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Molecular characterization of pediatric spindle cell tumors Knezevich, Stevan Robert 2000

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MOLECULAR CHARACTERIZATION OF PEDIATRIC SPINDLE CELL TUMORS by  STEVAN ROBERT KNEZEVICH B.Sc. The University of British Columbia, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A 2000 © S T E V A N ROBERT K N E Z E V I C H ,  2000  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference and  thesis by  this  for  his  scholarly  or  thesis  study.  her  for  of I  I further  purposes  gain  shall  requirements  agree  that  agree  may  representatives.  financial  the  be  It not  is  the  that  Library  permission  granted  by  understood be  for  allowed  of  Pfrrmi-OQY  The University of British Vancouver, Canada  Date fl-pr-s ' /  DE-6  (2/88)  5~/  to*l> C/j&aRAWZY'  Columbia  OQ  for  that without  M£PTCZW£-  advanced  shall  the  permission.  Department  an  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Congenital fibrosarcoma (CFS) is a cellular, mitotically active neoplasm of soft tissues. It affects infants less than two years of age, has a low metastatic rate and a relatively high propensity for local recurrence. One of the predominant clinical issues surrounding CFS is its distinction from other histologically identical and virtually indistinguishable pediatric spindle cell tumors  including adult-type  fibrosarcoma (ATFS) and infantile fibromatosis (IFB). ATFS is a malignant lesion that is treated more aggressively than CFS, while IFB is a benign lesion which is treated less aggressively. Reliable distinction between these entities is therefore clinically very important.  We therefore wanted to identify a diagnostic tool to  distinguish CFS from other fibroblastic tumors such as ATFS and IFB. Cytogenetic analysis of CFS cases has shown a nonrandom gain in chromosomes 8, 11, 17, and 20 with trisomy for chromosome 11 being present in most cases. Cytogeneticists at the Department of Pathology of B.C.C.H. recently identified recurrent cytogenetic alterations involving chromosome 12pl3 and 15q25 in three CFS cases, which were not  present in ATFS,  IFB, and  aggressive  fibromatosis.  Cloning  of  the  chromosomal breakpoints revealed a novel fusion between the ETS transcription factor member, ETV6, and the gene encoding the neurotrophin-3 receptor, NTRK3.  cell surface  This fusion results in the juxtaposition of the H L H dimerization  domain of ETV6 to the protein tyrosine kinase (PTK) domain of NTRK3.  We  hypothesized that this molecule acts as an aberrant PTK signaling molecule i n which the H L H domain mediates ligand independent dimerization resulting i n  Ill  constitutive P T K activation. The fusion protein exists as a 70-80 k D a doublet and was found to undergo homodimerization  as w e l l as heterodimerization  with  E T V 6 . Furthermore, we were able to show that the E T V 6 - N T R K 3 protein acts as a P T K that was capable of interacting w i t h P L O y l , but not w i t h other k n o w n N T R K 3 interactors including S H C , SH2Bp\ GRB2 and PI3K. Moreover, E T V 6 - N T R K 3 was shown to localize mainly i n the cytoplasm. O u r data support the notion that CFS is a biologically distinct entity, and ETV6-NTRK3  detection provides a diagnostic  screening tool potentially useful i n the clinical evaluation of children w i t h spindle cell tumors.  iv TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xiv  Chapter I  1 1 2  INTRODUCTION 1.1 Synopsis and Rationale for the Thesis 1.2 Pediatric Spindle C e l l Sarcomas 1.2.1 Pathologic W o r k u p , Radiology, and Clinical Features 1.2.2 Congenital Fibrosarcoma 1.3 General Aspects of N o r m a l G r o w t h Regulation 1.3.1 Signal Transduction Involved i n C e l l Proliferation The P D G F receptor and Ras pathway The phosphatidylinositol-3 kinase and protein kinase B pathway 1.3.2 Signal Transduction Involved i n L i m i t i n g G r o w t h 1.3.3 Cell Cycle 1.4 Mechanisms of Oncogenesis 1.4.1 Oncogenes P r o v i r a l Insertion Gene Amplification Point Mutations C h r o m o s o m a l Rearrangements 1.4.2 Tumor Suppressor Genes R B I p53 1.5 Genetic Aspects of Pediatric Solid Tumors 1.5.1 A n e u p l o i d y 1.5.2 Tumor Specific Translocations 1.5.3 Tumor Specific Translocations Result In Functional Gene Fusions In Solid Tumors  3 5 7 8 9 12 16 20 24 25 25 26 28 28 29 29 31 32 34 36 41  V  1.6  Chapter II  42 45 46 47 49  MATERIALS A N D METHODS  51  2.1  51 51 52  2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14  Chapter III Ewing's Sarcoma Family Of Peripheral Primitive NeuroectoDermal Tumors Myxoid Liposarcoma Alveolar Rhabdomyosarcoma Synovial Sarcoma A i m s and Objectives  Clinical Features of Index Case 2.1.1 Pathology of Index Case 2.1.2 Cytogenetic Analysis of Index Case Clinical Samples, Tissue Culture Techniques and Cytogenetic Analysis Y A C and Cosmid Probes D N A and R N A Isolation Southern and Northern Blot Analysis Fluorescence In Situ Hybridization (FISH) Studies 3' and 5' Rapid Amplification of c D N A Ends (RACE) R T - P C R Analysis of Tumor Samples Preparation of Protein Lysates for Immunoprecipitation and Immunoblotting Immunoprecipitation Immunoblotting Generation of G S T - E T V 6 - N T R K 3 Fusion Proteins In Vitro Protein Association Studies Subcellular Localization by Immunofluorescence  A N O V E L t(12; 15)(pl3; q25) IN CONGENITAL FIBROSARCOMA 3.1 3.2  3.3  Introduction Results 3.2.1 Cytogenetic Analysis 3.2.2 Fish Analysis Identifies a C o m m o n Derivative Chromosome 3.2.3 Identification of the Breakpoint Region by Y A C Mapping 3.2.4 Micromapping the Breakpoint with C o s m i d Probes Discussion  52 59 59 61 62 63 64 65 67 67 68 70 71  73 73 74 74 74 76 81 83  vi Chapter IV  CLONING A N D CHARACTERIZATION OF T H E t(12;15) IN CFS 4.1 Introduction 4.2 Results 4.2.1 C l o n i n g the t(12;15) Breakpoint i n C F S 4.2.2 Reciprocal Fusions were N o t Detected 4.2.3 R T - P C R Analysis of CFS and Other Morphologically Similar Tumors 4.2.4 Northern A n d Southern Blot Analysis 4.3 Discussion  91 91 92 92 94 94 95 95  ETV6-NTRK3  Chapter V  GENE FUSIONS A N D TRISOMY 11 ESTABLISH A HISTOGENETIC LINK BETWEEN MESOBLASTIC N E P H R O M A A N D CONGENITAL FIBROSARCOMA 104 5.1 Introduction 104 5.2 Results 106 5.2.1 Clinical History and Cytogenetics 106 5.2.2 R T - P C R Analysis of C M N Cases 108 5.2.3 Northern Blot Analysis 110 5.2.4 FISH Analysis 110 5.3 Discussion 110  Chapter VI  M O L E C U L A R STUDIES OF THE ETV6-NTRK3 FUSION PROTEIN 6.1 Introduction 6.2 Results 6.2.1 Expression and Phosphorylation Status of E T V 6 N T R K 3 and E T V 6 - N T R K 3 Mutant Proteins i n N I H 3 T 3 Cells 6.2.2 E T V 6 - N T R K 3 Homodimerizes and Heterodimerizes w i t h E T V 6 6.2.3 Downstream Interactors Affected by the ETV6N T R K 3 Molecule 6.2.4 Subcellular Localization 6.3 Discussion  Chapter VII S U M M A R Y A N D CONCLUSIONS 7.1 Identification of a Recurring t(12;15) i n Congenital Fibrosarcoma  115 115 116  116 119 121 123 123  132 133  vii 7.2 7.3  7.4 7.5  REFERENCES  The ETV6-NTRK3 Gene Fusion Characterizes Congenital Fibrosarcoma Trisomy 11 and the ETV6-NTRK3 Gene Fusion L i n k Congenital Fibrosarcoma to Congenital Mesoblastic Nephroma Molecular Studies of the E T V 6 - N T R K 3 Fusion Protein General Comments  134  134 135 138  141  viii  LIST OF TABLES Page Table 1.  Various Classes of Oncogenes and their Mode of Action Within Tumors  30  Table 2.  Tumor Suppressors and the Tumors Affected by their Loss  33  Table 3.  Summary of the Various Recurring Chromosomal Abnormalities Found in Pediatric Soft Tissue Tumors  35  Table 4.  Summary of Cytogenetic Analysis of Initial B C C H C F S Cases  58  Table 5.  Summary of the Various Constructs Used to Transfect NIH3T3 Cells Summary of Various Antibodies Used for Immunoblotting,  66  their Source and Required Concentrations  69  Table 7.  Summary of ETV6  82  Table 8.  Clinical Characteristics and Molecular Genetic Findings in C M N Cases Summary of ETV6-NTRK3 (TEL-TRKC) Analysis  Table 6.  Table 9.  Rearrangements in H u m a n Neoplasia  107 136  ix LIST OF FIGURES  Page(s) Figure 1. Figure 2.  Ras Signaling in the Eukaryotic Cell Partial Schematic of Signaling Mechanisms Involved in Growth Control  13-14 18-19  Figure 3.  The Cell Cycle  21  Figure 4.  The Possible Outcomes of Chromosomal Translocations  38-40  Figure 5.  Schematic Representation of EWS-ETS Fusions  43  Figure 6.  Histologic Analysis of CFS and IFB  53-54  Figure 7.  G-Banded Karyotype of Index Case  55-56  Figure 8.  Mapping of Chromosomal 12pl3 and 15q25-26 Breakpoints in CFS  75  Figure 9.  Dual-Coloured FISH of CFS  77-78  Figure 10. Figure 11.  FISH Analysis for CFS Breakpoints Schematic Representation of the cDNA for ETV6 as well as Some of the More Common Rearrangements Involving the  79-80  ETV6 Gene  87-88  Gene Fusions in CFS  93  Figure 12.  ETV6-NTRK3  Figure 13.  Northern Analysis of CFS Cases  96  Figure 14.  Southern Analysis of CFS Cases  97  Figure 15.  Schematic Representation of the Predicted ETV6-NTRK3 Protein  102  Figure 16.  ETV6-NTRK3  Detection in CMN  109  Figure 17.  Northern Analysis of CMN Cases  111  Figure 18.  FISH Analysis for Trisomy 11  112  Figure 19.  Western Blot Analysis of NIH3T3 Cells Expressing ETV6NTRK3 and Various Mutants Using CC-NTRK3 (C-14)  118  X  Figure 20.  Figure 21.  Figure 22.  Figure 23.  Figure 24.  Immunoprecipitation and Western Blot Analysis Demonstrates E T V 6 - N T R K 3 Tyrosine Phosphorylation  120  Immunoblot Analysis Demonstrating H L H - D o m a i n Dependant H o m o - and Heterodimerization of E T V 6 - N T R K 3  122  E T V 6 - N T R K 3 Interacts w i t h P L C y but not w i t h S H C , GRB2, or PI-3K p85 Subunit  124  Analysis of P L C y Binding Mutants for A b i l i t y to Associate w i t h PLCyl  125  Confocal Microscopy of E T V 6 - N T R K 3 and A H L H Expressing N I H 3 T 3 Cells  126  xi LIST O F A B B R E V I A T I O N S A AEL AFB AJ ALD ALL AML APC ARMS ATFS ATP BAD BCCH BCR B-NHL bp BSA C CCHTN CCSK CDK CDKI  cDNA CFS CGH CHLA Ci CID  adenine acute eosinophilic leukemia aggressive fibromatosis adherens junction activation loop dead acute l y m p h o i d leukemia acute myeloid leukemia adenomatosis polyposis coli alveolar rhabdomyosarcoma adult-type fibrosarcoma adenosine triphosphate Bcl-2 antagonist of cell death British C o l u m b i a Children's Hospital B cell antigen receptor B-cell non-Hodgkin's lymphoma base pair bovine serum albumin cytosine Cooperative H u m a n Tissue N e t w o r k clear cell sarcoma of the kidney cyclin dependent serine/threonine kinases cyclin dependent serine/threonine kinases inhibitor complimentary deoxyribonucleic acid congenital fibrosarcoma comparative genomic hybridization Children's Hospital Los Angeles Curie chemically-induced dimerization  OP/KIP CML CMML CMN CS DAG DAPI  DBD DDIT3 Del Der DFSP DAG AHLH dmin DMEM DNA DNA-PK Drk ECD EDTA EGF EGFR ERK ERMS ES ETS ETV6  CDK-interacting proteins chronic m y e l o i d leukemia chronic myelomonocytic leukemia congenital mesoblastic nephroma calf serum diacylglycerol diamidino-2phenylindole dihydrochloride hydrate D N A b i n d i n g domain D N A damage-inducible transcript 3 deletion derivative dermatofibrosarcoma protuberans diacylglycerol deleted H L H domain double minute Dulbecco's Modified Eagle M e d i u m deoxyribonucleic acid D N A - d e p e n d e n t protein kinase Drosophila receptor kinase extracellular ligand binding domain ethylene-diaminetetraacetic acid epidermal growth factor epidermal growth factor receptor extracellular signal regulated kinase embryonal rhabdomyosarcoma Ewings sarcoma E-26 transforming specific Ets variant gene 6  xn FCS FGF FGFR FISH FKHRL1  G GADD153  G6PD GAP GSK3 GST GTPase H&E HLH HPV HSR HUVE IFB IGF2 InsP  3  IPs ITF JAK kb KD kDa KRAB LOH LPS MAPK  fetal calf serum fibroblast growth factor fibroblast growth factor receptor fluorescence in situ hybridization forkhead i n rhabdomyosarcoma-like 1 guanine growth arrest and D N A damage-inducible gene 153 glucose-6-phosphate dehydrogenase GTPase activating protein glycogen synthase kinase*J  glutathione S-transferase guanosine triphosphatase hematoxylin and eosin helix-loop-helix human papillomavirus heterogeneously staining region h u m a n umbilical vein endothelial cells infantile fibromatosis insulin growth factor 2 inositol (1, 4, 5)triphosphate inositol triphosphate inducible transcription factor Janus family of tyrosine kinases kilo-base kinase dead kilo-Daltons Kruppel-associated box loss of heterozygosity liposarcoma mitogen-activated protein kinase  MDS MEK MFB mRNA MSCV NF NFTP nM NSE nt NT-3 NTRK3 NWTSG PBS PCNA PCR PDGF PDGFR PI-3K PKC PLCy PMSF pPNET PTB Ptdlns PtdInsP PTP  2  myelodysplastic syndrome M A P kinase/ERKactivating kinase infantile myofibromatosis messenger ribonucleic acid murine stem cell virus neurofibromatosis neurofilament triplet protein nanomolar neuron-specific enolase nucleotide neurotrophin-3 neurotrophic tyrosine kinase receptor type 3 National Wilms' Tumor Study G r o u p phosphate buffered saline proliferating cell nuclear antigen polymerase-chain reaction platelet derived growth factor platelet derived growth factor receptor phosphoinositol-3' kinase protein kinase C phospholipase-Cy Phenylmethylsulfonyl Fluoride Peripheral p r i m i t i v e neuroectodermal tumors phosphotyrosine b i n d i n g domain phosphatidylinositol phosphatidylinositol diphosphate protein tyrosine phosphatases  Xlll PSF PTK RACE RAEB  RB RMS RNA RNP RTK SDS SH2 SH3 SNT  Sos T TAD  penicillin streptomycin fungazone protein tyrosine kinase rapid amplification of c D N A ends refractory anemia w i t h excess blasts (with basophilia) retinoblastoma rhabdomyosarcoma ribonucleic acid ribonucleoprotein receptor tyrosine kinase sodium dodecyl sulfate Src homology 2 Src homology 3 sucl-associated neurotrophin factor target Son of Sevenless thymine transactivation d o m a i n  TBS Tcf4 TCR TEL TF TLS TNFSF6  TRKC TSG UTR VEGF WT YAC ZNF  tris-buffered saline T-cell transcription factor-4 T cell antigen receptor translocation, Ets, leukemia transcription factor translocated i n liposarcoma tumor necrosis factor ligand superfamily member 6 tropomyosin receptor kinase C tumor-suppressor gene untranslated region vascular endothelic growth factor W i l m s ' tumor yeast artificial chromosome zinc finger  xiv ACKNOWLEDGEMENTS First and foremost, I w o u l d like to thank Dr. P o u l H B Sorensen for his expertise, patience, leadership, relaxing personality and for h a v i n g been an integral part i n m y maturation  over the four years I spent under  his  supervision.  Moreover, this thesis and the work represented w i t h i n w o u l d never have  been  completed without his help and instruction. From the Sorensen laboratory, I w i s h to thank Jerian L i m for her immense technical knowledge and close friendship.  In  addition, I w i s h to thank Beth Lawlor, Jessica Palmer, W e n Tao, and Daniel W a i for their friendship and never ending support.  In addition, I w i s h to thank  the  members of m y supervisory committee, Dr. D o u g Horsman, D r . Keith H u m p h r i e s , and D r . Janet Chantler, for their interest and continued enthusiasm.  Finally, I  thank m y parents w h o have given me the opportunity and continuous support i n following m y dreams.  1  CHAPTER I INTRODUCTION  1.1  S Y N O P S I S A N D R A T I O N A L E F O R T H E THESIS The studies to be described i n this thesis were initially performed o n a series  of congenital fibrosarcoma cases, w h i c h belong to the family of spindle cell lesions of  childhood.  This  family  also  includes  infantile  fibromatosis,  aggressive  fibromatosis and adult-type fibrosarcoma, all of w h i c h appear histologically s i m i l a r under  the microscope.  significant  Pediatric spindle cell lesions of early childhood pose  diagnostic challenges  differentiate  for the  pathologist,  from one another due to their  as they  are  similar morphologic  difficult  to  appearance.  Differentiating these tumors is of great importance clinically as they show different clinical behaviors and require distinct treatment protocols.  We were  therefore  interested i n finding a specific recurring genetic anomaly w h i c h w o u l d provide the pathologist w i t h a molecular tool for accurately diagnosing these tumors. studied and identified a recurrent  t(12;15)(pl3;q25) i n congenital  We  fibrosarcoma,  w h i c h lead to the identification of a novel gene fusion between ETV6 (also k n o w n as TEL) and NTRK3 respectively.  (also k n o w n as TRKC) from chromosomes  12 and 15,  Further studies showed that the fusion gene encoded a chimeric  tyrosine kinase protein w h i c h we hypothesized functioned by dysregulating n o r m a l signaling pathways w i t h i n the malignant cell. G i v e n these findings, the remainder of this chapter w i l l deal w i t h pediatric spindle cell tumors (with an emphasis  on  congenital fibrosarcoma and other morphologically similar lesions), general aspects  2 of cancer biology and genetics, normal and abnormal growth (signal transduction and cell cycle), pediatric solid tumors, as well as signal transduction as it relates to tyrosine kinase receptors.  1.2  PEDIATRIC SPINDLE CELL S A R C O M A S H u m a n malignant tumors can be categorized into sarcomas, carcinomas and  hematopoietic (including lymphoid) malignancies.  Sarcomas are tumors  which  have arisen from mesenchymal tissue while carcinomas derive from epithelial tissue. Hematopoietic malignancies arise from blood forming and l y m p h o i d cells w h i c h originate from mesoderm.  During embryogenesis there are three p r i m a r y  germ layers; endoderm, mesoderm and ectoderm. The ectoderm gives rise to the epithelium, the entire nervous system, the lens and retina of the eye as w e l l as a broad range of structures i n the head and pharynx.  The mesoderm gives rise to  mesenchyme w h i c h is responsible for forming the skeletal muscles, vertebrae and skull, connective tissues and blood vessels of the body w a l l , the skeletal elements of the body w a l l , girdles and limbs, the smooth muscles and connective tissue of the digestive tract, the heart, the blood vessels of the viscera and blood. The e n d o d e r m gives rise to the epithelium of the digestive tract and to the epitheliod components of all organs that arise as evaginations from the embryonic foregut, midgut, or hindgut.  Sarcomas are malignant mesenchymally-derived tumors w h i c h exhibit  local recurrence  and metastatic behavior, and have  a h i g h proliferative rate.  Sarcomas are categorized on the basis of cell of origin. rhabdomyosarcomas  (derived  from  skeletal  For example, there are  muscle  precursor  cells),  3  leiomyosarcomas liposarcomas  (fat  (smooth cells),  muscle  cells), chondrosarcomas  hemangiosarcomas  (cartilage cells),  (blood vessels),  fibrosarcomas  (fibroblasts), osteosarcomas (bone cells), synovial sarcomas (synovial cells), and sarcomas of unknown origin. Several of the sarcomas mentioned above can also have spindle cell morphology. The word "spindle" refers to the shape of the cells, being long and drawn out in the shape of a spindle. Some examples of spindle cell tumors include fibrosarcoma, leiomyosarcoma, synovial sarcoma and some forms of rhabdomyosarcoma. One of the predominant issues in tumor pathology is the difficulty i n differentiating malignant sarcomas from each other as well as from benign lesions such as fibromatoses (benign lesions involving fibroblast cells).  These benign  lesions can be virtually indistinguishable from their malignant counterparts by morphological criteria, but have no metastatic behavior, have a low recurrence rate, and are less aggressive than sarcomas. A variety of approaches commonly used by the pathologist in diagnosing tumors are discussed below.  1.2.1  Pathologic W o r k u p of Pediatric Sarcomas  Histology is the primary method for evaluating tumors and is based on the study of the morphology of the various tumors. Briefly, the tumor specimen is fixed in formalin and embedded in paraffin. A 5pm section of the tumor is then placed onto a microscope slide and stained with hematoxylin and eosin. This is known as an H&E section and the characteristic staining patterns it produces is the primary diagnostic modality used in evaluating tumors.  