ D , O ' D o h e r t yM J , e ta l . E v a l u a t i o no positron emission tomography ((18)FDG PET) in the detection of malignant peripheral nerve sheath tumours arising from within plexiform neurofibromas in neurofibromatosis 1. J Neurol Neurosurg Psychiatry 2000;68:353-7. 4 . L o r e e T R , N o r t h J H J r , W e r n e s s B A , etal. M a l i g n a n t p e r i p h e r a l n e r v e s tumors of the head and neck: analysis of prognostic factors.Otolaryngol Head Neck Surg 2000;122:667-72. 5. K i n g A A , D e b a u n M R , R i c c a r d iV M , G u t m a n n D H . M a l i g n a n t p e r i p h e r a l n e r v e sheath tumors in neurofibromatosis 1. Am J Med Genet 2000;93:388-92. 6. F r i e d m a n J M , R i c c a r d i V M . C l i n i c a la n d e p i d e m i o l o g i c a l f e a t u r e s . I n : G u t m a n n D H , M a c C o l l i n M , R i c c a r d i V M , e d i t o r s .N e u r o f i b r o m a t o s i s : p h e n o t y p e , n a t u r a l h i s t o r y ,a n d p a t h o g e n e s i s . 3 r d e d . B a l t i m o r e : J o h n H o p k i n s U n i v e r s i t y P r e s s ; 1999. p.26-86. 7. RiccardiVM. An Overview of NF-1: Dysplasia and Neoplasia. In:Riccardi VM, editors.Neurofibromatosis: Phenotype, Natural History and Pathogenesis. 2nd ed. Baltimore: The Johns Hopkins UniversityPress; 1992. p.18-39. 8. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann celloriginand role of tumor environment. Science 2002;296:920-2. 9. Harrisingh MC, Lloyd AC. Ras/Raf/ERK signallingand NF1. Cell Cycle 2004;3:1255-8. 1 0 . M a r c h u k D A , S a u l i n oA M , T a v a k k o l R ,e ta l . c D N A c l o n i n g o ft h e neurofibromatosisgene: complete sequence of the NF1 gene product. Genomics 1991;11:931-40. 1 1 . I n g r a m D A , Y a n g F C , T r a v e r sJ B , e ta l . G e n e t i ca n d b i o c h e haploinsufficiencyof the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo. J Exp Med 2000;191:181-8. 1 2 . G u t m a n n D H , L o e h r A , Z h a n g Y , etal. H a p l o i n s u f f i c i e n c y f o r t h e neurofibromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation.Oncogene 1999;18:4450-9. 1.3. Kim HA, Rosenbaum T, Marchionni MA, Ratner N, DeClue JE. Schwann cells f r o m n e u r o f i b r o m i n d e f i c i e n t m i c e e x h i b i t a c t i v a t i o n o f p 2 1 r a s , i n h i b i t i o no f c e l l proliferationand morphological changes. Oncogene 1995;11:325-35. 14. Tonsgard JH, YelavarthiKK, Cushner S, Short MP, Lindgren V. Do NF1 gene deletions result in a characteristic phenotype? Am J Med Genet 1997;73:80-6.  108  1 5 . 1 6 . 1 7 .  L e p p i g K A , K a p l a n P , V i s k o c h i l D , etal. F a m i l i a l n e u r o f i b r o m a t o s i s 1 m i c r o d e l e t i o n s : c o s e g r e g a t i o n w i t h d i s t i n c tf a c i a l p h e n o t y p e a n d e a r l y o n s e t o f cutaneous neurofibromata. Am J Med Genet 1997;73:197-204. J o h a n n e s s e n C M , R e c z e k E E , J a m e s M F , etal. T h e N F 1 t u m o r s u p p r e s s critically regulates TSC2 and mTOR. Proc Natl Acad Sci U S A 2005;102:8573-8 F e n gL ,Y u n o u e S , T o k u o H , e f a l . P K A p h o s p h o r y l a t i o n a n d 1 4 - 3 - 3 i n t e r a regulate the function of neurofibromatosis type Itumor suppressor, n e u r o f i b r o m i n .F E B S L e t t 2 0 0 4 ; 5 5 7 : 2 7 5 - 8 2 .  109  

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