4 Immunohistochemistry helps determine the cell of origin. A thin section of the tumor is placed onto a slide and is then incubated w i t h antibodies against specific antigens such as neuron-specific enolase (NSE), Leu7, neurofilament triplet protein (NFTP), desmin, muscle-specific actin, v i m e n t i n , S100, keratin, leukocyte c o m m o n antigen (CD45), and the surface antigen M I C 2 .  For example, a n e u r a l  tumor w o u l d be positive for N S E , Leu7, and N F T P , while myogenic tumors w o u l d be positive for desmin, muscle-specific actin, and M Y O D (a transcription factor that appears early i n myogenesis and activates later gene expression).  Therefore,  the  combination of positive and negative staining for the above antigens helps the pathologist form an accurate diagnosis. Another analytical technique often used is electron microscopy. The electron microscope is used to evaluate  ultrastructural  features.  Neuroblastomas,  for  example, contain dense core granules, rhabdomyosarcomas contain myofilaments, and acute megakaryocytic leukemia contains platelet granules each of w h i c h can be identified b y electron microscopy. Cytogenetics is a technique w h i c h involves the analysis of chromosomes from short term cultures of tumors. Recurrent chromosomal abnormalities can be used as a diagnostic tool, especially i n pediatric tumors where there are m a n y examples  of  recurring  chromosomal  deletions,  translocations  and  whole  chromosome gains and losses (discussed i n further detail below). The results from histologic, immunohistochemistry, electron microscopic and  cytogenetic  information.  analysis  provide  the  pathologist  with  useful  diagnostic  This information needs to be correlated w i t h the clinical history and  5 radiological features of the tumor.  In summary, histology and other  pathologic  modalities, coupled w i t h the clinical history (including radiological results) is the initial route taken i n the accurate diagnosis of many soft tissue pediatric tumors. Often however, it is extremely difficult if not impossible to distinguish different sarcomas from each other or sarcomas from benign lesions as they may be v i r t u a l l y identical morphologically. This has lead to the relatively new field of molecular pathology, i n w h i c h the tumor is further analyzed by cytogenetic and molecular techniques to make an accurate diagnosis (discussed i n further detail below).  1.2.2  Congenital Fibrosarcoma  Congenital (infantile) fibrosarcoma (CFS) arises from fibroblasts and is a mitotically active spindle cell lesion affecting soft tissue.  CFS acquired its n a m e  because of its histologic similarity to adult fibrosarcoma [1]. Fibrosarcomas tend to be categorized into two distinct age groups (the upper and lower limits of w h i c h are poorly defined): those occurring before the age of 2 years (most under one year of age), k n o w n as CFS, and those occurring i n patients aged 10 years or older, k n o w n as adult-type fibrosarcoma (ATFS). differences i n clinical behavior.  Correlating w i t h these age groups are distinct W h i l e C F S has a metastatic and recurrence rate of  10% and up to 40%, respectively [2-4], it is unique among h u m a n sarcomas for its excellent prognosis w i t h an 80-90% overall s u r v i v a l rate [5, 6]. O n the other h a n d , A T F S is an aggressive lesion w i t h a poor prognosis fibrosarcoma [7].  similar to that of adult  6 Under the microscope, C F S is morphologically similar to other fibroblastic tumors such as aggressive fibromatosis (AFB), A T F S and infantile fibromatosis (IFB) and has, historically, been difficult to diagnose [8-10].  This is evidenced by C F S  having been misdiagnosed i n the past for a lymphatic malformation  [11, 12],  hydrops fetalis [13], or congenital hemangioma [11,14]. C F S was also postulated as being histogenetically related to infantile myofibromatosis (MFB) and congenital hemangiopericytoma [1,15,16]. CFS is predominantly found i n the extremities (71%) and is p r i m a r i l y treated by surgical excision [17], often w i t h adjuvant  preoperative  and  postoperative  chemotherapy as w e l l as combination chemotherapy [8, 18-22]. The propensity for CFS tumors to metastasize seems to depend on the primary tumor site: primary C F S tumors located i n the extremities metastasize  7-8% of  w h i l e 26% of those  located w i t h i n the abdomen, pelvic and chest area metastasize [9, 23]. The benign counterpart to fibrosarcoma is infantile fibromatosis (IFB). IFB is a lesion that arises from fibroblasts a n d / o r myofibroblasts and exhibits lower cellularity and mitotic activity than fibrosarcomas [1]. Infantile fibromatosis (IFB) primarily occurs i n children aged 2 years or younger and, i n addition to the above characteristics, contains more collagenous matrix than CFS.  A variant of IFB  k n o w n as aggressive fibromatosis (AFB) is clinically more aggressive than IFB and is inclined to increased local invasion and recurrence [1]. Cytogenetic analyses of C F S cases to date have s h o w n non-random chromosome gains for chromosomes 2, 8, 11, 17 and 20 [6, 24-26].  whole  Other t u m o r s  w h i c h share some of these specific chromosomal abnormalities include: +20 and +8  7 i n desmoid tumors [27-29], +8 i n Dupuytren's contracture, Peyronie's disease and hematologic malignancies [30-33], +20, +17, +11, and +8 i n congenital mesoblastic nephroma  ( C M N ) [34, 35], and  +11 i n acute m y e l o i d leukemia  ( A M L ) [36].  Currently, there have been no reports of any recurring chromosomal abnormalities for IFB and A T F S .  A s w i l l be discussed i n later chapters, we have n o w identified a  recurrent t(12;15)(pl3;q25) translocation i n CFS.  1.3  GENERAL ASPECTS OF N O R M A L GROWTH REGULATION Understanding  the mechanisms  of oncogenesis  (see section  1.4 below),  requires the familiarization w i t h the way an extracellular "message" is transferred from the cell membrane into the nucleus.  Once i n the nucleus the initial message  results i n the activation or suppression of transcription of certain genes required either for growth and progression through the cell cycle or differentiation.  A cell  maintains a normal rate of growth by a variety of important biological mechanisms. The following discussion w i l l briefly cover three mechanisms pertaining to n o r m a l growth regulation: 1. Signal Transduction i n v o l v e d i n C e l l Proliferation: the concept of how a cell receives an extracellular "message" and the intracellular ( i n c l u d i n g nuclear) consequences. 2. Signal Transduction Involved i n L i m i t i n g C e l l G r o w t h : the concept of how  a cell  environment.  "knows" w h e n  to stop growing i n the  context  of  its  8  3.  C e l l C y c l e : the n u c l e a r m a c h i n e r y responsible for d e t e r m i n i n g w h e n it is appropriate for a cell to proliferate a n d d i v i d e .  1.3.1  Signal Transduction Involved in Cell Proliferation  In general, signal t r a n s d u c t i o n refers to the specific m o l e c u l a r  interactions  a n d subsequent m o d i f i c a t i o n s i n response to a n external s t i m u l u s , s u c h as  growth  factors a n d cytokines. Messages f r o m extracellular g r o w t h regulatory m o l e c u l e s are transferred into the cell v i a receptors that b i n d these m o l e c u l e s .  T h e r e are s e v e r a l  different classes of receptors i n c l u d i n g g r o w t h factor receptors (eg., fibroblast g r o w t h factor receptor), steroid receptors (eg., g l u c o c o r t i c o i d receptor), n e u r o t r a n s m i t t e r receptors (eg., acetylcholine receptor), seven t r a n s m e m b r a n e - s p a n n i n g or s e r p e n t i n e receptors (eg., gastrin releasing peptide receptor), B-cell receptors (eg., B cell a n t i g e n receptor (BCR)), a n d T - c e l l receptors (eg., T C R ) .  Since o u r studies r e s u l t e d i n t h e  d i s c o v e r y of a c h i m e r i c f u s i o n p r o t e i n c o n t a i n i n g  a p o r t i o n of a tyrosine  receptor (a m e m b e r of the g r o w t h factor receptor f a m i l y ) , the r e m a i n d e r d i s c u s s i o n w i l l focus o n signal t r a n s d u c t i o n as it pertains to the p r o t e i n  kinase of this tyrosine  kinase f a m i l y of g r o w t h factor receptors. M a n y of the signals responsible for i n d u c i n g p r o l i f e r a t i o n or d i f f e r e n t i a t i o n act t h r o u g h g r o w t h factors b i n d i n g to cell surface receptor tyrosine kinases  (RTKs).  T h e process of t r a n s d u c i n g a signal f r o m R T K s o n the cell surface to the n u c l e u s  can  be d i v i d e d into 4 steps (other receptors, s u c h as the steroid receptors, c o n t a i n slightly different series of events):  a  9 1. ligand binding 2. receptor dimerization and P T K activation 3. phosphorylation of cytoplasmic proteins 4. phosphorylation of nuclear proteins (i.e. transcription factors responsible for the activation or inhibition of genes involved i n growth control) These steps w i l l now be discussed below i n the context of the P D G F receptor and the well established R A S pathway [37-39]. The P D G F receptor and R A S pathway The way many  extracellular hormone  or growth factors  deliver  their  messages to the intracellular environment of a cell is by b i n d i n g to a specific protein tyrosine kinase (PTK) receptor.  Each P T K molecule has an extracellular ligand  binding domain, a transmembrane d o m a i n [37]. extracellular transduction.  domain, and an intracellular tyrosine kinase  The ligand (e.g. E G F , F G F or PDGF), w i l l b i n d to the portion of the  receptor,  thus  beginning  the  appropriate  process  of signal  A n example of this is the platelet derived growth factor  (PDGF)  binding to P D G F R . U p o n ligand binding, the P D G F receptor undergoes h o m o d i m e r i z a t i o n [37] bringing the kinase domains i n close proximity.  This interaction results i n the  auto- or cross-phosphorylation of specific tyrosine moieties w i t h i n the intracellular d o m a i n of the receptor.  The structure responsible for this is k n o w n as  activation loop and consists of tyrosine residues  within  the  kinase  the  domain.  Phosphorylated tyrosine residues outside the tyrosine kinase d o m a i n act as anchors for downstream molecular interactions (see below) [40, 41].  In addition to these  10 tyrosine residues are tyrosines w h i c h do not get phosphorylated and act as structural amino acids. Other types of receptors w h i c h undergo ligand induced d i m e r i z a t i o n (and even oligomerization) include B-cell receptors, T-cell receptors, hormone  and  cytokine receptors [42]. Once the ligand interaction has taken place, the P D G F / P D G F R complex acts as a tyrosine kinase and phosphorylates interacting proteins (including other tyrosine kinases) w h i c h can then interact w i t h other molecules and phosphorylate them [37]. The P D G F receptor contains several different phosphorylated tyrosines responsible for its interaction w i t h associating molecules such as Src [43], phosphatidylinositol-3 kinase (PI-3K) [44], G A P [45], Syp [46], S H C [47], growth factor receptor-bound protein 2 (GRB2) [48, 49] and P L C - y l [44]. These downstream interacting molecules possess an SH2 d o m a i n (for Src homology 2) w h i c h allows for the specific interaction w i t h phosphorylated tyrosines  [50].  The specificity for a particular  phosphorylated  tyrosine is determined by the 3 dimensional shape of the S H 2 d o m a i n i n the downstream  molecule and the 3 dimensional context of the  tyrosine moiety o n the receptor.  Another  phosphorylated  d o m a i n w h i c h has recently  implicated i n phosphorylated tyrosine binding is k n o w n as the  been  phosphotyrosine  binding d o m a i n (PTB) [51, 52]. GRB2 is the 217 amino acid homologue of drk i n Drosophila  and Sem-5 i n Cuenorhubditis  [53, 54].  It contains an S H 2 d o m a i n  responsible for interacting w i t h one of the phosphorylated  tyrosines  intracellular portion of the activated E G F receptor and a SH3 d o m a i n  on  the  which  interacts w i t h proline rich sequences on downstream molecules such as Sos [55]. GRB2 interaction w i t h Sos (the 1596-residue product of the Son of Sevenless  gene,  11 so named because Sos interacts w i t h the Sevenless gene product, a P T K receptor that regulates  the  development  of the  R7 photoreceptor  cell i n  the  Drosophila  compound eye [42]) results i n the relocalization of Sos to the plasma  membrane  where it can convert inactive R A S (RAS-GDP) into active R A S (RAS-GTP) [56, 57]. One of the pathways affected by R A S is the mitogen activated protein kinases ( M A P K ) pathway w h i c h is a series of cytosolic serine/threonine kinases [58]. One of the molecules w h i c h leads to the activation of the M A P K pathway is R A F 1 , w h i c h is a serine/threonine (MAPKKK)) activates  [59].  protein kinase (also k n o w n as M A P kinase kinase Phosphorylation of multiple serine  R A F 1 allowing  it to interact  with  and threonine  and phosphorylate  kinase residues  MEK(MAP  kinase/ERK-activating Kinase or M A P kinase kinase ( M A P K K ) ) [39, 60]. Activated M E K then interacts w i t h and phosphorylates a family of proteins, k n o w n as the MAP  kinases  or  ERKs  (extracellular-regulated  kinases)  which  require  the  phosphorylation of both serine/threonine and tyrosine residues i n order for proper activation. Before proliferation or differentiation can occur, certain genes i n v o l v e d i n growth regulation need to be turned on or off. phosphorylation  One mechanism involves the  of particular transcription factors  found  i n the  activated cytoplasmic kinases that have migrated into the nucleus.  nucleus  by  In the R A S  pathway, activated (phosphorylated) E R K migrates from the cytoplasm into the nucleus and phosphorylates various transcription factors, such as J u n / A P l , Fos, M y c , and S a p l w h i c h subsequently turn their respective target genes on or off [6062]. These target genes are most likely responsible for controlling the cell cycle and  12 proliferation  associated signaling pathways, but more  research  is needed  to  elucidate the exact targets and functions of these transcription factors (see Figure 1). In addition to controlling the R A F kinase cascade, R A S has been s h o w n to either directly or indirectly interact w i t h a number of other molecules including PI3K [63], Bcl-2 [64-66], protein kinase CC, (PKCCJ [67, 68], Rin, the guanine nucleotide exchange factors for R a l , A F 6 and p l 2 0  G A P  w h i c h can result i n a variety of cellular  responses including cell survival, mitogenesis, and differentiation [69]. Since the purpose of this introduction is to familiarize  the reader w i t h the  important  mechanisms involved i n growth regulation, a brief discussion of the PI-3K pathway w i l l n o w be included as it is important i n activating cell survival pathways. The phosphatidylinositol-3 kinase and protein kinase B pathway PI-3K is a heterodimeric enzyme composed of a p85 and a p l l O subunit (other isoforms exist for both of these molecules) [70]. detachment-induced  programmed  cell death  R A S can protect a cell f r o m  (apoptosis)  and other  forms  of  apoptosis through the activation of the PI-3K pathway [57]. R A S activation of PI-3K is mediated through the p l l O subunit of PI-3K [63]. The PI-3K pathway is i m p l i c a t e d i n apoptosis, cell motility, and vesicle trafficking and secretion [71].  Its role i n  apoptosis w i l l be discussed further. Activated PI-3K (either through the interaction w i t h an activated tyrosine kinase  receptor  such  as  PDGF  receptor  or  through  RAS)  leads  to  the  phosphorylation of phosphatidylinositol (Ptdlns) [71]. In addition, activated PI-3K can phosphorylate Ptdlns 4-P and Ptdlns 4, 5-P [71]. Yao and Cooper found that the 2  13  F I G U R E 1. R A S signaling i n the eukaryotic cell. The initial event, w h i c h ultimately results i n the activation of nuclear transcription factors, is l i g a n d induced dimerization of the cell surface receptor. This results i n the autophosphorylation of specific tyrosine residues of w h i c h only a few are i n v o l v e d i n attracting cytosolic molecules to the receptor. A n adaptor molecule, G R B 2 , attaches itself to one of these phosphotyrosines and is responsible for recruiting Sos (son of sevenless) to the plasma membrane, w h i c h facilitates the dissociation of G D P from R A S , thus activating R A S . This process is reversed by another m o l e c u l e k n o w n as GTPase activating protein (GAP). R A S activation can result i n the activation of mitogen activated protein kinase pathways like R A F , M E K (mitogen activated, E R K activating protein), and E R K (extracellular signal-regulated protein kinase). Phosphorylated E R K can phosphorylate and thus activate nuclear transcription factors such as Elk, M y c and c-Jun, w h i c h results i n physiological changes i n the cell (ie. Cell cycle activation).  14  15 PI-3K inhibitor, wortmannin, caused apoptosis i n PC12 cells suggesting that the PI3K pathway is involved i n cell survival [72]. One of the downstream interactors for PI-3K responsible for cell survival is the protein kinase A K T (also k n o w n as "protein kinase B " (PKB)) [73]. A K T is homologous to the protein kinase A and C families and to the retroviral oncogene v-akt and encodes a serine/threonine  protein kinase that is  ubiquitously expressed [74]. A K T differs from P K A and P K C i n that the a m i n o terminal portion contains an A K T homology domain part of w h i c h is related to the Pleckstrin homology domain.  This domain is found i n a number of signaling  molecules including the phospholipase family of tyrosine kinases (see chapter 6) and are thought to mediate protein-lipid and/or protein-protein interactions [75, 76].  Serine and threonine  phosphorylation of A K T by PI-3K activated P D K 1  activates A K T . This leads to the phosphorylation by A K T of the Bcl-2 antagonist of cell death (BAD). B A D may also be phosphorylated by an as of yet u n k n o w n kinase w h i c h does not involve PI-3K or A K T [77]. In its unphosphorylated form, B A D can interact w i t h the Bel family members Bcl-XL and Bcl-2 i n d u c i n g apoptosis.  There  has been a report o n n o n - B A D mediated apoptosis, however further elucidation of the pathways i n v o l v e are required [78]. Once phosphorylated by A K T h o w e v e r , B A D associates w i t h another protein called 14-3-3 (a group of scaffolding proteins that bind to phosphorylated serine residues and are thought to be implicated i n cell cycle control and various signal transduction pathways) and is no longer able to interact w i t h Bcl-XL or Bcl-2 and apoptosis is abrogated [79, 80]. Expression of B A D  16 is not ubiquitous however, suggesting that other cell s u r v i v a l proteins must exist for A K T [81]. Recently, Brunet et al. showed that A K T can directly phosphorylate  and  inactivate the forkhead i n rhabdomyosarcoma-like 1 transcription factor ( F K H R L 1 ) [82]. F K H R L 1 is a member of the forkhead family of transcription factors and is thought to transcribe genes responsible for cell death.  Phosphorylation causes  F K H R L 1 to associate w i t h 14-3-3 proteins [83, 84]. This association causes F K H R L 1 to remain i n the cytoplasm thus inhibiting its transcriptional activity. absence  of  survival  factors,  the  FKHRL1  transcription  factor  In the becomes  dephosphorylated, translocates to the nucleus and transactivates target genes critical for cell death, such as the tumor necrosis factor ligand superfamily member  6  (TNFSF6) gene [85].  1.3.2  Signal Transduction Involved i n L i m i t i n g G r o w t h  Cells need to k n o w w h e n to stop growing. It is thought that w h e n a n o r m a l cell is i n contact w i t h a neighboring cell, certain proteins o n the surface of the cell recognize similar proteins o n the neighboring cell, sending signals to the nucleus to stop proliferation. Adherens junctions (AJ or zonula adherens) mediate between cells, communicate  a signal that neighboring cells are present  inhibition), and anchor the actin cytoskeleton.  adhesion (contact  Beta-catenin (p-catenin) is an A J  protein w h i c h is critical for the establishment and maintenance of epithelial layers, such as those lining organ surfaces [86]. AJs are therefore responsible for regulating normal cell growth and behavior.  17 The A J is a multiprotein complex assembled around calcium-regulated cell adhesion  molecules  called cadherins  [86].  Cadherins  are membrane  spanning  proteins that mediate interactions w i t h neighboring cells also containing cadherins. The intracellular domain of the cadherin molecule transmits the adhesion signal resulting i n the anchoring of the A J to the actin cytoskeleton.  The cytoplasmic  proteins responsible for transmitting the signal include the a-, [}-, and y-catenins [87]. Korinek et al. and M o r i n et al. showed that the adenomatosis polyposis coli (APC) gene (mutated i n adenomatosis polyposis of the  colon), is a  negative  regulator of beta-catenin signaling [88, 89]. The A P C protein normally binds to (3catenin, w h i c h interacts w i t h the Tcf and Lef transcription factors.  Studies by  Korinek et al. showed nuclei of A P C - / - colon carcinoma cells contained a stable (3catenin/Tcf4 (T-cell transcription factor-4) complex that was constitutively active. Reintroduction of A P C removed beta-catenin from Tcf4 and ablated transcriptional activation.  They concluded that A P C loss of function  leads to constitutively  activated Tcf4 and may be an important step towards early transformation of colonic epithelium. Some colorectal tumors have been found to contain intact A P C genes, while the (3-catenin gene contains an activating mutant.  These studies suggest that  regulation of |3-catenin is critical for normal growth, that the A P C gene acts as a growth inhibitor (tumor suppressor gene; see below), and that mutations A P C or (3-catenin can lead to tumorigenesis (see Fig. 2).  i n either  18 FIGURE 2. Partial schematic of signaling mechanisms involved in growth control. In order for a cell to become tumorigenic and/or malignant, the cell must acquire immortality, increase its growth rate, and even develop motility in order to become metastatic. A number of these requirements are met by the constitutive activation of RAS. R A S can become constitutively activated by a point mutation or by another aberrantly regulated molecule which could lead to increased proliferation through the activation of certain M A P K pathways. R A S can also activate N F K B (responsible for the expression of anti-apoptotic proteins) and PI-3K, which can activate A K T which stimulates B A D and ultimately results in the activation of anti-apoptotic pathways. Activated A K T can phosphorylate and block the activity of another molecule, namely GSK3 (glycogen synthase kinase-3) to block either gene transcription mediated through p-catenin or motility regulated by A P C (adenomatous polyposis of the colon). Some of these pathways are duplicated by the cell surface receptor itself. For example, when a ligand binds to the extracellular ligand binding domain (ECD), the protein tyrosine kinase domain (PTK) can activate PI3-K or RAS. Moreover, the activated cell surface receptor can activate acatenin, which is involved in cytoskeletal modifications.  19  20 Cell Cycle  1.3.3  Since many of the pathways discussed above converge on the cell cycle, regulation of the cell cycle will now be summarized. The process of cell division and differentiation is essentially determined by the impact of external stimuli (e.g., growth factors, lack of nutrients, stress, DNA damage) on the cell cycle machinery within the cell [90].  The cell cycle machinery is composed of cyclins, cyclin  dependent serine/threonine kinases (CDKs) and their regulatory kinases and phosphatases. Briefly, the cell cycle consists of four phases known as G S, G , and v  2  M. Gj (GAP1) refers to the first growth phase of the cell cycle and represents the time frame during which the various growth factors can act upon the cell. The synthesis of DNA occurs during S phase, where the normal diploid content, 2n, becomes tetraploid, 4n [90].  G (GAP2) follows S phase and represents the 2  termination of DNA synthesis and the continuation of cell growth (organelles and proteins). Mitosis (M phase) is the point when the cell divides and distributes the newly synthesized DNA into two identical daughter cells. Another phase exists in which the cell is static.  There is neither growth nor differentiation and this is  known as G . When nutrients are in short supply or the cells are touching one 0  another (contact inhibition), the cell enters G . Surgical removal of tissue, on the 0  other hand, will cause the surrounding cells to re-enter the cell cycle and begin the regeneration process until growth is arrested due to contact inhibition (see Fig. 3). The cell cycle is tightly controlled by cyclins (regulatory subunits) [91], cyclin dependent kinases (CDKs) (enzymatic subunits) [91, 92], and cyclin-dependent kinase inhibitors (CDKIs) [93,94]. Briefly, the level of expression of cyclins and their rapid  21  Cyclin D Cyclin E  Cyclin A ^ - ^ •  •• •• ••  *  m  Gl  s  *  *  Cyclin B \  A"" \ \  ^  N N  \ \  s  v  x  G2  •  N  \ \  \  x x  M  Figure 3. The cell cycle. The chart (top panel) displays the variation of expression of some of the cyclins which are important in regulating the transition from one phase to another. There are 4 phases in the cell cycle (bottom) including G l , S, G2 (which comprise interphase) and M (mitosis). The amount of time spent in each phase and the amount of D N A present within a phase is also shown. Quiescent cells are in a phase known as GO and can re-enter the cell cycle with certain stimuli such as growth factors.  22  d e g r a d a t i o n , determines w h e n a transition f r o m o n e phase to another w i l l occur. a d d i t i o n , the cyclins c a n interact w i t h C D K proteins  to f o r m  complexes  In  whose  p h o s p h o r y l a t i o n status determines if it is active o r not, thus a u g m e n t i n g the c o n t r o l of the cell cycle [90]. P h o s p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n p l a y a major the cell cycle. product, R B  T h e G j to S transition  [95]. W h e n  role i n the c o n t r o l o f  is c o n t r o l l e d by the r e t i n o b l a s t o m a  R B is h y p o p h o s p h o r y l a t e d , it c a n interact w i t h  t r a n s c r i p t i o n factor w h i c h c a n activate t r a n s c r i p t i o n of a n u m b e r When  gene E2F, a  of genes [96-98].  the R B p r o t e i n is h y p e r p h o s p h o r y l a t e d b y the c y c l i n - D / C D K 4/6 c o m p l e x , it  n o longer has the ability to interact w i t h E2F. H y p e r p h o s p h o r y l a t i o n of R B needs to take place i n o r d e r f o r the t r a n s i t i o n c o m m o n l y r e f e r r e d to as the G  1  Once phosphate  the signal w h i c h groups  need  from  G  5  to S to o c c u r  [99-102].  initiated  the p h o s p h o r y l a t i o n  to be r e m o v e d  A n additional level  in  order  of c o n t r o l is a c h i e v e d  molecules. C D K inhibitors, s u c h as p ^ 27  CAI™  p  and  inhibiting  K I P 1  dependent  /  a  n  d p57  r a p 2  their  kinase  is  to S checkpoint.  to  is  terminated, the  terminate  downstream  activations [103-107]. T h i s is carried o u t b y proteins k n o w n as phosphatases CDC25.  This  4  8  pltf ™*, 1  ,  pl8  s u c h as  b y directly i n h i b i t i n g I N K 4 C  , p ^  4  0  , p21  C I P 1  '  CDK  W A F 1  -  S D n  -  are responsible for directly c o u p l i n g w i t h C D K m o l e c u l e s  regulative  inhibitor)  D / C D K 4 / 6 complexes, w h i l e  role i n the cell proteins  are  the C I P / K I P  cycle  [93].  responsible  for  (CDK-interacting  inhibit C y c l i n A , B, a n d E / C D K 2 complexes [93].  The INK4  (cyclin-  inhibiting  Cyclin  proteins)  molecules  23 Another important gene w h i c h is involved i n the regulation of the cell cycle is p53. Like RB, p53 is also a tumor suppressor gene whose activity increases w h e n there is damage to D N A [108-111]. p53 is a nuclear phosphoprotein w i t h two D N A binding domains [112], two SV40 large T-antigen b i n d i n g sites [113, 114], a nuclear localizing  signal  [115], an  oligomerization  domain  [116,  117], and  several  phosphorylation sites [118]. It is thought to act as a transcription factor for other growth regulatory genes either activating or inhibiting their transcription [109, 119]. In addition to acting as a transcription factor, p53 can interact w i t h proteins such as p21 (CIP1) [120-122].  CIP1 inactivates G  2  c y c l i n / C D K complexes and i n  addition, binds to the D N A polymerase cofactor, proliferating cell nuclear antigen ( P C N A ) , thus preventing D N A replication, but allowing for D N A repair [123]. T h i s event effectively blocks the G^ to S transition and again acts as a checkpoint w i t h i n the cell cycle.  Bunz et al. demonstrated  that u p o n D N A damage, cells enter a  sustained arrest i n the G phase only w h e n p53 was present i n the cell and capable of 2  transcriptionally activating the cyclin-dependent kinase inhibitor CD?1 [124].  After  disruption of either the p53 or the CIP1 gene, gamma-radiated cells progressed i n t o mitosis, but failed to undergo cytokinesis.  The cells therefore  exhibited a G  2  (tetraploid) D N A content. In addition, Shieh et al. showed that D N A damage leads to the phosphorylation of p53 and that this event reduces the ability of p53 to interact w i t h M D M 2 . M D M 2 is a negative regulator of p53 that normally binds to p53 inhibiting  its function  [125].  Furthermore,  they  demonstrated  phosphorylation of p53 by purified DNA-dependent protein kinase impairs the ability of M D M 2 to inhibit p53-dependenf transactivation.  that  the  (DNA-PK)  24 Finally, p53 has been implicated i n apoptosis [126-128]. One group, Polyak et al, examined i n detail the transcripts induced by p53 expression before the onset of apoptosis [129]. Of the 7,202 transcripts identified, only 14 (0.19%) were found to be markedly increased i n p53-expressing cells compared w i t h controls.  Strikingly,  many of these genes were predicted to encode proteins that could generate or respond to oxidative stress. p53 levels are normally very l o w , but have been s h o w n to rapidly increase after D N A damage or viral infection.  It is k n o w n that p53  induces B A X transcription, a member of the Bcl-2 gene family [130]. The exact mechanism of p53 associated apoptosis, however, is not k n o w n and further research is needed to elucidate this pathway.  1.4  MECHANISMS OF ONCOGENESIS The activation or inactivation of specific molecules or cellular pathways, s u c h  as those discussed above, can result i n the transformation of n o r m a l cells i n t o oncogenic ones. For example, inactivating mutations of p53 results i n the tolerance of D N A damage throughout the cell cycle leading to increased genetic instability and possibly an oncogenic advantage. Tumor cell metastasis is thought to be a result of a disrupted (3-catenin pathway where contact inhibition is no longer present thus allowing the cell to continue growing i n the presence of neighboring cells and invade other tissues i n the body [131].  V i r a l oncoproteins (such as the S V 4 0 ,  adenovirus and papillomavirus) have the capability to bind R B and p53 and thus disrupt their ability to inhibit growth [132-134]. It is important to note that cancer is not due to a single aberration, but the disruption of multiple pathways. A n increase  25 i n the expression of an oncogene or the loss of tumor suppressor factor expression alone, for example, is insufficient to produce a malignant tumor.  Genes w h i c h are  responsible for enhancing the proliferative rate of a cell are k n o w n as oncogenes while growth inhibiting genes are k n o w n as tumor suppressor genes (discussed i n further detail below).  1.4.1  Oncogenes  Oncogenes are mutated forms of their normal cellular counterparts, oncogenes.  proto-  Briefly, these proto-oncogenes can be converted to an oncogene by a  chromosomal rearrangement,  proviral insertion, gene amplification or a point  mutation (discussed i n further detail below). Proto-oncogenes have been found at virtually every level of the proliferation associated signal transduction pathway and include: growth factors, growth factor receptors, guanine nucleotide like proteins, guanine nucleotide exchange factors (GNEFs), cytoplasmic serine/threonine  or  tyrosine protein kinases, or nuclear proteins (transcription factors). Oncogenes were first discovered as the transforming elements i n the R N A viruses responsible for causing sarcomas i n fowl [135]. The following discussion w i l l detail some of the mechanisms b y w h i c h a proto-oncogene is converted into an oncogene. Proviral Insertion W h e n a retrovirus inserts itself adjacent to a proto-oncogene, it places the expression of the proto-oncogene under the control of the enhancer elements of the retrovirus.  This type of conversion  (proto-oncogene  to oncogene)  was  first  identified w i t h avian leukosis virus-induced bursal lymphomas where the level of  26 transcription of c-myc was 50 to 100 fold higher than n o r m a l due to p r o v i r a l insertion upstream of the c-myc proto-oncogene locus [136]. Gene A m p l i f i c a t i o n Genomic amplification has been seen i n many solid tumors and represents another mechanism by w h i c h an oncogene may be overexpressed [137]. The actual mechanism by w h i c h this amplification occurs is not clear but the end result may be seen cytogenetically as structures homogenously  staining  referred  regions  (HSRs)  extrachromosomal, circular structures  to as double minutes [138,  139].  (dmins)  dmins  are  and small  that occur i n pairs and contain specific  chromosomal regions, but lack telomeres [140]. These regions (amplicons) contain a number of genes (typically proto-oncogenes) determines  and the number of dmins per cell  the level of amplification of the genes i n v o l v e d .  Tumorigenesis  usually results w h e n one or more of the genes amplified are responsible for cell proliferation [141,142]. HSRs are dmins w h i c h have integrated into the genome. They appear as homogenously chromosome.  staining regions  (hence  the  name)  within  a  The mechanism(s) by w h i c h a cell acquires dmins and H S R s is not  fully understood.  The fact that both forms of gene amplification may be seen i n a  particular cancer is interesting. For example, one cell (or subline) may have an H S R while another cell (or subline) w i t h i n the same tumor may have a d m i n .  It has  been postulated that the dmins represent the unstable form of H S R s [143].  These  dmins however, w i l l integrate and form HSRs i n culture [144]. One of the most extensively studied tumors where gene amplification plays an important role i n tumorigenesis is neuroblastoma.  The NMYC  oncogene is  27  located on chromosome  2p24.1 and  is amplified  25- to 700-fold i n  human  neuroblastomas by means of dmins and HSRs [145-149]. The h i g h levels of N M Y C protein production is thought to deregulate transcription and lead to a proliferative advantage for the cell.  NMYC  amplification i n neuroblastoma  is associated w i t h  advanced stage disease and a poor prognosis [146,150,151]. A recent review has summarized sequence  copy  number  the various reports of recurrent D N A  amplifications  in  human  neoplasms  detected  by  comparative genomic hybridization ( C G H ) [152]. C G H is a technique similar to F I S H which  allows for the  detection  of deletions,  duplications  and  amplifications.  A m p l i c o n s have been identified for almost every chromosome.  One of these  regions, namely 12ql3-q21, encompasses the GU, CHOP, CDK2, MDM2  SAS,  WNT1,  WNTlOb,  and CDK4 genes, and has been shown to be amplified i n a variety of  tumors. Portions of this region have also been shown to be amplified i n a n u m b e r of tumors i n c l u d i n g osteosarcoma and  SAS),  chondrosarcoma  specifically i n v o l v i n g  (12ql3-ql4, specifically i n v o l v i n g CDK4,  (12cen-ql5  the CDK4  and  and MDM2  embryonal and alveolar rhabdomyosarcoma  12q24.1),  liposarcoma  MDM2  (12ql4-q21  genes), synovial sarcoma  (12ql5),  (12ql3-ql5), breast carcinoma (12ql5),  hereditary ovarian cancer (12ql3-q21), colon carcinoma (12ql3), bladder carcinoma (12ql3-ql5), diffuse large cell l y m p h o m a (12ql3-ql4), follicular l y m p h o m a (12ql3ql4, specifically i n v o l v i n g the GLI gene), neuroglial tumors (12ql3-ql5), n o n - s m a l l cell l u n g cancer (12ql4-q21), and squamous cell carcinomas of the head and neck (12ql3-ql4) [153-176].  28 Point Mutations Another mechanism by w h i c h a proto-oncogene  can be converted to a n  oncogene is by single base pair substitutions (mutations), w h i c h can have effects o n the translated protein.  These mutations  drastic  can arise due to replication  errors, or from direct D N A damage such as ultraviolet radiation [42]. The RAS gene family (HRAS, KRAS, and NRAS)  have all been found to contain point m u t a t i o n s  i n various malignancies [177-179]. These point mutations result i n the constitutive activation of the R A S molecule by ablating its need for a guanine  nucleotide  exchange factor thus m i m i c k i n g a constitutively activated growth factor receptor, such as E G F R or P D G F R [177-182]. second exon of the HRAS1 residue i n a melanoma  Sekiya et al. found a point mutation i n the  gene substituting an adenine residue to a t h y m i n e  [183].  Activation of the NRAS  gene by point m u t a t i o n  occurs i n about 15% of all h u m a n melanomas [184]. In these cases, mutated  NRAS  was found to contribute to tumor growth b y enhancing cellular proliferation and by blocking apoptosis. Other changes observed include: g l y l 2 v a l (bladder carcinoma), glyl2asp (mammary carcinosarcoma), gln611eu (lung carcinoma), and gln61 to arg (renal pelvic carcinoma) for the HRAS  gene, and gln61 to arg (lung carcinoma) for  the NRAS gene [185]. C h r o m o s o m a l Rearrangements Finally, rearrangement, promoter  proto-oncogene  activation  can  arise  when  a  chromosomal  such as a translocation, places a proto-oncogene downstream of a  of an IgG locus.  This results i n the  constitutive  expression of a n  otherwise tightly regulated gene. A n example of this is seen w i t h the M Y C gene and  29 its translocation to an IgG locus i n Burkitt's l y m p h o m a due to a t(8;14) [186]. Alternatively a translocation can produce a fusion gene where part of gene A is fused to part of gene B (this topic w i l l be discussed i n further detail below). Table 1 summarizes the more w e l l k n o w n oncogenes.  1.4.2  T u m o r Suppressor Genes  Generally, tumor suppressor genes are responsible for inhibiting cellular growth. M u l t i p l e molecular approaches that have typically been used to identify tumor suppressor genes, including: cytogenetic analysis to determine the extent of chromosomal loss (deletions) or rearrangements;  linkage analysis to  determine  w h i c h region-specific markers are linked to the disease (which helps lead to the identification of the gene); loss of heterozygosity (LOH) analysis, w h i c h detects the loss of an allele or other molecular marker. Oncogenic transformation of a cell can also occur if both copies of a tumor suppressor gene are inactivated. Inactivation of tumor suppressor genes can occur v i a a variety of mechanisms i n c l u d i n g loss of the gene (deletion) or mutation (point, missense or nonsense) and must include both copies. Some well characterized examples of tumor suppressor genes include APC (discussed above), RBI, and p53. A brief discussion o n their inactivation w i l l n o w be discussed using RBI and p53 as examples. RBI The retinoblastoma gene, RBI (see cell cycle above), is a tumor  suppressor  since it is responsible for controlling the transition from G l to S i n the cell cycle. Retinoblastoma occurs w h e n both copies of the RBI gene have been inactivated  30  TABLE 1.  Various classes of oncogenes and their mode of action w i t h i n tumors.  From Vogelstein, 1998 [90].  Oncogene  Mechanism of Activation  Neoplasm  DNA transfection studies DNA transfection studies  Glioma/fibrosarcoma Kaposi's sarcoma Stomach carcinoma  Growth Factors vsis  KS3 HST  Tyrosine Kinases: Integral Membrane Proteins, Growth Factor Receptors EGFR v-fins  Amplification  TRK NEU  Rearrangement Point mutation Amplification  v-kit v-ros  Squamous cell carcinoma Sarcoma  Sarcoma Sarcoma Colon carcinoma Neuroblastoma Carcinoma of breast  Tyrosine Kinases: Non-receptor SRC  Colon carcinoma  v-yes v-fgr v-fes  BCR/ABL  Sarcoma Sarcoma Sarcoma Chromosome translocation  Chronic myelogenous leukemia  Membrane Associated G Proteins H-RAS K-RAS N-RAS  Point mutation Point mutation Point mutation  Colon, lung, pancreas carcinoma AML, thyroid carcinoma, melanoma Carcinoma, melanoma  Rearrangement  Diffuse B-cell lymphoma Osteosarcomas  GEF Family of Proteins Dbl Ost  Serine/Threonine Kinases: Cytoplasmic v-mos v-RAF Pim-1  Sarcoma Sarcoma Proviral insertion  T-cell lymphoma  Gene amplification Gene amplification  Neuroblastoma: Lung carcinoma Carcinoma of lung  Nuclear Protein Family v-myc N-MYC L-MYC  v-myb v-fos v-jun v-ski v-rel v-ets v-erbA  Carcinoma myelocytomatosis  Myeloblastosis Osteosarcoma Sarcoma Carcinoma Lymphatic leukemia Myeloblastosis Erythroblastosis  31 [187-192]. Patients w i t h germline mutations i n one allele of RBI are predisposed to other malignancies i n c l u d i n g osteosarcomas, soft tissue sarcomas and m e l a n o m a later i n life [90].  L o h m a n n et al. investigated  a series of isolated u n i l a t e r a l  retinoblastomas from 119 patients for the frequency and nature of germline  RBI  gene mutations [193]. Of the 119 patients studied, 99 (83%) contained mutations for the RBI  gene.  The types of mutations  found included large deletions  (15%),  translocations (26%), and base substitutions (42%). p53 The p53 protein exists as a tetramer and acts as a tumor suppressor  gene  because it can inhibit tumor growth w h e n introduced into a variety of transformed cells by blocking cells from entering the S phase of the cell cycle [194-197].  Other  evidence has shown that p53 also blocks the transition from G2 to M v i a binding to CIP1 [128,198-200]. Mutations i n the p53 gene represent the most frequently encountered  genetic  aberrations i n human malignancies [90, 201-204], Vogelstein and Kinzler outlined 5 mechanisms for p53 inactivation [109]. Deletion of one or both p53 alleles reduces the expression of tetramers, resulting i n decreased expression of growth i n h i b i t o r y genes such as CIP1. Nonsense or splice site mutations that result i n truncation of the protein inhibit oligomerization, thus resulting i n a similar reduction of p53 tetramers. Mutations of this type are fairly c o m m o n i n l u n g [205], esophagus [206], and other cancers [207]. A third mechanism involves missense mutations resulting i n dominant-negative effects w i t h an even greater reduction of functionally active tetramers. Such missense mutations are c o m m o n i n colon [208, 209], brain [210],  32 lung  [205], breast [207], skin  [211, 212], and bladder cancers  [213].  A  fourth  mechanism by w h i c h p53 is involved i n oncogenesis is common i n cervical cancers where the expression of the E6 gene of human papillomavirus ( H P V ) results i n the functional inactivation of p53 through binding and degradation [214]. Patients w i t h germline p53 mutations are predisposed to breast cancers, sarcomas, brain tumors, lymphomas and Li-Fraumeni syndrome [215-217]. The p53 pathway may also be disrupted by alteration of its negative regulator, MDM2  (see above).  This  gene was  originally  identified by virtue  amplification i n a spontaneously transformed mouse cell line [218]. The  of  its  MDM2  gene is amplified (seen cytogenetically as HSRs and dmins) i n a significant fraction of the most c o m m o n h u m a n  sarcomas and the consequent  overexpression of  M D M 2 is likely to interfere w i t h p53 activity [219]. Table 2 summarizes the t u m o r suppressor genes discussed above as w e l l as a few others including their f u n c t i o n and localization w i t h i n cells.  1.5  G E N E T I C A S P E C T S O F P E D I A T R I C SOLID T U M O R S The presence of activated oncogenes and the deletion of tumor  suppressor  genes as discussed above has been reported i n many pediatric solid  tumors.  Pediatric solid tumors are k n o w n to contain various chromosomal  aberrations,  many of w h i c h can be detected by conventional cytogenetics.  aberrations  These  include whole chromosome losses and gains (aneuploidy), translocations, HSRs and dmins (discussed above), deletions, duplications, ring chromosomes, and marker chromosomes.  inversions  Aneuploidy, amplifications (dmins and HSRs) and  33  Table 2. Tumor suppressors and the tumors affected by their loss. F r o m Vogelstein, 1998 [90].  SYNDROME Retinoblastoma  GENE RBI  TUMORS Ret, Ost  Li-Fraumeni  p53  Familial adenomatous polyposis  APC  Sar, breast and brain tumors Adenomatous polyps, C C  N F Type I  NF1  Neurofibromas, sar, gli  Nucleus  N F Type II  NF2  Schwannomas, meningiomas  Cytoplasm,  Familial breast cancer  BRCA1  LOCALIZATION F U N C T I O N Nucleus T F / c e l l cycle control T F Nucleus Cytoplasm  Possible Bcatenin regulator p21RASGTPase activator Cytoskeleton membrane link D N A repair  Breast and ?Nucleus ovaries BRCA2 Breast and ?Nucleus D N A repair ?other W i l m s ' tumor WT1 Nephroblastoma N u c l e u s TF Abbreviations. Ret; retinoblastoma, Ost; osteosarcoma, TF; transcription factor, sar; sarcoma, g l i ; glioma, N F ; neurofibromatosis, C C ; colon cancer, ?= not k n o w n .  34 translocations  are the  most common  pediatric soft tissue tumors.  recurrent  abnormalities  associated  with  Since gene amplification was discussed i n section above, the following discussion w i l l concentrate o n aneuploidy and translocations i n pediatric solid tumors. Table 3 summarizes the common recurring abnormalities found i n various soft tissue tumors.  1.5.1  Aneuploidy  Every cell i n the h u m a n  body (except for sperm and ova) contains  46  chromosomes. There are 22 autosomes (1 through 22) and two sex chromosomes (X and Y). Every normal cell contains two copies of each of the autosomes chromosomes) and either a pair of X chromosomes chromosome (male).  (44  (female) or an X and a Y  Since there are two copies of each chromosome  (except i n  male individuals w h o have only one X and one Y chromosome) the D N A content is said to be d i p l o i d or 2n (n equals the h a p l o i d content of the cell).  Aneuploidy  refers to the abnormal amount of genetic material o n the chromosome level [90, 144]. W h o l e chromosomes or their arms may be lost or gained and this is easily detected using conventional cytogenetics. Some examples of soft tissue tumors that display aneuploidy are embryonal rhabdomyosarcomas (gains i n chromosomes 2, 8, 12, 13 and 20 as well as a loss of material from H p l 5 . 5 ) [220], leiomyosarcomas (frequent losses i n lOq and 13q and frequent gains i n 17p) [221], mesotheliomas (frequent deletions of specific regions w i t h i n chromosome arms l p , 3p, 6q, 9p, 15q and 22q) [222], prognostically poor neuroblastomas (deletions i n the short arm of  35 T A B L E 3. Summary of the various recurring chromosomal abnormalities found i n pediatric soft tissue tumors. Adapted from Enzinger, 1988 [1].  HISTOLOGY Clear cell sarcoma  CYTOGENETICS t(12;22)(ql3;ql2) t(7;18)(pll.2;q21.3), +der(7)t(7;18)(pll.2;q21.3) +8, +der (8;17)(ql0;ql0), t(12;22)(ql3;ql2.2-12.3) t(17;22)(q22;ql3), ring derived from t(17;22) t(ll;22)(q24;ql2) t(l;16)(qll;qll.l) t(21;22)(q22;ql2) t(7;22)(p22;ql2) t(9;22)q(22-31;qll-12), -Y +2, +8, +11, +17, +20 Translocation at 12ql3 t(ll;22)(pl3;ql2) /  /  Dermatofibrosarcoma protuberans E w i n g sarcoma  /  /  /  Extraskeletal m y x o i d chondrosarcoma Infantile fibrosarcoma Hemangiopericytoma Intraabdominal desmoplastic small round cell tumor Leiomyosarcoma Malignant fibrous histiocytoma H i g h grade Myxoid Malignant peripheral nerve sheath tumor Neuroblastoma  Rhabdomyosarcoma Alveolar Embryonal Schwannoma Synovial Sarcoma D e s m o i d tumor  Deletion of l p Complex R i n g Chromosomes Complex del(l)(p32-36), der(l)t(l;17)(p36;?), dmins and HSRs (amplification of NMYC)  t(2;13)(q35;q34), t(l;13)(p36;ql4) +2q, +8, +20 -22 t(X;18)(pll;qll) +8, +20 deletion of 5q Lipoblastoma t(7;8)(q31;ql3) Lipoma t(l;12)(p33-34;ql3-15), t(2;12)(p2223;ql3-15), t(3;12)(q27-28;ql3-15), t(5;12)(q33;ql3-15), t(ll,T2)(ql3;ql315),t(12;21)(ql3-15;q21), t(ll;22)(q24;ql2), del(12)(ql3ql5), del(13)(ql2-q22) Liposarcoma (myxoid) t(12;16)(ql3;pll) Uterine l e i o m y o m a t(12;14)(ql5;q24), deletion of 7q, +20 Abbreviations. Del= deletion and der= derivative.  36 chromosome  1), desmoid tumors (gains i n chromosomes 8 and 20) [27-29] and  uterine leiomyomas (deletions of the long arm of chromosome 7) [223]. One possible role of an extra copy of a chromosome is to introduce an extra copy of a growth related gene (growth factors or their receptors) or oncogenes (such as RAS, PDGF, and MYC), leading to an increase i n the proliferative rate of the cell. Loss of a chromosome  or a chromosome  region could lead to a proliferative  advantage for a cell if tumor suppressor genes (such as APC, p53, and RBI) were deleted. In addition to the examples provided above, there are other tumors that may have many extra copies of several chromosomes. A n example of this is uterine leiomyosarcoma w h i c h can have up to 8 copies (8n) of almost every chromosome i n contrast to two copies (2n) i n normal cells [224].  1.5.2  Tumor Specific Translocations  A translocation refers to the exchange of chromosomal material between two chromosomes.  A translocation can involve either a part of a chromosome arm or  the entire arm itself. Robertsonian translocations result i n the fusion of the l o n g arms of two acrocentric chromosomes (chromosomes 13-15 and 21 and 22) w i t h the subsequent loss of the short arms [144]. Translocations can occur i n either transcriptionally active or inactive regions. The translocation results i n the formation of two derivative chromosomes (if the translocation is reciprocal). A derivative chromosome is a structurally rearranged chromosome  generated  either  by a rearrangement  involving  two  chromosomes or b y multiple aberrations w i t h i n a single chromosome.  or  more  37 The majority of reciprocal translocations result i n derivative chromosomes that give rise to no visible phenotypic change [225]. This can be explained by the fact that the breakpoint may fall w i t h i n D N A that does not contain any genes or is n o t expressing any genes. The expression of the genes around the breakpoint therefore, are not affected. Alternatively, there are two possibilities where the translocation can give rise to derivative chromosomes associated w i t h a phenotypic change. One possibility involves  the  breakpoint  occurring i n  transcriptionally  inactive D N A , as above, except that this region is responsible for the expression of nearby genes. The derivative chromosomes now contain a part of chromosome ' A ' fused to a part of chromosome ' B ' . Since the translocation w i l l affect the expression of nearby genes, the function of these genes w i l l determine the viability of this rearrangement.  For example, if the translocation results i n the overexpression of  nearby genes and one of these genes is an oncogene (e.g., MYC, PDGF or RAS)  then  the cell w i l l most likely have an increased proliferative rate. O n the other hand, i f the expression of the genes is suppressed and one of the genes i n v o l v e d is a t u m o r suppressor gene (e.g., ARC, pl6 or p53), then the proliferative rate may increase as above. If the gene(s) involved i n the latter case is i n v o l v e d i n cell s u r v i v a l such as the glucose-6-phosphate  molecule (necessary for glucose metabolism), then  the  phenotype may be lethal (see Fig. 4a). The most interesting result of a translocation occurs w h e n the breakpoints occur w i t h i n expressed sequences (see Fig. 4b). T h i s type of translocation is relatively c o m m o n i n soft tissue tumors [90, 224, 226, 227], where a chimeric gene fusion is formed due to the translocation and w i l l n o w be discussed i n more detail below.  38  FIGURE 4. The possible outcomes of chromosomal translocations. a) Translocations can occur in either transcriptionally inactive or active DNA. The phenotype of the former is usually nothing, unless the transcriptionally inactive region is involved in regulating the expression of nearby genes. The latter possibility can result in a deletion of a gene, the introduction of a stop codon, a non functional protein or a functional chimeric protein which fuses part of gene one to part of gene 2. b) Chimeric gene fusions can form as a result of an inter-exonic gene fusion or an intra-exonic gene fusion. The fusion gene can be oncogenic if the regulation of specific domains involved in the fusion are lost.  39  41 1.5.3  Tumor Specific Translocations Result in Functional Gene Fusions in  Solid Tumors Hematopoietic tumors helped pave the w a y to understanding chimeric genes as a result of specific chromosomal translocations [228-230]. These translocations almost always result i n a proliferative advantage for the malignant cell and this is accomplished by one of two mechanisms. The first possibility is that the translocation splices an oncogene to a positive regulatory element  of the partner gene.  The result is overexpression of the  oncogene leading to an increased growth rate for the malignant cell. A n example of this is seen i n Burkitt's l y m p h o m a due to the t(8;14) w h i c h places the M Y C oncogene under the control of the IgH locus promoter [229, 231, 232]. A s i m i l a r situation exists i n the solid tissue tumor dermatofibrosarcoma protuberans (DFSP). In this tumor the expression of the P D G F - B chain molecule is placed under control of the COL1A1  gene promoter  [233].  constituent of the connective tissue matrix.  the  The C O L 1 A 1 product is a major  The increased expression of P D G F - B  contributes to tumorigenesis since it has transforming activity and is a potent mitogen for a number of cell types [234-237]. Its role i n the oncogenic process however is not fully understood. The second possibility involves the juxtaposition of a portion of one gene to a portion of another gene. The breakpoints can occur w i t h i n an intron or an exon, but the end result is usually the i n frame fusion between the exons of two genes. These chimeric gene fusions contain functional domains from both genes and are largely responsible for the increased proliferation seen i n many soft tissue t u m o r s  42 and leukemias  [238].  Many  sarcomas  express  chimeric  transcription  factors  resulting from gene fusions [229]. Since our studies have identified a gene fusion in  congenital fibrosarcoma  as a result  of a chromosomal  translocation,  the  remainder of this chapter w i l l concentrate on a few examples of solid tumors  and  their associated gene fusions. Ewing's Sarcoma Family of Peripheral P r i m i t i v e N e u r o e c t o - D e r m a l Tumors E w i n g sarcoma, peripheral neuroepithelioma and A s k i n tumor are a group of malignancies w h i c h are poorly differentiated  and belong to the  family of  peripheral Primitive Neuroectodermal Tumors (pPNETs) [5, 239]. Ewings sarcomas (ES) and peripheral neuroepithelioma contain a t(ll;22)(ql2;q24) w h i c h juxtaposes the FLU gene from chromosome l l q l 2 to the EWS gene from chromosome 22q24 i n approximately 85% of cases [240-246]. The FLI1 gene is a member of the ETS family of transcription factors and is k n o w n to contain at least two functional  domains  [247, 248]. One of the domains, a helix loop helix d o m a i n ( H L H D ) , is responsible for protein-protein  dimerization while the other  domain, the  domain, is responsible for binding to D N A . The EWS binding  domain  translocation  and  results  a transcriptional i n der(22) giving  activation  ETS D N A b i n d i n g  gene contains an R N A  domain  rise to a fusion  [249, 250].  gene containing  The the  transcriptional activation domain from E W S as the 5'end of the fusion gene and the ETS D N A binding domain from the FLI1 gene as the 3' end (see Fig. 5) [244, 251]. Studies have shown the fusion product to be a transcriptional activator due to the swapping of the R N A binding domain from the EWS gene w i t h the D N A binding  43  •rt  H  cu  c  S S  H  Q CQ  Q  S  H  S  fi Q  ttJ  rou.c^uxs^g rocS-&£w>c^ crogrorofi-S^ o " Ji ^ * u  M  DC  £ bp| £ § < |  c  cn  j_  s  £  'y  H  lie |  H  n  O  a 11  roo3~(tla»' ro'ii >  o _ , r o - t c y j j < 2 i  2  <y CO n fi  o  CA CU  I  J3 v 1  v  £Txs v  w  cn  H w  O  ro  WO  £ M •§ 3  -a cu  o  2 £ u ts o, ° fi -3  <u  w  ro LO  ^  .2 X JB rM £ 53 fi  cn  H w  cn  •Ti  ro CO  ^ ro ro yg o  ,  ^  -;H  cu  S o . fi o .fi •a y -2 fi o ro  S Sa S §  44 d o m a i n from the FLU gene [252]. This places the D N A binding d o m a i n from F L U under the promoter elements of the E W S gene, w h i c h effectively disrupts normal activity of the D N A binding domain.  It is therefore  thought  the  that this  altered D N A b i n d i n g activity somehow gives rise to an increased proliferative rate. One possibility is that the chimeric fusion protein may be able to activate c - M Y C , since both E W S / F L I 1 and F L U are capable of activating the c - M Y C promoter [253]. The elucidation of the exact mechanism of pathogenesis, however, w i l l require further investigation. The majority of the remaining 15% of p P N E T s appear to harbor a variant t(21;22)(q22;ql2), w h i c h has been shown to express an EWS-ERG chimeric transcript [247, 254, 255]. The fusion protein acts as a transcriptional activator and requires the amino terminal domain of E W S [256]. In addition, since the clinical behavior of the p P N E T s w i t h an EWS-FLI1 or an EWS-ERG fusion appear similar [254, 257], it is thought that the fusion proteins may be acting along similar oncogenic pathways. Another rare variant translocation has been discovered i n E w i n g sarcoma where a t(7;22)(p22;ql2) results i n the fusion of the 5' portion of the EWS gene to the 3' D N A binding domain (ETS domain) of the ETV1 gene, another member of the ETS family of transcription factors.  It is still not k n o w n whether this variant functions i n  similar ways as do the EWS-FLI1 and E W S - E R G chimeras. The EWS gene has been i n v o l v e d i n at least two other translocations i n v o l v i n g the ATF1 and WT1 genes, however  these fusions occur i n malignant melanoma  of soft parts and intra-  abdominal desmoplastic small round cell tumor, respectively.  45 M y x o i d Liposarcoma Liposarcomas are malignant tumors of fat cells that primarily occur i n the extremities and retroperitoneum of adults [1]. Of the various types of liposarcomas, m y x o i d liposarcoma (myxoid LPS) is the most common.  Several independent  groups characterized the t(12;16)(ql3;pll) i n myxoid L P S and found that the FUS gene from chromosome 1 6 p l l is fused to the CHOP gene from chromosome [258-262].  In addition, round cell liposarcoma, a poorly differentiated  12ql3  form of  m y x o i d LPS, was also found to harbor the same gene fusion [263]. The FUS gene (also k n o w n as TLS (for "translocated i n /iposarcoma") is a nuclear R N A - b i n d i n g protein w i t h extensive homology to EWS  [259]. M o r e o v e r ,  the amino terminus of F U S appears to contain a strong transcriptional activation domain as found i n EWS [264]. A m a n et al. found that the size of the FUS gene is 11 kb and consists of 15 exons [261]. The CHOP gene is a nuclear protein and acts as a dominant-negative transcription factor inhibitor for C / E B P and L A P [265].  CHOP  stands for " C / E B P - / i o m o l o g o u s protein", but it has also been referred to as DDIT3 and GADD153 for " D N A damage-inducible transcript 3" and "growth arrest and D N A damage-inducible  gene", respectively.  C H O P inhibits the  transcriptional  activity of C / E B P and L A P by heterodimerizing w i t h them through the leucine zipper domain,  thus interfering  w i t h their  D N A binding capabilities.  This  interaction is mainly seen i n response to cellular stress, such as D N A damage [265]. In addition, C H O P is thought to function i n pathways of terminal and growth arrest i n fat cells [266].  differentiation  46 The t(12;16) results i n the fusion of the F U S amino terminus (FAT) to the entire C H O P coding region [258, 259]. Transformation of N I H 3 T 3 cells requires both the F A T portion as well as the entire C H O P region [264]. Furthermore, the carboxy terminal leucine zipper d o m a i n from C H O P was essential for transformation. Based o n these results it w o u l d be reasonable to assume that the genes regulated by C / E B P and L A P are tumor suppressor genes and the constitutive i n h i b i t i o n of their expression by F U S - C H O P could lead to oncogenesis. In addition to the FUS-CHOP  fusion, the FUS gene was found to be  rearranged i n an acute myeloid leukemia w i t h a t(16:21)(pll;q22) [267, 268].  The  fusion partner was identified as E R G , seen previously i n E w i n g sarcoma as part of an E W S - E R G gene fusion. E R G expression i n both acute m y e l o i d leukemia and i n E w i n g sarcoma is therefore under the control of either the FUS or EWS  promoter  elements, respectively [269]. A m a n et al. states that although the N - t e r m i n a l ends of F U S and E W S are different, they share extensive homology and are distinct f r o m the N-terminal regions of other ribonucleoprotein (RNP)-carrying proteins [269]. A l v e o l a r Rhabdomyosarcoma Rhabdomyosarcomas (RMS) are a heterogeneous group of malignant t u m o r s and are the most c o m m o n soft-tissue sarcoma of childhood [220]. There are three subtypes: embryonal R M S (ERMS) (~ 65% of R M S cases), alveolar R M S ( A R M S ) (~ 20% of R M S cases), and the less well-defined undifferentiated sarcomas (~15% of R M S cases) [5, 270]. Differentiation between these subtypes as w e l l as other poorly defined sarcomas poses a problem to the pathologist, once again illustrating the need for more accurate tools for diagnosing these tumors.  47 Alveolar R M S cases harbor either a t(2;13)(q35;ql4) (-60% of A R M S cases) or a t(l;13)(p36;ql4) (-10-20% of A R M S  cases) w h i c h fuses the paired box (PB) and  homeobox (HB) D N A binding domains  from either  the PAX3  respectively, to the acidic and proline rich domain of the FKHR  or PAX7  genes,  gene [271-273]. T h e  PAX3 and PAX7 genes are members of the P A X family of transcription factors w h i c h means the fusion gene is a chimeric transcription factor. The PAX genes appear to be important for myogenic differentiation during embryonic development  [274-276].  FKHR belongs to another transcription factor family k n o w n as the forkhead family of transcription factors [277]. F K H R is k n o w n to contain a D N A b i n d i n g d o m a i n as w e l l as a carboxy terminal transactivation domain.  The D N A binding d o m a i n of  F K H R is of the winged-helix type. The fusion product between P A X 3 or P A X 7 and FKHR  has the  D N A binding domain  transactivation domain from F K H R . oncogenic transformation  from  P A X 3 or P A X 7 attached to  the  The overexpression of PAX genes leads to  [278]. This has lead researchers to hypothesize that the  transcriptional disruption of normal PAX target genes due to the PAX-FKHR  gene  fusions plays a major role i n A R M S oncogenesis. Synovial Sarcoma Synovial  sarcoma  is  an  aggressive  soft  tissue  malignancy  occurring  predominantly i n the extremities of adolescents and young adults [1]. A recent case was misdiagnosed as a desmoplastic small round-cell tumor  providing  further  evidence for the need of a molecular based approach to diagnosing m o r p h o l o g i c a l l y similar tumors [279].  The recently characterized t(X;18)(pll.2;qll.2) is found  in  approximately 90% of h u m a n synovial sarcomas [280-282]. This results i n a der(X)  48 chromosome w h i c h gives rise to the fusion of the chromosome 18 SYT gene (also k n o w n as SSXT) located at 18qll.2, to either of 2 distinct genes (at least 2Mb apart from each other at the X p l l . 2 locus) from X p l l . 2 , SSX1 or SSX2 [283-286]. studies have shown that the SYT protein contains a transcriptional  Recent  co-activator  domain [287]. The N-terminus of the SYT protein has a novel conserved 54 a m i n o acid d o m a i n ( S N H domain) w h i c h has been observed i n a wide variety of species. The C-terminal domain is rich i n glutamine, proline, glycine and tyrosine Q P G Y domain), w h i c h harbors transcriptional  activator  sequences.  (the  Mutagenic  analysis of the SYT gene has shown an increase i n transcriptional activation along w i t h the deletion of the S N H domain, suggesting that this d o m a i n acts as a n inhibitor of the activation domain [287]. The mouse homologue of SYT, Syt, was isolated and sequenced i n full by de Bruijn et al. [288]. They found that during early embryogenesis, mouse Syt is ubiquitously expressed.  Later, expression becomes  confined to cartilage tissues, specific neuronal cells, and some e p i t h e l i u m - d e r i v e d tissues and i n primary spermatocytes. The SSX1 and SSX2 proteins are 81% homologous and are rich i n charged amino acids [289]. Due to the high homology between the molecules, it is likely that the function of SYT-SSX1 and SYT-SSX2 is similar.  The SSX molecules contain a  transcriptional co-repressor domain, k n o w n as the Kruppel-associated box ( K R A B ) , at their N-terminus [290-292]. K R A B domains have been previously identified o n l y i n Kruppel-type zinc finger proteins, e.g., zinc finger protein-117 and -83 (ZNF117 and ZNF83).  49 The translocation i n synovial sarcomas fuses the transcriptional  activating  domain from the SYT gene to the transcriptional repressor d o m a i n from the SSX gene. Crew et al. found these transcripts i n 29 of 32 (91%) synovial sarcomas, thus p r o v i d i n g pathologists w i t h a useful diagnostic tool [286]. Further analysis of the breakpoints lead to the identification of 2 distinct fusion junctions for each of SYTSSX2 and SYT-SSX1  fusion transcripts [286]. Since then, additional variants h a v e  been characterized, differing i n the placement addition, subcellular localization studies have  of the breakpoint shown  [293, 294].  In  that SYT and S Y T - S S X  proteins co-localize w i t h the human homologue of the S N F 2 / B r a h m a protein B R M i n the nucleus. The function of SNF2 i n mammals is u n k n o w n , but evidence i n S. cerevisiae  and D. melanogaster  transcription [295].  In vitro  suggests that it may act as a global activator of studies have further  s h o w n that these molecules  interact w i t h each other [287]. This implies that the SYT-SSX fusion protein may have a role i n activating or inhibiting the normal role of h u m a n homologue of SNF2.  1.6  A I M S A N D OBJECTIVES We focussed on the genetics of congenital fibrosarcoma (CFS) and adult-type  fibrosarcoma (ATFS) for three reasons. Firstly, C F S and A T F S are considered to be malignant lesions. Secondly, CFS and A T F S (but particularly CFS) are very difficult to differentiate from benign fibroblastic proliferations of childhood such as infantile fibromatosis (IFB) and aggressive fibromatosis (AFB) due to significant phenotypic overlap and similar age distributions. Lastly, they appear to differ markedly f r o m  50 each other i n behavior and response to therapy, making a reliable marker for their distinction extremely important from a clinical perspective. W i t h this i n m i n d , o u r goal was to identify and characterize recurrent  genetic alterations  i n cellular  fibroblastic tumours of childhood that w o u l d be useful for the pathologic and prognostic classification of members of this tumor subgroup. Cytogenetic analysis of CFS cases to date have only shown non random abnormalities i n v o l v i n g chromosomes, such as trisomy of chromosome  11.  whole  The cytogeneticists at B . C .  Children's Hospital identified an apparently non-random abnormality i n v o l v i n g the chromosomal regions 12pl3 and 15q25. The specific objectives of the work described i n this thesis were to: 1. Characterize, at the molecular cytogenetic level, the consequences of the 12pl3 and 15q25 aberrations by fluorescence i n situ hybridization. 2. Clone  the  chromosomal  breakpoints  and  characterize  the  genes  involved. 3. Characterize the ETV6-NTRK3 4. Characterize the E T V 6 - N T R K 3  gene fusion at the c D N A level. protein product (molecular weight,  hetero- and homodimerization status, and phosphorylation status) 5. Elucidate the downstream protein interactions of E T V 6 - N T R K 3 .  51  CHAPTER II MATERIALS AND METHODS  2.1  CLINICAL FEATURES OFINDEX CASE  A  term baby girl  (TB) was found  discoloration i n the left lumbar region.  at birth to display a  quarter-sized  Over the course of four weeks the mass  grew i n size to 7 x 5 cm. Initial biopsy was diagnosed as a congenital fibrosarcoma by a dermatologist. This diagnosis was confirmed by pathologist review. A C T scan and ultrasound confirmed the absence of any deep tissue extension.  Surgery  revealed a fleshy grey colored mass w h i c h was resected w i t h overlying b l u i s h tinged skin.  The deep margin was at the posterior superior iliac spine and was  negative for tumor. There was no further treatment administered post surgery. T B was followed yearly and has since shown no recurrence at the local site and chest X rays have been clear.  2.1.1  Pathology of Index Case  The gross specimen consisted of a large tan-brown mass w i t h  minimal  evidence of hemorrhage or necrosis. Histologic analysis revealed a cellular spindle cell lesion i n w h i c h there was nuclear  pleomorphism  and moderate  mitotic  activity. Cells showed no obvious evidence of specific differentiation other t h a n having  the  appearance of possible fibroblastic origin.  immunohistochemical  There  was  positive  staining for v i m e n t i n , but not for muscle specific actin,  desmin, S100, histiocytic markers, or endothelial markers. Ultrastructural analysis  52 showed  no  rhabdomyoblastic  fibroblastic origin.  differentiation,  but  also  suggested  a  possible  Based o n these features, the specimen was diagnosed as a  congenital fibrosarcoma (see Fig. 6a). This appearance is contrasted w i t h infantile fibromatosis (see Fig. 6b), w h i c h shows similar, but less pleomorphic cells, lower mitotic activity and increased cellular matrix between cells.  2.1.2  Cytogenetic Analysis of Index Case  Cytogenetic analysis of cultured tumor tissue of the initial C F S case showed an abnormal karyotype i n all cells examined as indicated i n Figure 7.  A l l cells  examined showed additional copies of chromosome 11, w i t h a few cells i n c l u d i n g chromosomes  +20  and  +2.  There  were  structural  additional material on the long arm of chromosome  abnormalities  including  1, apparent deletion of the  distal portion of the short arm of chromosome 12, and a rearrangement of the l o n g arm of chromosome 15. The final cytogenetic assessment of 24 metaphases of this case was as follows: 46-49, X X , - X , add(l)(q43), +2, +11, del(12)(pl3), add(15)(q25), +20.  2.2  C L I N I C A L S A M P L E S . TISSUE C U L T U R E T E C H N I Q U E S A N D CYTOGENETIC ANALYSIS A l l CFS, C M N , A T F S , and IFB cases analyzed i n this study were collected  from either British Columbia's Children's Hospital, Childrens Hospital of Los Angeles,  National Wilms'  Tumor  Study Group ( N W T S G )  tumor  bank,  or  Cooperative H u m a n Tissue Network ( C C H T N ) at Columbus Children's H o s p i t a l , Columbus, Ohio and presented during the period 1988 to the present. The cases  53  F I G U R E 6. Histologic analysis of CFS and IFB. A, 40X magnification of a C F S case stained w i t h hematoxylin and eosin (H&E). Note the characteristic highly cellular spindle morphology (bluish-purple), mitotic figures and nuclear pleomorphism. B, 40X magnification of an IFB case stained w i t h H & E . Note the lower density of spindle cells (bluish-purple) and an increase i n the amount of extracellular collagenous matrix (pink) relative to C F S .  55 F I G U R E 7. G-banded karyotype of index case. A total of 24 metaphases were analyzed by B.C. Children's Hospital's cytogeneticists. The following karyotype displays 48 chromosomes, X X , add(l)(q43), +11, del(12)(pl3), add(15)(q25), and +20. Three of four C F S cases analyzed showed recurring anomalies including addition of chromosome 11 (vertical arrow), apparent deletion of 12pl3 (angled arrow), and a rearrangement of 15q25-qter (horizontal arrow).  57 were analyzed by short term culturing and cytogenetic analysis by cytogeneticists at either  B.C. Children's Hospital or Childrens Hospital of L . A . according  established protocols [296, 297].  Briefly, excised tumor  to  tissue was minced i n  collagenase (200 u n i t s / m l , Sigma) and incubated for 2 hours.  Washed cells were  incubated i n 60 m m plastic petri dishes i n R P M I 1640 m e d i u m w i t h L - g l u t a m i n e (Gibco BRL) supplemented w i t h 15 or 20% fetal bovine serum (FBS, Sigma), 5% antibiotic-antimycotic solution (Gibco BRL), and maintained i n this m e d i u m  at  i  37°C i n a 5% C 0 incubator. Short-term cultures used for cytogenetic analysis were 2  arrested i n metaphase w i t h Colcemid (1 n g / m l final concentration, Gibco BRL) for 3 to 4 hours prior to harvesting. After swelling the cells i n a hypotonic solution, the cells were fixed i n a 3:1 solution of methanol to acetic acid and dropped onto glass slides. G-banding techniques were used to stain metaphases previously fixed, dried, and treated overnight at 60°C on the petri dish surface.  Cells were harvested at  various passages and were used for cytogenetic analysis and as a source of D N A or RNA.  Frozen primary tumor specimens for molecular studies were stored at -70°C  prior to analysis. Karyotypes are described i n accordance w i t h the System for H u m a n Cytogenetic Nomenclature, 1995.  International  Karyotypes and  clinical  features for the 4 C F S cases initially analyzed cytogenetically are summarized i n Table 4. C e l l lines expressing N T R K 3 (kindly provided by Drs. B. N e l k i n  and D.  Kaplan) were g r o w n i n R P M I 1640 supplemented as above w i t h the addition of 200  58 T A B L E 4. Summary of cytogenetic analysis of initial B C C H CFS cases. Initial karyotype refers to the initial cytogenetic assessment of the cases while final karyotype refers to the cytogenetic interpretation after F I S H analysis. A final cytogenetic assessment on case 2 was not possible due to the absence of material for FISH.  CASE  INITIAL CYTOGENETICS  FINAL CYTOGENETICS  1  46-49, X X , - X , +11, +20, +2,  46-49, X X , - X , +11, +20, +2,  add(l)(q43), del(12)(pl3),  t(l;12;15)(q44;pl3;q25)  add(15)(q25) [cp 24] 2  48, X Y , +11, add(15)(q26), +21 [cp 16]  NA  / 48, X Y , +8, +11, add(15)(q26) [cp2] / 47, X Y , +11, add(15)(q26) [cp 2] 3  48, X Y , +11, +8, t(4;12)(pl0;ql0), add  48, X Y , +11, +8,  (15)(q25), t(12;15)(pl3;q26) [cp 21]  t(4;12;15)(ql2;pl3;q25)  Abbreviations. cp= number of cells evaluated, N A = not available, add= addition, del= deletion.  59 u g / m l of G418 (Gibco/BRL).  2.3  Y A C A N D COSMID PROBES Yeast artificial chromosomes  (YACs), mapping  w i t h i n specific areas of  interest were k i n d l y provided by Dr. S. Scherer from the Canadian Genome  Centre  Y A C Core Facility at the Hospital for Sick C h i l d r e n , Toronto,  Ontario.  These  included  738_B_11  890_E_3, 854_E_6,  chromosome  924_H_12,  817_H_1, 954_G_10,  from  12pl3, and 882_H_8, 895_H_10, 932_F_12 (chimeric), and 802_B_4  from chromosome 15q25-26. A l l Y A C s were either previously confirmed to be n o n chimeric [298, 299], or were confirmed to be non-chimeric normal metaphases i n our laboratory.  by F I S H analysis of  C o s m i d contig probes spanning the  ETV6  locus, i n c l u d i n g 179A6, 171H6, 45E12, 163E7, 54D5, and 148B6 [300], were generous gifts of Dr. Peter Marynen, University of Leuven. Y A C s and cosmids were g r o w n and maintained  i n our laboratory using standard methods [301].  labeled w i t h either biotin or digoxygenin using a commercially  Probes were available  kit  according to the manufacturer's instructions ( G i b c o / B R L ) .  2.4  D N A A N D R N A ISOLATION Primary C F S , IFB, or C M N tumor tissue, normal fibroblasts and N I H 3 T 3 cells  infected w i t h ETV6-NTRK3  retroviral constructs (kindly provided by Daniel W a i i n  our laboratory) were used as sources of D N A or R N A . Several other p r e v i o u s l y established cell lines were used as either positive or negative controls i n c l u d i n g the leukemia cell lines K562 [302] and Jurkat [303], the neuroblastoma  cell line  SAN2  [304], a E w i n g tumor cell line TC-71 [305], and the rhabdomyosarcoma cell line Birch  60 (established at St. Jude's Hospital, M e m p h i s , T N , Piper S, unpublished data). D N A was extracted from these cells using standard methods and an A p p l i e d Biosystems DNA  Extractor, M o d e l 340A [306].  Total R N A was extracted using the  acid  guanidium thiocyanate phenol/chloroform method [307]. D N A used for F I S H was isolated from either Y A C or cosmid clones (see section above). D N A from cosmids was extracted using a Plasmid M i d i K i t (Qiagen) according to the manufacturer's  instructions.  D N A from Y A C s was isolated by  previously established methods (D. W a r d , personal communications).  Briefly, a n  A H C plate was innoculated w i t h a Y A C clone and allowed to grow at 30°C for at least two days or until colonies were visible.  One red colony (the red colony  indicates the presence of insert w i t h i n the Y A C ) was then used to innoculate either 50 m l of minimal media or yeast extract, peptone, dextrose (YPD) media, w h i c h was subsequently shaken overnight at 30°C at 225 rpm. Cells were then centrifuged at 2000 r p m for 3 minutes and the pellet was resuspended i n 0.5 m l of I M sorbitol (Difco)/0.1M N a 2 E D T A ( p H 8) and 20 u l of Sigma Lyticase (Sigma)(12.5 m g / m l of Sigma Lyticase, prepared fresh, i n I M s o r b i t o l / O . l M N a 2 E D T A ( p H 8)).  The  solution was then transferred to an eppendorf tube, incubated at 37°C for 60 m i n , and centrifuged for 1 minute.  The pellet was resuspended (gently w i t h a pipet) i n  0.5 m l 50 m M Tris-Cl ( p H 7.4)/20 m M N a E D T A ( p H 8) before the addition of 50 u l 2  10% SDS. This mixture was incubated at 65°C for 30 minutes.  The SDS was  removed by adding 0.2 m l 5 M potassium acetate, incubating o n ice for 60 m i n u t e s and then centrifuging at 14,000 r p m at 4°C for 5 minutes. The supernatant was t h e n transferred  to a fresh  eppendorf  tube and  the  D N A was precipitated  with  61 isopropanol according to previously established methods [306].  The D N A was  RNase treated prior to hapten labeling for use as a F I S H probe  2.5  SOUTHERN A N D NORTHERN BLOT ANALYSIS Southern  and  Northern  blot  analyses  were  performed  as  previously  described [306]. For Southern analysis, lOug of genomic D N A from each case was digested w i t h Hindlll, BamHI, EcoRI, or Xhol. total R N A was used.  For N o r t h e r n analysis, at least 20ug  Briefly, a 0.8% agarose gel was used for Southern analysis,  while a 1.2% formaldehyde-agarose  gel was used for N o r t h e r n analysis.  D N A or  R N A was transferred onto n y l o n membrane filters ( H Y B O N D ) by capillary blotting and fixed to the membrane length ETV6  by baking at 80°C for 2 hours.  Probes included: f u l l  c D N A (a generous gift of Dr. Peter M a r y n e n , U n i v e r s i t y of L e u v e n ) ;  E T V 6 - 5 / 6 , consisting of ETV6 exons 5 and 6 (nt 825-1137) and generated by digesting ETV6  full length c D N A w i t h BamHI  and Pvul);  full  length NTRK3  generous gift of Dr. Barry N e l k i n , Johns H o p k i n s University); and consisting of NTRK3  followed  NTRK3-PTK,  nt 1740-2715 (including the P T K region), and generated by Xbal  digestion of full length NTRK3 (50uCi) using  c D N A (a  random  primer  cDNA.  Probes were radiolabeled w i t h [a- P]dCTP  extension  32  (Oligo Labeling K i t , P H A R M A C I A )  by n i c k - c o l u m n purification ( P H A R M A C I A ) .  The  D N A and R N A  membranes were hybridized overnight w i t h the radiolabeled probes after w h i c h they were washed and autoradiographed at -70°C using standard methods [306]. T o control for equal loading of R N A for N o r t h e r n analysis, membranes were stripped and reprobed w i t h a fi-actin D N A probe.  62 2.6  FLUORESCENCE  IN SITU H Y B R I D I Z A T I O N  (FISH) STUDIES  Cell suspensions of normal cultured lymphocytes, fibroblasts, and p r i m a r y C F S tumor cells were processed according to standard cytogenetic procedures [296] and stored at -20°C i n methanol/acetic acid fixative (3:1) until used.  Metaphase  chromosome spreads and interphase nuclei were prepared on glass microscope slides [296]. Slides for FISH were prepared by applying a drop of fixed cells onto a slide from approximately 1 foot above the slide, w h i c h was held at an angle of 45°. The slide was allowed to air dry and then aged by storing the slide i n a dessicating chamber overnight at room temperature.  Prior to use, the slides were passed  through a series of room temperature ethanol washes (2 minutes each i n 70%, 90% and 100% to dehydrate  the  slides).  The  chromosomes  were  denatured  by  submerging them i n 70% Formamide (Sigma)/2X SSC ( p H 7.0) for 2 minutes.  The  slides were then immediately r u n through a -20°C ethanol series as above.  The  slides were then allowed to air dry before the probe was applied. The D N A probes for FISH ( Y A C , cosmid, a n d / o r a-centromeric) were labeled with  either  Mannheim)  biotin-14-dATP  (Gibco/BRL) or d i g o x i g e n i n - l l - d U T P  using BioNick Labeling K i t (Gibco/BRL) and purified  precipitation according to the manufacturer's instructions,  (Boehringer by e t h a n o l  a-centromeric probes  were purchased prelabeled w i t h biotin or digoxigenin (Oncor). The addition of 5 Units of D N A Polymerase I (New England Biolabs) per labeling reaction was necessary for proper labeling of Y A C and cosmid D N A .  The purified probe (500 u g  of labeled Y A C or 50 ng of cosmid probe) was then dissolved i n Hybrisol V I I (Oncor), denatured at 75°C for 10 minutes and allowed to preanneal at 37°C for 30  63 minutes (for cosmids) or 1 hour (for Y A C s ) .  The preannealed probe was t h e n  applied directly to the denatured slides, sealed w i t h a coverslip and rubber cement (Canadian Tire) and allowed to hybridize for at least 16 hours at 37°C. After hybridization, the slides were washed i n 50% f o r m a m i d e / 2 X SSC ( p H 7.0) at 45°C for 5 minutes, and another wash i n 2X SSC at 45°C for 5 m i n u t e s . Detection of the signal then proceeded according to the manufacturer's instructions (Oncor).  The chromosomes were counter-stained i n d i a m i d i n o - 2 - p h e n y l i n d o l e  dihydrochloride hydrate (DAPI) (2ul of a 2 0 0 m g / u l stock i n 200ul of antifade; 20ul of this solution was applied to each slide) and visualized immersion  objective  through  a  Zeiss A x i o p l a n  using a 100X o i l  Universal  epifluorescence  microscope. Images were captured w i t h a C O H U H i g h Performance camera w i t h PSI Scientific Systems software (League City, TX).  Images were converted to TIFF  files and then processed through Adobe Photoshop 5.0 prior to printing.  2.7  3 ' A N D 5' R A P I D A M P L I F I C A T I O N O F c D N A E N D S (RACE) T w o micrograms of total R N A were used as starting material for 3 ' - R A C E .  ETV6 primers 541 and 701 [308] were used sequentially as sense primers i n 3 ' - R A C E experiments performed according to the manufacturer's  instructions  (3'-RACE  System; G i b c o / B R L ) . P C R conditions for both primers were as follows: 94°C for 1.5 minutes followed by 34 cycles of 94°C for 45 seconds, 58°C for 2 minutes, and 72 °C for 3 minutes, and a final extension of 72°C for 10 minutes. Products were analyzed on agarose gels, cloned using the Invitrogen T A C l o n i n g System (Version 1.3), and  64 sequenced o n an A B I A p p l i e d Biosystems 373A D N A Sequencer.  Sequences were  analyzed using D N A S T A R Sequence Analysis software. To determine if the 3' end of the ETV6 gene (the ETS D N A b i n d i n g d o m a i n ) was i n v o l v e d i n a gene fusion, 5 ' - R A C E was utilized using five micrograms of R N A as starting material. Primers used included: TELOUT:  5'-GCTGGGTAGTTTGTCTAAGGTGC-3'  TELMID:  5'-TGGTCTGCAAGAGAAGTGTCCCT-3'  TELIN:  5'-CAGGGCTCTGGACATTTTCTCATA-3'  Products were analyzed as described above. To determine whether the 5' end of the NTRK3 binding domain i n c l u d i n g the transmembrane  gene (extracellular l i g a n d  domain) was i n v o l v e d i n a gene  fusion, 3 ' - R A C E was utilized using 2 ug of R N A as starting material and primers: TRKC1044: 5 ' - G G A G T C C A A G A T C A T C C A T G T G G - 3 ' TRKC1329:  5'-TGCTGCTTTTGCCTGTGTCCTG-3'  Products were analyzed as described above.  2.8  RT-PCR A N A L Y S I S OF T U M O R SAMPLES Total R N A (2ug) was isolated from primary tumor samples and used to  make  cDNA  as previously described [309].  Olignucleotide primers  included: ETV6 primers 541 and 701 [308] 352  (5'-GGTGATGTGCTCTATGAACTCC-3')  199 ( 5 * - A T T T A C T G G A G C A G G G A T G A C - 3 ' )  for P C R  65 used i n combination w i t h NTRK3  primer:  NTRK3-2 (TRKC nt 1816-1838:  5-CCGCACACTCCATAGAACTTGAC-3').  P C R conditions were as follows: 94°C for 1.5 minutes, followed by 33 cycles of 94°C for 45 seconds, 60°C for 1 minute and 72°C for 1 minute and a final extension of 72°C for 10 minutes.  Products were analyzed as described above o n agarose gels.  The presence of amplifiable R N A i n all samples was confirmed by R T - P C R using (3A c t i n primers as a control. breakpoint  by Southern  A l l samples were confirmed for the presence of the  blot analysis  as described above using a D N A oligo  spanning the breakpoint: 5'-GGGAGAATAGCAGATGTGCAGCAC-3'.  2.9  PREPARATION OF PROTEIN LYSATES FOR IMMUNOPRECIPITATION A N D IMMUNOBLOTTING Cells infected w i t h the various retroviral constructs i n c l u d i n g  ETV6-NTRK3,  along w i t h the i n d i v i d u a l mutants (see Table 5) were grown until 80-90% confluent at w h i c h point the media was decanted and the cells were rinsed twice w i t h ice-cold PBS. Briefly, 1ml of Lysis Buffer (1.5 m M M g C l (Fisher), 150 m M N a C l (Fisher), 50 2  m M Hepes (Sigma), 10 m M N a F (Sigma), 10 m M N a P 0 (Sigma), 2 m M N a V 0  4  (Sigma), 2 m M ethylene-diamine-tetraacetic acid (EDTA) (Fisher), 2 m M N a M o 0  4  4  2  7  3  2PLO (Sigma), 10% Glycerol (Fisher), 0.5-1.0% Nonidet P-40 (Fisher), L e u p e p t i n (1:1000 dilution of 2 m g / m l stock made i n H 0)(Sigma), A p r o t i n i n (1:1000 d i l u t i o n 2  of l O m g / m L stock made i n H Q)(Sigma), Phenylmethylsulfonyl Fluoride (PMSF) 2  66 T A B L E 5. Summary of the various constructs used to transfect N I H 3 T 3 cells. T h e vector used to make these constructs was M S C V p a c and contained the v a r i o u s mutants listed below. M a n y of the mutations involved replacing a tyrosine residue (Y) w i t h a phenylalanine (F). The Kinase Dead mutant (KD) consisted of a mutation replacing a lysine residue to an arginine. The A H L H mutant contained a deletion encompassing nucleotides 191 - 347 of E T V 6 - N T R K 3 . Corresponding residues i n N T R K 3 are given for comparison.  M U T A T I O N IN ETV6-  CORRESPONDING  NTRK3  R E S I D U E I N NTRIC3  ETV6-NTRK3  -  AHLH  A191-347  -  ALD  Y513F, Y517F, Y518F  Y705, Y709, Y710  KD  K380N  K572  PLCyQ  Y628Q  Y820  PLOyT  Y628T  Y820  PLOyE  Y628E  Y820  CONSTRUCT  Abbreviations. A= deletion, Y= tyrosine, F= phenylalanine, K = lysine, N = asparagine, Q= glutamine, T= threonine, and E= glutamate.  67 (1:200 dilution of a lOOmM solution made i n dimethyl sulfoxide)(Sigma)) was t h e n added to the rinsed cells and incubated for 15 minutes on ice [310, 311].  Lysates  were then centrifuged at 12000 r p m for 10 minutes, at w h i c h point the supernatant was transferred into a fresh tube for further analysis.  2.10  IMMUNOPRECIPITATION One milliliter of lysate was incubated w i t h gentle agitation for two hours at  4°C w i t h either N T R K 3 antibody (20ul) (Santa C r u z Biotechnology) or a - E T V 6 : H L H (3ul) (generous gift of Dr. P. Marynen) along w i t h lOul of Protein A-Sepharose (Pharmacia).  The tubes were centrifuged at 2500 r p m for 5 minutes  and  the  supernatant discarded. The pellet was washed 2 to 3 times i n Lysis Buffer (except the concentration of Nonidet P-40 was 0.1% instead of 1%), boiled i n L a e m m l i buffer [306], loaded onto a Protean E / x i Cell electrophoresis system (Bio-Rad) and electrophoresed o n a 7.5, 10 or 15% polyacrylamide gel overnight at 70 - 100 V o l t s according to standard methods [306].  2.11  IMMUNOBLOTTING Transfer  of the proteins  from the  gel to Immobilon-P (Millipore)  was  accomplished w i t h the Bio-Rad Trans-Blot SD Semi-Dry Transfer cell at 25 volts for 45 minutes using T o w b i n Transfer Buffer (25 m M Tris (Fisher), 192 m M glycine (Fisher), 20% methanol (Fisher).  The membranes  were blocked w i t h  Blocking  Buffer ( I X TBS, 5% S k i m M i l k Powder (Safeway), 0.05% Tween-20 (Fisher)) or (IX TBS, 1% B S A , 0.05% Tween-20) (for the RC-20 anti-phosphotyrosine  antibody  68 (Transduction Laboratories)) for one  hour  at room  temperature  with  gentle  agitation. The membrane was then incubated w i t h one of the following antibodies: anti-TrkC (C14) [ l u g / m l ] (Santa C r u z Biotechnology), RC20-Horse Radish Peroxidase conjugated [1:2500], anti-SHC [1:250], anti-PI3-K [1:5000], anti-GRB2 [1:5000], anti-PLCy [1:1000] (Transduction Laboratories) (see Table 6). The membrane was washed three times for 5 minute intervals i n TBS/0.05% Tween-20 and then incubated i n the secondary antibody: anti-mouse-horse radish peroxidase conjugated [1:7000] or antirabbit-horse radish peroxidase conjugated one hour w i t h gentle agitation. visualization  by enzymatic  [1:7000] (Transduction Laboratories) for  The blot was then washed as above prior to  chemiluminescence  according to the manufacturer's  instructions.  (ECL) ( A m e r s h a m / P h a r m a c i a )  After E C L detection, the  membrane  was placed i n between two i n d i v i d u a l Saran wrap sheets and placed into an X-ray cassette and exposed to a X A R - 5 film for 10 seconds up to 20 minutes.  2.12  G E N E R A T I O N OF GST-ETV6-NTRK3 FUSION PROTEINS The BaculoGold Starter Package (Pharmingen) was used for the p r o d u c t i o n  and purification of recombinant virus encoding the fusion GST-ETV6-NTRK3  gene  and subsequent infection of SF9 cells for the production of large quantities of recombinant protein.  A l l materials mentioned were supplied i n the kit unless  otherwise stated. The recombinant protein was purified by affinity chromatography. Briefly, the lysate (prepared according to the  manufacturer's  instructions)  was  applied to a 0.8 x 4 cm Poly-Prep Chromatography C o l u m n (Bio-Rad) equipped w i t h a 2-way Stopcock (Bio-Rad) containing 1 m l of glutathione beads. The beads were  69 T A B L E 6. Summary of various antibodies used for immunoblotting, their source and required concentrations.  ANTIBODY  MANUFACTURER FACTOR  Anti-ETV6-HLH  Dr. Peter M a r y n e n  1:5000  A n t i - T r k C (C14)  SCBT  2.0 u g / m l  Dr. D a v i d Kaplan  1:2500  RC20-HRPO  TL  1:2500  Anti-Rabbit-HRPO  TL  1:7500  Anti-Mouse-HRPO  TL  1:7500  Anti-GRB2  TL  1:5000  Anti-PI3K  TL  1:5000  Anti-SHC  TL  1:250  Anti-PLCy  TL  1:1000  Dr. Lyiangyou R u i  1:15000  Anti-TrkC  Anti-SH2BfJ  Abbreviations. SCBT= Santa C r u z Biotechnology, TL= Transduction Laboratories, Inc., H R P O = horse radish peroxidase.  70 washed w i t h washing buffer and the protein was eluted w i t h 3 m l of elution buffer (both of w h i c h are supplied i n the kit).  The concentration  of protein  was  determined using the BioRad Protein Determination Assay K i t according to the manufacturer's instructions. The fusion protein was then aliquoted and frozen at -20°C until use.  2.13  IN VITRO P R O T E I N  A S S O C I A T I O N STUDIES  To determine whether there is heterodimerization between E T V 6 - N T R K 3 and w i l d type E T V 6 , both ETV6 and E T V 6 - N T R K 3 were co-translated w i t h the T N T T 7 / T 3 C o u p l e d Reticulocyte Lysate System (Promega). The ETV6-NTRK3  construct  was contained w i t h i n pBluescript II K S (kindly provided by Daniel W a i i n the laboratory) and required the T3 promoter for the production of m R N A , while the ETV6 construct was contained w i t h i n the pQE9 vector (generous gift of Dr. P. Marynen) and requiring the addition of 1 U n i t  of R N A Polymerase, E. coli  (Boehringer M a n n h e i m ) for proper m R N A production.  The in vitro  translated  materials were then immunoprecipitated w i t h the N T R K 3 (C-14) antibody (Santa C r u z Biotechnology), electrophoresed o n 10% polyacrylamide gels, blotted onto Immobilon-P and immunoblotted  w i t h the E T V 6 : H L H antibody, as described  above. To  determine  if  the  ETV6-NTRK3  properties, 5ug of the G S T - E T V 6 - N T R K 3 mixed w i t h  35  protein  recombinant  had  homodimerization  protein (see above) was  S - M e t h i o n i n e (Amersham) labeled in vitro translated E T V 6 - N T R K 3  (labeled according to the instructions provided w i t h the T N T T 7 / T 3 C o u p l e d  71 Reticulocyte Lysate System), immunoprecipitated  w i t h glutathione  beads, and  electrophoresed as described above. The resolving gel was then dried using a G e l Drier (Labconco) for 1.5 hours prior to exposing the gel to X A R - 5 film for 4 to 24 hours.  2.14  S U B C E L L U L A R L O C A L I Z A T I O N BY I M M U N O F L U O R E S C E N C E Green fluorescent protein constructs of E T V 6 - N T R K 3 ,  H L H , and K D were  made using the p E G F P - N 3 vector (Clonetech). Since the G F P portion was placed o n the 3' end of the various constructs, the stop codon was replaced w i t h a glycine codon to allow for the continued translation of GFP. This was accomplished using the Q u i k C h a n g e ™ manufacturer's  Site-directed mutagenesis  instructions.  kit (Stratagene) according to  the  Primers used to mutate the stop codon (TAG) to a  glycine (GGG) for the various constructs were: 5 ' - G A C A T T C T T G G C G G G T G G T G G C T G G T G G T C - 3 ' (forward) 5 ' - G A C C A C C A G C C A C C A C C C G C C A A G A A T G T C - 3 ' (reverse) N I H 3 T 3 cells were then transfected LipofectAMINE™  w i t h 2 ug of the  (Gibco/BRL) according to  the  purified construct  manufacturer's  and  instructions.  D u r i n g transfection, the cells were grown i n O p t i M E M ™ I Reduced Serum M e d i a (Gibco/BRL). T w o days post-transfection, the cells were passaged 1:10 into selective m e d i u m (RPMI 1640 w i t h L-Glutamine, 10% calf serum, l x P S F , 400 ug G e n e t i c i n (Gibco/BRL)) and grown i n this m e d i u m for 5 days. The cells were then onto Fisherbrand Superfrost/Plus  seeded  (Fisher) glass slides and allowed to grow i n  regular growth m e d i u m (RPMI 1640 w i t h L-Glutamine, 10% calf serum and lxPSF)  72 until 50-80% confluent. The slides were then quickly rinsed w i t h PBS and fixed i n 4% paraformaldehyde ( p H 7.4) (Sigma) i n PBS for 10 minutes at r o o m  temperature.  The slides were then washed i n PBS (3 x 5), counterstained w i t h D A P I (as above for F I S H studies) and briefly rinsed i n PBS. One drop of VectaShield™ was t h e n applied and the slide was covered w i t h a coverslip and v i s u a l i z e d by confocal microscopy using a Bio-Rad MRC-600 Laser Scanning Confocal Microscope. In addition, NIH3T3 cells infected w i t h the various constructs i n c l u d i n g N I H 3 T 3 cells transfected w i t h vector alone (for use as a negative control) (provided by D . W a i i n our lab) were analyzed by immunofluorescence.  Cells were g r o w n o n  Fisherbrand Superfrost/Plus (Fisher) glass slides as described above until  the  confluency reached approximately 80%. The slides were then rinsed quickly w i t h PBS and fixed i n 4% paraformaldehyde ( p H 7.4) i n PBS for 10 minutes at r o o m temperature and permeabilized w i t h 0.1% TritonX-100 (Sigma) for an additional 10 minutes at room temperature.  A n antibody towards the carboxy terminal p o r t i o n  of N T R K 3 was used as a primary antibody [1:2500] (a generous gift of Dr. D . Kaplan). The slide was incubated w i t h the primary antibody for 1 hour at 37°C, at w h i c h point the slide was run through a series of four 5 minute washes i n I X phosphatebuffered saline (PBS). Rhodamine anti-rabbit (Boehringer M a n n h e i m ) was used as the secondary antibody at a concentration of 30 u g / m l and the slide was incubated at 37°C for 30 minutes.  The slides were then r u n through another  PBS series,  counterstained, mounted and visualized by confocal microscopy as described above.  73  CHAPTER III A NOVEL t(12:15)(pl3: q25) IN CONGENITAL FIBROSARCOMA  3.1  INTRODUCTION Subtle chromosomal translocations, inversions, and other rearrangements  are often missed by conventional cytogenetics [312].  Molecular cytogenetics has  overcome these barriers by p r o v i d i n g more sensitive techniques offering higher resolution  [297].  Fluorescence in situ hybridization (FISH), for example, is a  powerful molecular tool, w h i c h can be used to determine if a certain c h r o m o s o m a l region has been deleted or rearranged or to finely map a newly discovered gene. Conventional  cytogenetics  along w i t h molecular  cytogenetics  has,  therefore,  permitted scientists to investigate genetic alterations i n cancer cells, thus a l l o w i n g for the detection of recurrent genetic abnormalities i n certain tumors [238, 313]. The  diagnosis  of CFS has  historically been  very  pathologist due to its morphologic overlap w i t h other tumors i n c l u d i n g A T F S , IFB, myofibromatosis  challenging for  the  childhood spindle cell  (MFB), and A F B [314, 315]. W e  therefore performed cytogenetic analysis on a group of CFS cases at B C C H , as well as a series of A T F S , IFB, M F B , and A F B cases i n an attempt to identify genetic markers of these entities.  recurrent  Of these cases, only the C F S cases displayed a  recurring chromosomal abnormality i n v o l v i n g chromosome 12pl3 and 15q25. T o determine if this aberration was a translocation, whole chromosome F I S H of a C F S case using a chromosome  12 painting probe was performed.  This revealed a  74 portion of chromosome 12 o n a smaller acrocentric chromosome.  W e therefore  wanted to elucidate the exact consequences of this rearrangement.  3.2  RESULTS 3.2.1  Cytogenetic Analysis  To screen for recurrent genetic markers of CFS, cytogenetic analysis was performed o n a series of C F S (n=4), IFB (n=15) and A T F S (n=2) cases diagnosed i n patients either from B C C H or C H L A . C F S Cases 1-3 each demonstrated a b n o r m a l metaphases w i t h a subtle rearrangement of chromosome 15q25-26. T w o of these cases had additional abnormalities of chromosome 12pl3. Cytogenetic analysis of A T F S (n=2) and IFB (n=15) cases showed no rearrangements  involving  either  chromosome 12 nor 15 (see Fig. 7 (chapter 2)).  3.2.2  Fish Analysis Identifies a Common Derivative Chromosome  We next performed fluorescence in situ hybridization (FISH) analysis of 12pl3 and 15q25-26 alterations using the FISH probes depicted i n F i g . 8. O n l y C F S cases 1 and 2 had tumor metaphases available for FISH. To map each breakpoint, we screened C F S metaphases using a series of non-chimeric 12pl3 and 15q25-26 yeast artificial chromosomes  (YACs) to identify those spanning the breakpoints.  Initially, we started w i t h Y A C s representing the telomeric portions of 12p and 15q. To test for a translocation of 12p material to chromosome 15q, we used either Y A C 890_E_3  or  854_E_6  (from  12p  telomere)  along  with  an  oc-centromeric  chromosome 15 probe. Conversely, we tested either Y A C 882_H_8 or 895_H_10  75  12pl3 D12S380E  D12S99 D12S381E  D12S89  D12S358  D12S1275  D12S308  12q  Cen. Tel. 890E3  854E6  924H12  954G10 738B11  r  817H1  TEL locus 40 kb D12S89  D12S98  1A  IB  3 4 56 78  179A6  163E7  148B6  45E12 54D5  171H6  802B4  932F12  895H10  882H8  Cen. D15S151  D15S202  D15S963  CHLC.ATA28G05  D15S120  D15S157  D15S203  15q25-26  F I G U R E 8. Mapping of chromosome 12pl3 and 15q25-26 breakpoints in CFS. Chromosomes 12pl3 and 15q25-26 are schematically represented at the top and bottom, respectively, of the diagram. The positions of the ETV6 (TEL) and KIP1 loci are indicated by boxes. Relative YAC positions are indicated by solid lines; neither chromosome or YAC sizes are to scale. The ETV6 locus is drawn to scale, and exons are indicated by numbers and letters above the solid line. Cosmid positions are indicated by open lines. The position of the NTRK3 (TRKQ locus relative to 802B4 is based on the present study.  76 (from 15q telomere) along w i t h an a-centromeric chromosome 12 probe. W h e n the telomeric 12p Y A C 890_E_3 and the chromosome 15 a-centromeric probe were used i n a dual colored metaphase preparation, one of the chromosomes hybridized both probes  (see Fig. 9a).  This study demonstrated  that the  rearrangement  involving 12p represented a translocation of material to 15qter. O n the other h a n d , material from chromosome  15q25 d i d not translocate back to 12pter, but rather to  another  (later identified as chromosome 1 by D A P I banding) (see Fig. 9b).  FISH using the above Y A C s together w i t h a series of chromosome-specific probes demonstrated complex three-way translocations for both cases i n w h i c h material from distal 12p was translocated to distal 15q and material from distal 15q was translocated to either lq43 or 4ql0, respectively.  3.2.3  Identification of the Breakpoint Region by Y A C M a p p i n g  To narrow d o w n the breakpoint, we " w a l k e d " along chromosome 12p and 15q using various Y A C clones until we found one w h i c h spanned the breakpoint. We found that the 12pl3 Y A C , 817_H_1, and the 15q25-26 Y A C , 802_B_4, were each split i n C F S cells, giving 3 signals as opposed to only 2 i n IFB and other controls. When  these Y A C s were used together  i n dual-coloured F I S H ,  a derivative  chromosome hybridizing a fusion signal was detected i n both C F S cases (see F i g . 10a). This derivative was shown to represent a der(15)t(12;15) as it s i m u l t a n e o u s l y hybridized both an a-centromeric chromosome 15 probe and 817_H_1 by dual-color F I S H (data not shown). Similar experiments o n 3 A T F S cases and 6 IFB cases  77 F I G U R E 9. Dual-coloured FISH of CFS. A, A metaphase from a C F S case was used i n a dual colored F I S H experiment using the 12p telomeric Y A C 890_E_3 (green), and a 15 a-centromere (red). A der(15) chromosome hybridizing both probes shows the translocation of material from 12pter to 15qter. B, Another metaphase preparation from the same CFS case was subjected to dual colored F I S H using the 15qter Y A C , 882_H_8 (green), along w i t h a 12 a-centromere (red), h o w e v e r , material from 15qter, as seen by the 15qter Y A C , was translocated to another chromosome, later identified as chromosome 1 by computer generated D A P I banding.  78  79 FIGURE 10. FISH analysis for CFS breakpoints. A, Dual-coloured F I S H of C F S case 2 metaphase using the 12pl3 breakpoint-spanning Y A C , 817_H_1 (green), and the 15q25-26 breakpoint-spanning Y A C , 802_B_4 (red). The arrowhead shows a yellow fusion signal indicating the der(15)t(12;15)(pl3;'q25-26). B, Dual-coloured F I S H of CFS case 1 interphase cell using ETV6 exon 1-containing cosmid 179A6 (green) and ETV6 exon 8-containing cosmid 148B6. The arrowhead shows a fusion signal representing one copy of normal chromosome 12 i n each cell, while the arrows show separate signals indicating disruption of the ETV6 gene.  80  81 revealed normal FISH patterns only with these probes.  FISH using 802_B_4  together with an a-centromeric 12 probe failed to detect reciprocal der(12)t(12;15) chromosomes in either case 1 or 2 (data not shown). In each case, FISH identified derivative chromosomes that had not been previously detected cytogenetically (see Table 7).  3.2.4  Micromapping the Breakpoint with Cosmid Probes  The 12pl3 breakpoint was narrowed to the telomeric end of 817_H_1 as the distal overlapping 12pl3 Y A C , 924_H_12, was also split in CFS (data not shown).  This  region contains the ETV6 gene (TEL), a member of the ETS family of transcription factors and has been involved in a varietv of translocations which have given rise J  to gene fusions in human leukemias [308, 316-320]. To test for the involvement of ETV6  in CFS, we performed dual-coloured FISH using 802_B_4 together with  either cosmid 179A6, which contains ETV6 exon 1, or cosmid 148B6; containing ETV6  exon 8 [300]. This revealed a fusion signal with 179A6 but not with 148B6  (data not shown).  Dual-coloured FISH of CFS using 179A6 and 148B6 together  revealed one fusion signal as expected for normal chromosome 12, as well as two widely separated signals  (see Fig. 10b).  This indicates that the breakpoint lies  between exon 1 and 8 of ETV6 in CFS, and that the telomeric portion of ETV6 translocated to chromosome  15q25-26.  is  Further mapping using cosmids 171H6,  45E12, 163E7, and 54D5 (see Fig. 8), localized the ETV6  breakpoint to the region  between exons 5 and 7 of ETV6. Fusion signals were not detected by FISH using  82 Table 7. Summary of ETV6 rearrangements in human neoplasia.  Gene Fusion  Translocation  Protein  Phenotype  Ref.  MDS preB c A L L , a C M L CMML  [319] [320,321] [316] [322] [318]  (ETV6-- Partner Gcne)  E TV6-MDS/E Vll ETV6-JAK2 ETV6-PDGF/3R ETV6-STL ETV6-ABL  t(3;12)(p26;pl3) t(9;12)(p24;pl3) t(5;12)(q33;pl3)  NFD - ND H L H - PTK H L H - PTK  t(6;12)(q23;pl3) t(9;12)(q34;pl3)  H L H , D B D - N D B-cell A L L cell line H L H - PTK A M L , A L L , aCML  [308,323H L H - DBD, cALL 326] TAD [317] t(12;22)(pl3;q22) DBD - ND A M L and M D S [327,328] t(12;13)(pl3;ql2) NFD - ND AML t(5;12)(q31;pl3) ND R A E B , A M L , A E L [329] [330] t(4;12)(qll-ql2;pl3) ND AML t(l;12)(q25;pl3) HLH-SH2, SH3, A M L [331] PTK [332] ETV6-? t(7;12)(q22;pl3) ND MDS [332] • ETV6-? t(7;12)(q36;pl3) ND AML [332] ETV6-? t(9;12)(qll;pl3) ND ALL [333] ' ETV6-? t(10;12)(q24;pl3) ND MDS [328] ETV6-? t(6;12;17)(p21;pl3;q25) N D AML ETV6-? [328] t(7;12)(pl4;pl3) ND AML [328] ETV6-? t(12;17)(pl3;qll) ND B-NHL ETV6-? [328] t(7;12)(pl2;pl3) ND AML [334] ETV6-? t(X;12)(q28;pl3) ND MDS [334] ETV6-? t(l;12)(q21;pl3) ND AML [334] ETV6-? t(9;12)(p23-24;pl3) ND ALL ETV6-? [335] t(12;14)(pl3;qll) ND ALL ETV6-? [335] t(7;12)(q35;pl3) ND ALL Abbreviations. AML= acute myelogenous leukemia, CMML= chronic myelomonocytic leukemia, BNHL= B-cell non-Hodgkin's lymphoma, SH2(3)= Src homology 2(3), MDS= myelodysplastic syndrome, ALL= acute lymphoblastic leukemia, cALL= childhood acute lymphoblastic leukemia, aCML= atypical chronic myeloid leukemia, ? or ND= not determined, NFD= no functional domain, HLH= helix loop helix domain, PTK= protein tyrosine kinase domain, TAD= transactivation domain, DBD= ETS DNA binding domain, RAEB= refractory anemia with excess blasts (with basophilia), AEL= acute eosinophilic leukemia. ETV6-CBFA2 (AML1) ETV6-MN1 ETV6-CDX2 ETV6-ACS2 ETV6-BTL ETV6-ARG  t(12;21)(pl3;q22)  83 802_B_4 in conjunction with 148B6, confirming the absence of the der(12) t(12;15)(pl3;q25-26) in tumor cells.  3.3  DISCUSSION In this report we describe for the first time  the  association  of a  t(12;15)(pl3;q25-26) translocation with congenital fibrosarcoma. All CFS cases with abnormal karyotypes in this study demonstrated alterations of the distal long arm of chromosome 15 (15q25-26), while 2 also showed subtle alterations involving chromosome 12pl3.  The normal karyotype of case 4 likely represents in vitro  overgrowth by normal fibroblasts. In contrast, similar abnormalities were not detected in a series of IFB, MFB, or AFB cases. FISH analysis using region-specific YAC probes confirmed the presence of a der(15)t(12;15)(pl3;q25-26) in 2 of 2 CFS cases with metaphases available for FISH studies. Moreover, when we screened with a series of 12pl3 and 15q25-26 specific YACs against CFS metaphases, we found that the same region-specific YAC was split in both CFS cases, i.e. YAC 817_H_1 spanning the chromosome 12pl3 breakpoint and YAC 802_B_4 spanning the 15q2526 breakpoint.  Therefore the breakpoints in each chromosome are contained  within identical region-specific YACs in both of the CFS cases tested, providing further evidence that this represents a non-random rearrangement in CFS. We also tested 3 ATFS, 3 IFB, and 3 AFB cases using the identical probes. None of the 9 non-CFS  cases  demonstrated  similar  findings,  suggesting  that  the  der(15)t(12;15)(pl3;q25-26) is specific for CFS. We are currently accumulating and  84  testing additional cases of C F S , A T F S , IFB, and A F B to more rigorously test this hypothesis. Finer FISH mapping of the breakpoint using cosmid probes localized the breakpoint within the ETV6 gene. ETV6  This is the first known involvement  gene fusion in solid tumors,  as such rearrangements  of the  were  previously  The ETV6 (ETS variant gene 6) gene is located on chromosome  12pl3 and  observed only in leukemias [308, 316-320, 333].  was cloned as a result of a t(5; 12)(q33; pl3), fusing it to the platelet-derived growth factor B receptor (PDGFfiR) gene in chronic myelomonocytic  leukemia  (CMML)  [316]. ETV6 (also known as T E L : translocation ETS /eukemia) is a member of the large family of transcription factors known as the E26 transformation-specific (ETS) transcription factors first discovered as part of the E26 avian erythroblastosis v i r u s genome [336]. The ETS family of transcription factors recognize the core motif C / A G G A A / T [337]. The (with  one  ETV6 gene is approximately 240 kb in size and is made up of 8 exons alternatively  spliced exon)  which  are  oriented  from  telomere  to  centromere (see Fig. 8) [300]. It encodes a nuclear phosphoprotein with a helix loop helix dimerizing domain ( H L H D ) encoded within exons 3 and 4 and a D N A binding domain (DBD) encoded within exons 6-8 [300, 338]. The ETS D N A binding domain is known  to contain the nuclear  localization  signal and sequence specific D N A  binding activity [339]. The m R N A encoding the ETV6 protein contains two possible initiation sites and results i n two species of transcripts.  One of the E T V 6 proteins  migrates around 50 kDa and the other around 57 kDa. Both contain the H L H D and  85 the ETS D B D , but only the full length species encodes a M A P K  consensus site  (amino acids 20-23), w h i c h is phosphorylated i n v i v o [338]. This suggests that the regulation of expression of these two proteins are different.  Poirel et al. noted that  most of the cell lines they examined showed greater expression of the  higher  molecular weight ETV6 species [338]. Since its discovery i n 1994, the ETV6 gene has been implicated i n a large number of hematopoietic malignancies. Fluorescence in situ hybridization (FISH) analysis of the 12p chromosomal  region has shown numerous  translocations  i n v o l v i n g the ETV6 gene, w i t h some cases showing a deletion i n addition to the translocation  [308, 324, 340-343].  Approximately 50% of the  rearrangements  i n v o l v i n g the ETV6 gene w i t h i n these neoplasms have been characterized  (see  Table 7). Cytogenetic and molecular genetic analysis of these rearrangements has led to the discovery of chimeric gene fusions i n v o l v i n g ETV6 gene as a result of chromosomal translocations.  specific exons from the  In C M M L , for example, the  helix loop helix dimerization domain ( H L H ) from the ETV6 gene is fused to the protein tyrosine kinase domain (PTK) from the PDGFPR gene [316].  The H L H  d o m a i n acts as a protein-protein dimerizing domain and constitutively activates the PTK  domain  by ligand-independent  dimerization.  The  t(12;22)(pl3;qll)  in  myeloproliferative disorders (MDS) results i n the fusion of nearly the entire M N 1 protein to the ETS D N A binding domain from the ETV6 gene [317]. In this case, it is thought that oncogenesis is due to the altered transcriptional activity of the ETV6 gene. The ETV6-CDX2 and the ETV6-STL gene fusions i n A M L and a B-cell A L L cell-line, respectively, only contain the first two exons from the ETV6 gene fused to  86 u n k n o w n 3' sequences from the partner gene [322, 327].  There are no k n o w n  functional domains i n the first two exons of ETV6 and it is thought that the STL and CDX2 genes are under transcriptional control from the ETV6 promoter. w e l l characterized ETV6-CBFA2  The  ( T E L - A M L 1 ) is usually accompanied by a deletion  of the residual ETV6 gene o n the normal chromosome [308]. It is possible that the normal E T V 6 protein dimerizes w i t h the E T V 6 - C B F A 2 protein interfering w i t h its oncogenic potential  or that E T V 6 - C B F A 2 functions  as a dominant  negative  inhibitor of normal ETV6 function. Protein analysis led to the discovery of up to 5 different species due to alternative  translational initiation and postranslational  modification [338, 344]. Other ETS family members are k n o w n to be phosphorylated and it has been shown that these postranslational modifications play an i m p o r t a n t role i n the function of the protein [345-348]. The mechanism by w h i c h a chromosome participates i n a translocation is still unclear.  In a recent study, however, the ETV6-CBFA2  breakpoint was cloned at  the genomic level and almost all breakpoints i n the ETV6 gene were found near a p u r i n e / p y r i m i d i n e rich sequence w i t h i n  intron 5 of E T V 6 (most  i n v o l v i n g ETV6 occur i n intron 5) [349].  This suggests that this region may be  susceptible to D N A breakage and re-ligation, i n c l u d i n g translocations. summarizes  the  k n o w n breakpoints  within  the  ETV6  breakpoints  Figure 11  gene as w e l l  as  the  corresponding fusion genes. ETV6 expression is seen i n almost all tissues during development and i n the adult.  E T V 6 has recently been s h o w n to be important i n angiogenesis and its  transcription is downregulated by an angiogenic growth factor, vascular endothelic  87 F I G U R E 11. Schematic representation of the c D N A for ETV6 as w e l l as some of the more c o m m o n rearrangements i n v o l v i n g the ETV6 gene. The ETV6 gene is located on chromosome 12pl3 and consists of 8 exons. The top panel shows the coding sequence (cDNA) of E T V 6 along w i t h the nucleotide position of each exon (exons are separated by a vertical line w i t h the nucleotide position s h o w n below). A number of translocation breakpoints have been found i n the E T V 6 gene resulting i n its i n frame fusion to other genes such as PDGFfiR (in C M M L ) , ABL (in A M L , A L L , and a C M L ) , AML1 (in c A L L ) and MN1 (in A M L and M D S ) . The fusion proteins generated juxtapose either the H L H dimerization domain from exons 3 and 4 of E T V 6 to the protein tyrosine kinase (PTK) d o m a i n of P D G F B R , A B L and the r u n t and transactivation domain (TAD) of the A M L 1 gene or the ETS D N A b i n d i n g d o m a i n (ETS domain) from exons 6, 7 and 8 to the M N 1 region of the M N 1 gene (shown as protein structures i n the bottom 5 panels). The location of some of the more c o m m o n breakpoints found i n various hematologic malignancies are s h o w n i n the c D N A sequence (arrows) (see Table 7 for more information).  89  growth factor (VEGF) [350].  Angiogenesis refers to the process by w h i c h new  vascular elements emerge from pre-existing vasculature.  This group, however,  found that only the lower molecular weight species was expressed i n  human  umbilical v e i n endothelial cells ( H U V E ) . In addition, they were able to demonstrate the loss of one of the m R N A ETV6 species u p o n treatment of the cells w i t h V E G F . The precise role of ETV6 i n blood vessel formation and maturation under investigation.  is presently  It has been suggested that the E T V 6 protein may act as a  transcriptional repressor (Golub et al., unpublished data).  Since V E G F promotes  vascular growth and inhibits ETV6 transcription, it is possible that the transcription of genes inhibited b y ETV6, are involved i n blood vessel formation and maturation. ETV6 knockout mice showed embryonic lethality and failed to maintain yolk sac blood vessel formation. Furthermore, studies have shown E T V 6 to be necessary for bone marrow hematopoiesis but not essential for liver hematopoiesis [351]. We observed complex three-way translocations i n both C F S cases studied by F I S H (see Table 4 (chapter 2)), suggesting that these alterations might be favored i n CFS.  Three-way  translocations  generating  typical EWS-FLI1  gene fusions  described i n E w i n g tumors [352, 353], as are complex translocations i n positive leukemia cell lines [354]. Moreover, ETV6-CBFA2  are  ETV6-CBFA2  positive cases w i t h the  t(12;21) show a high frequency of variant derivative 21 chromosomes and absence of CBFA2-ETV6  expression, while other ETV6-CBFA2  the n o r m a l ETV6 allele [355].  positive cases commonly delete  This suggests that reciprocal fusion products  normal ETV6 itself may inhibit functional ETV6 chimeric oncoproteins.  or  Further  90 rearrangements of the der(12)t(12;15) i n C F S may therefore  be selected for as a  mechanism of eliminating expression of an inhibitory N T R K 3 - E T V 6 molecule. In summary, cytogenetic analysis coupled w i t h detailed F I S H m a p p i n g of two CFS cases w i t h available metaphases resulted i n the identification of a complex three-way rearrangements for both cases, interpreted  as t(l;12;15)(q44;pl3;q25-26)  and t(4;12;15)(ql0;pl3;q25-26), respectively [356]. We now report an apparently n o n random  association  between  the  presence  of  a  der(15)t(12;15)(pl3;q25-26)  chromosome and the diagnosis of CFS. This rearrangement cases of A T F S , IFB, M F B nor i n A F B , and therefore differentiation of these entities from CFS.  Furthermore,  was not detected i n  may be useful  in  the  our findings p r o v i d e  additional evidence that CFS is biologically distinct from fibrosarcomas occurring i n older children. These data suggest that CFS may be characterized by an ETV6 gene fusion; the identification of the partner gene is the topic of the next chapter.  91  CHAPTER IV CLONING AND CHARACTERIZATION OF THE t(12:15) IN CFS  4.1  INTRODUCTION Translocations leading to specific gene fusions are a common event i n cancer  [238]. Molecular analysis of these gene fusions has led to the discovery of chimeric oncogenes.  Most of these chimeras appear to act as aberrant transcription factors,  likely functioning i n transformation by dysregulating, ablating or introducing n e w gene expression profiles w i t h i n the cell [238].  Bone and soft tissue sarcomas of  childhood have provided an abundant resource for studying various types of gene fusions [238, 357]. The detection of these fusion transcripts i n tumor  specimens,  specific for given tumor subtypes, has become an extremely useful diagnostic t o o l for the pathologist. appearance  and  morphologically  Childhood  therefore [5].  very  sarcomas difficult  tend to be extremely p r i m i t i v e i n to  differentiate  from  each  other  Accurate diagnosis of two morphologically similar, yet  distinct, tumors is extremely important since initial diagnosis often  determines  w h i c h treatment protocol a patient is enrolled i n . Accurate pathologic classification is, therefore, a critical prognostic factor for these patients. The identification of the breakpoint w i t h i n the ETV6 gene led us to believe there may be a gene fusion i n v o l v i n g this gene i n CFS. The ETV6  gene has o n l y  been rearranged i n leukemias to date (see Table 7 i n Chapter 3), but this does not rule out its potential involvement i n a solid tumor as well. ERG and FLU are two other ETS family members found to be fused w i t h EWS or TLS/FUS  as a result of  92 chromosome translocations i n h u m a n solid tumors and leukemias [254, 258, 259, 268]. We  therefore hypothesized that the t(12;15) rearrangement  may s i m i l a r l y  give rise to an oncogenic gene fusion i n CFS. Because the der(15)t(12;15)(pl3;q25-26) was c o m m o n to both C F S cases analyzed by molecular cytogenetics, we further reasoned that a functional gene fusion, if present, might be expected to be expressed from this derivative chromosome and w o u l d involve the ETV6 gene.  4.2  RESULTS 4.2.1  C l o n i n g the t(12;15) Breakpoint i n C F S  The 8 exon ETV6 locus is oriented i n a telomere to centromere direction, w i t h exons 3 and 4 encoding a helix-loop-helix ( H L H ) protein dimerization d o m a i n and exons 6-8 contributing to the ETV6 ETS D N A b i n d i n g d o m a i n [300, 316]. Because the E T V 6 5 ' - H L H region is fused to other  partner  genes i n  human  leukemias [308, 316-320], we performed 3'-rapid amplification of c D N A ends ( R A C E ) using ETV6 exon 5 primers 541 and 701 along w i t h a poly-dT-linked primer (see Chapter 2). R A C E using primer 701 generated similar ~1.5 kb fragments i n 3/3 C F S cases but not i n 3 A T F S , 3 IFB, or other control cases.  C l o n i n g and sequencing of  these fragments revealed that 333 bp of ETV6 sequence were fused in-frame to 1115 bp of u n k n o w n sequence. Database analysis revealed this to represent the t e r m i n a l 1115 bp of the h u m a n NTRK3 gene encoding the neurotrophin-3 surface receptor [358-360] (see Fig. 12a). The fusion point i n all 3 cases was after nt 1033 of ETV6,  93  E T V 6 (nt 1033) ««-  > N T R K 3 (nt 1601)  tec ccg cct gaa gag cac gec atg ccc att ggg aga ata gca gat gtg cag cac art aag agg aga gac ate gtg ctg aag cga  S P P E E  H A M P I G  R I A D V Q H I K R R D I  V L K R  B  M 1 2  3 4 5  6 7 8 9  10  872 bp 603 bp 310 bp  F I G U R E 12. ETV6-NTRK3 gene fusions in CFS. A, Junctional nucleotide (small case) and single letter amino acid sequence of P C R fragments generated by 3 ' - R A C E of c D N A from CFS cases 1-3, using sense primers 541 or 701 from ETV6 exon 5 i n combination w i t h a poly-dT primer. Sequence analysis revealed an in-frame fusion after ETV6 nt 1033 w i t h nt 1601-2715 of the human NTRK3 gene. B, R T - P C R using ETV6 primer 541 and primer NTRK3-2 demonstrates a 731 bp fragment i n C F S (lanes 3-5) but not i n normal fibroblasts (lanes 1 and 2), IFB (lanes 6-8), A T F S (lane 9), or the Jurkat leukemia cell line (lane 10).  94 w h i c h is the last nt of ETV6 exon 5 [300]. The ETV6 breakpoints therefore appear to be localized to intron 5. The NTRK3  portion originated at NTRK3  included the entire protein tyrosine kinase  (PTK) domain  nt 1601 and  and remaining  C-  terminus of N T R K 3 [358-360].  4.2.2  Reciprocal Fusions were not Detected  5 ' - R A C E w i t h 5'-NTRK3  and 3 ' - R A C E w i t h 3'-ETV6  ETS region  failed to detect fusion transcripts that might be encoded by functional or der(l)t(l;15) chromosomes,  primers  der(12)t(l;12)  thus r u l i n g out additional ETV6 or NTRK3  gene  fusions i n v o l v i n g the 3'-ETS region and the 5 ' - N T R K 3 extracellular ligand b i n d i n g domain, respectively (data not shown).  4.2.3  R T - P C R A n a l y s i s of C F S and Other M o r p h o l o g i c a l l y S i m i l a r Tumors  R T - P C R using ETV6 primer 541 and the T R K C - 2 primer from the P T K region amplified the expected 731 bp ETV6-NTRK3  NTRK3  fusion transcripts i n all 3  CFS cases, while A T F S , IFB, and other controls were negative (see Fig. 12b). R T P C R using ETV6 primer 114 located 5' to the H L H region, together w i t h T R K C - 2 generated the expected 1158 bp product only i n CFS samples, and sequencing of this product  confirmed  the  presence of the  transcripts (data not shown).  entire  ETV6  H L H region  in  fusion  95 4.2.4  Northern and Southern Blot A n a l y s i s  N o r t h e r n blot analysis using a full length c D N A probe for ETV6 hybridized to three transcripts w i t h sizes of 6.2, 4.3, and 2.4 kb and was found to be ubiquitously expressed i n C F S , A T F S , IFB, and other control cells. W h e n we used a c D N A or a partial c D N A probe including the NTRK3  NTRK3  P T K motif, only the 4.3 kb  transcript i n CFS cells hybridized (see Fig. 13). Southern blot analysis of genomic D N A isolated from C F S primary tumor tissue showed the disruption of both ETV6 and NTRK3 genes w h e n hybridized w i t h the ETV6 5/6 and NTRK3-PTK Fig. 14). The multiple bands seen i n the CFS lane are due to the  probes (see chromosomal  rearrangement i n v o l v i n g the ETV6 gene.  4.3  DISCUSSION By cloning the chromosome  fuses the ETV6  breakpoints we show that the rearrangement  (TEL) gene from 12pl3 w i t h the 15q25 NTRK3  receptor gene (TRKC).  Analysis of m R N A revealed the expression of  chimeric transcripts i n 3 of 3 C F S tumors. infantile  fibromatosis  neurotrophin-3  (IFB),  ETV6-NTRK3  These were not detected i n A T F S or  a histologically similar  but  benign  fibroblastic  proliferation occurring i n the same age group as CFS. The NTRK3  gene (also k n o w n as TRKC  and neurotrophin 3 receptor) is the  third member of the T R K family of tyrosine kinase receptors. Lamballe et al. f o u n d the NTRK3 gene to contain up to 97 and 98% homology to the rat and porcine T r k sequences, respectively [358, 359]. The human NTRK3 gene was cloned and  96  TEL  TRKC  p-Actin  FIGURE 13. Northern analysis of CFS cases. Blots were probed sequentially w i t h full length ETV6 c D N A (top panel), a 3' NTRK3 partial c D N A probe encoding the P T K domain (NTRK3-PTK; middle panel), and a p-actin c D N A probe (bottom panel) to test for equal loading. The ETV6 c D N A probe detected p r e v i o u s l y described 6.2, 4.3, and 2.4 kb transcripts i n multiple samples (9), including CFS, while NTRK3-PTK detected a 4.3 kb species only i n CFS cases (lanes 3 and 4; arrow). Identical results were obtained using full length NTRK3 c D N A (data not shown). Lanes 1 and 2, normal fibroblasts; 5-7, three IFB cases; 8 and 9, leukemia cell lines K562 and fnrkat, respectively; 10, neuroblastoma cell line SAN2; 11, rhabdomyosarcoma cell line Birch.  97  FIGURE 14.  Southern analysis of CFS cases. Hindlll digests probed w i t h ETV6-5/6 (left panel) and NTRK3-PTK (right panel) revealed rearranged bands i n CFS case 3 and 1 (lanes 1 and 2, respectively), indicated by arrows. Germline bands only were observed i n IFB (lane 3), SAN2 (lane 4), Birch (lane 5), and normal fibroblasts (lane 6).  98 mapped to chromosome 15q25 by McGregor et al. [360]. Expression of NTRK3  in  adult tissues is predominantly restricted to the central and peripheral n e r v o u s systems, but detection of transcripts i n non-neural  cells including  intestinal  glandular cells, adrenal medullary cells, ovarian granulosa and thecal cells, k i d n e y tubular cells, as w e l l as skeletal muscle, lung, testis, prostate, and heart has also been reported [361, 362]. Fetal tissues show strong expression i n brain, kidney, l u n g , and heart tissues; however, the role of N T R K 3 i n non-neural  tissues is not  presently k n o w n . Studies of N T - 3 knockout mice showed multiple heart defects as w e l l as the ablation of proliferation and survival of neural crest cells suggesting a n important role for N T - 3 mediated pathways i n cardiogenesis and  neurogenesis  [363]. A d d i t i o n a l studies have shown that mechanical injury to the hippocampus results i n the increased expression of N T R K 3 and inducible transcription factors (ITFs) such as Fos, c-Jun, and Krox-24 [364, 365]. The results of this study suggest that the expression of N T R K 3 may be controlled i n part by ITFs. The h u m a n NTRK3  gene is organized w i t h its 20 exons from telomere to  centromere [366]. The extracellular d o m a i n consists of a signal peptide, 2 cysteine clusters, a leucine rich motif, and 2 Ig-like domains. These structures are encoded by exons 1 through 8. The transmembrane d o m a i n is encoded by exons 11-13 and the protein tyrosine kinase d o m a i n (intracellular domain) by exons 13-18. Exons 9 and 16 encode the alternative inserts found i n the extracellular and kinase d o m a i n s and exons 13b and 14b encode the terminal d o m a i n of the truncated isoform [361]. The truncated and full length isoforms have a carboxy terminal tail and a 3' U T R w h i c h are encoded by exons 14b and 18, respectively.  The isoform without any  99 inserts represents the active tyrosine kinase receptor, w h i l e the isoforms w h i c h are truncated or have inserts w i t h i n the kinase domain are inactive tyrosine kinase receptors [367-369]. The function of the inserts is not yet k n o w n , however, there is evidence that the truncated N T R K 3 receptors are important i n the m o d u l a t o r y development  of certain neural cell populations  [370].  Palko et al. found that  overexpression of a truncated form of the N T R K 3 receptor lacking the tyrosine kinase domain, resulted i n a phenotype w h i c h closely resembled N T - 3 deficient cells, suggesting that the truncated isoforms serve a role as N T - 3 sequestering molecules preventing the activation of the functional N T R K 3 isoform [370]. O u r studies showed no evidence of truncated forms of N T R K 3 nor of inserts i n the N T R K 3 portion of E T V 6 - N T R K 3 . Studies of knockout mice defective for the NTRK3 gene displayed n u m e r o u s anomalies i n their neuroanatomical development [371, 372]. There was a 20% loss of neural cells from dorsal root ganglia, 100% loss of Ia muscle afferents from the spinal cord, 50% loss of myelinated axons from the spinal cord/dorsal roots and 30% loss of various fibers from the spinal cord/ventral roots.  In addition, these  mice displayed highly unusual behavioral characteristics mainly i n v o l v i n g positioning of their limbs i n relation to their trunk. development  i n proprioception and  development of the nervous system.  the  This is suggestive of a faulty  can be mostly attributed  to the  faulty  Finally, these mice have a relatively short  lifespan (most die by the third week after birth (P21)) suggesting that these mice may have additional neural defects. U p o n N T - 3 activation, N T R K 3 molecules dimerize w i t h each other w h i c h  100 leads to the autophosphorylation of tyrosine moieties w i t h i n the intracellular domain [372-374]. The phosphotyrosines flanking the tyrosine kinase domain act as anchors for downstream signaling molecules including S N T , S H C , P L C - y l , r A P S , and SH2BB. These molecules, i n turn, activate other molecules ultimately leading to the activation or suppression of certain genes w i t h i n the nucleus.  These  signaling pathways are blocked by the binding of monoamine  activated  Macroglobulin (MA-oc M) to N T R K 1 , N T R K 2  M A - a M is a  2  or N T R K 3  [375].  a-2  2  ubiquitously expressed glycoprotein and may be i n v o l v e d i n neuronal regulation and certain neuropathologic conditions. NTRK1  and NTRK3  have been the only two members of the N T R K family  found to be i n v o l v e d i n a variety of h u m a n cancers. N T R K 1 is activated and has been implicated as a causative factor i n h u m a n prostate [376, 377], breast [378], thyroid [379-381], and colon cancer [382-385].  In colon cancer, for example,  the  N T R K 1 molecule is activated due to a chromosomal rearrangement fusing the coiled coil domain from the tropomyosin 3 (TPM3) gene from chromosome lq22 to the N T R K 1 kinase domain [385]. Papillary thyroid carcinomas, however, have been found to contain rearrangements of NTRK1 domains  to  either  TPR  on  chromosome  Alternatively, expression of either NTRK1  resulting i n its fusion of the kinase lq25  [381, 386], or  or NTRK3  TPM3  [387].  is a marker of favorable  prognosis marker i n neuroblastomas and medulloblastomas (reviewed i n [388]). The predicted E T V 6 - N T R K 3 chimeric protein consists of the E T V 6 H L H protein dimerization domain fused to the P T K domain of the N T R K 3  nerve  101 growth factor receptor (see Fig. 15). The E T V 6 - N T R K 3 fusion is similar to the T P R N T R K 1 fusion where the coiled coil protein dimerization d o m a i n from T P R is fused to the protein tyrosine kinase domain of N T R K 1 [381]. In h u m a n leukemias, the E T V 6 H L H d o m a i n is fused to the P T K domains of PDGFf} receptor, A B L , and J A K 2 [316, 318-320]. Resulting chimeric proteins have constitutively active P T K domains that stimulate corresponding PTK-mediated signal transduction pathways i n leukemic cells [318, 321]. mediated  cell  surface  Receptor PTKs, i n c l u d i n g N T R K 3 , require ligand-  oligomerization  leading  to  autophosphorylation  cytoplasmic tyrosine residues and consequent kinase activation [37].  of  In E T V 6 -  N T R K 3 fusions, the N T R K 3 ligand binding d o m a i n is replaced by the ETV6 H L H domain,  potentially  resulting  in  H L H - m e d i a t e d dimerization  and  ligand-  independent activation of the N T R K 3 protein tyrosine kinase domain. A l s o , since ETV6  is widely expressed i n m a m m a l i a n  tissues w h i l e NTRK3  expression is  primarily restricted to neuronal cells, an additional role of E T V 6 may be to p r o v i d e an  active  promoter  driving  ectopic  expression  of  NTRK3-induced  signal  transduction. In fact, while ETV6 was expressed i n normal fibroblasts, there was n o evidence of NTRK3 expression i n these cells (see Fig. 13). O u r studies indicate that a chimeric P T K is expressed i n CFS that may contribute dysregulation of N T R K 3  signal transduction  pathways.  to oncogenesis  We  have  previously unrecognized rearrangements giving rise to CFS-specific  by  identified ETV6-NTRK3  gene fusions. This is the first k n o w n involvement of the N T R K receptor family i n human oncogenesis. It is also the first ETV6 gene fusion described i n solid tumors, as such rearrangements were previously observed only i n leukemias. These data  102  n top  TO  re re TO  TO 5 •xi  53 S  £  TO  TO  w-T  m  g  m  c -~ > ^ h  w .a cn  .S  o  o  in  p ^  103 therefore provide a new example of a fusion gene partner implicated i n both leukemogenesis and solid tumor formation.  O u r data support the notion that C F S  is a biologically distinct entity, and ETV6-NTRK3  detection provides a diagnostic  screening tool potentially useful i n the clinical evaluation of children w i t h spindle cell tumors.  104  CHAPTER V ETV6-NTRK3  GENE FUSIONS AND TRISOMY 11 ESTABLISH A  HISTOGENETIC LINK BETWEEN MESOBLASTIC NEPHROMA AND CONGENITAL FIBROSARCOMA  5.1  INTRODUCTION Congenital mesoblastic nephroma ( C M N ) is a renal spindle cell tumor that  occurs predominantly i n newborns and very young infants, w i t h most cases being diagnosed before three months of age [224, 389]. This tumor is subdivided into socalled classical and cellular forms based o n histologic features.  Classical C M N  consists of a moderately cellular proliferation of loosely arranged bland fibroblastic cells, while  cellular  (or atypical) C M N is characterized  by h i g h cellularity,  numerous mitoses, and cellular pleomorphism [224]. M i x e d forms are also k n o w n to occur, and it has been suggested that cellular C M N may arise from classical C M N . Despite the infiltrative growth patterns seen i n all forms of C M N , these tumors are generally thought to have an excellent prognosis w i t h surgery alone being curative [390]. However, there are several reports of local recurrences and metastatic spread, and these are almost exclusively associated w i t h the cellular variant [391, 392]. It therefore remains to be determined whether cellular morphology is predictive of a more aggressive course. The histogenesis of C M N is u n k n o w n .  Several lines of evidence point to a  derivation from primitive nephrogenic mesenchyme and a possible relationship to  105 other pediatric kidney tumors  [393].  A link to W i l m s '  tumor  (WT) has been  proposed based on similar patterns of loss of heterozygosity ( L O H ) i n v o l v i n g chromosome l l p l 3 - 1 5 i n W T and C M N [394, 395]. However, other studies failed to detect L O H of this region i n C M N [396]. Moreover, the observed pattern i n C M N of abundant expression of insulin-like growth factor II (IGFII) coupled w i t h lack of W i l m s ' tumor gene 1 (WT1) expression is distinct from the documented expression of both transcripts i n W T [396, 397]. In fact, the pattern of expression of these genes i n C M N is reminiscent of that observed i n clear cell sarcoma of the kidney (CCSK), a highly aggressive pediatric renal neoplasm [398] and it has been proposed that C C S K may be the malignant counterpart of C M N [224]. Cytogenetic analysis of classical and cellular C M N has led to an alternate hypothesis for the derivation of these tumors.  The most consistent  non-random  karyotypic finding i n C M N is trisomy 11, w i t h additional copies of chromosomes 8, 10, 17, and 20 being less commonly reported  [34, 395, 399-401]. Moreover, trisomy  11 appears to correlate w i t h the cellular phenotype [34, 400, 401], whereas classical C M N cases are only rarely associated w i t h this finding [34, 400].  This is h i g h l y  reminiscent  in  of the pattern of trisomy 11 and other  trisomies  congenital  fibrosarcoma (CFS), a malignant tumor of fibroblasts that occurs i n patients aged 2 years or younger that has striking morphologic similarity to cellular C M N [402]. CFS is characterized by local recurrence but, like cellular C M N , has an excellent prognosis and a very low metastatic rate [402].  Its benign counterpart, infantile  fibromatosis (IFB), occurs i n the same age group as CFS but, like classical C M N , lacks trisomy 11 [6]. This, together w i t h ultrastructural similarities, has led to the  106 proposal that classical and cellular C M N are the renal counterparts of IFB and C F S , respectively [403]. A s described i n chapters 3 and 4, we have  recently identified a n o v e l  t(12;15)(pl3;q25) translocation i n CFS, and have shown that this rearrangement fuses the ETV6 (TEL) gene from 12pl3 w i t h the 15q25 neurotrophin-3 receptor gene, NTRK3  (TRKC) [356]. ETV6-NTRK3  fusion transcripts encoding the helix-loop-  helix ( H L H ) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) d o m a i n of N T R K 3 were identified i n CFS tumors but not i n adulttype fibrosarcoma or IFB. The CFS cases studied also showed trisomy 11 [404]. Several previous reports have described alterations of chromosomes  12 a n d / o r 15  i n C M N [399, 401, 405], including a t(12;15)(pl3;q25) [401]. W e therefore screened a series of classical and cellular C M N cases for both ETV6-NTRK3 trisomy 11. NTRK3  gene fusions and  W e found that cellular C M N was strongly correlated w i t h  ETV6-  expression and trisomy 11, but that classical C M N was negative for both  findings. These results suggest that cellular C M N is distinct from classical C M N and is histogenetically related to CFS.  5.2  RESULTS 5.2.1  C l i n i c a l History and Cytogenetics  The clinical features of the  15 C M N cases analyzed i n this study  are  summarized i n Table 8. These included 9 cellular C M N s , 2 mixed C M N s , and 4 classical C M N s i n 9 males and 6 females.  The diagnosis for each case was based o n  107 Table 8. Clinical characteristics and molecular genetic findings i n C M N cases.  ETV6-NTRK3  Trisomy 11  (RT-PCR)  (FISH)  F  +  +  16  M  +  +  cellular  1  F  +  +  4  cellular  2.5  M  +  +  5  cellular  14 days  M  +  +  6  cellular  1  F  +  +  7  cellular  2  F  +  +  8  cellular  1  M  +  ND  9  cellular  9  F  -  -  10  mixed  5 days  M  +  +  11  mixed  7 days  M  +  +  12  classical  36  F  -  -  13  classical  1 day  M  -  -  14  classical  2 days  M  -  -  15  classical  2  F  -  -  Case  CMN  Age  Subtype  (months)  1  cellular  2  2  cellular  3  Sex  +, present; -, absent; N D , not determined  fl  108 standard pathologic criteria [224] and was confirmed by N W T S G pathologic review.  or  CHTN  A l l cases were i n patients 3 years of age or younger, and the  majority were i n patients younger than 3 months as expected for C M N [224]. Two of the cellular C M N cases from this study (case 1 and 2 i n Table 8) h a d previous  cytogenetic analysis performed  on tumor  metaphases.  Case 1 was  previously published as having a t(12;15)(pl3;q25) i n addition to trisomy 11 and other trisomies [401].  Case 2 had a similar karyotype, w i t h a t(12;15)(pl3;q24.1),  trisomy 11, and other trisomies (data not shown).  These findings, coupled w i t h  k n o w n morphologic similarities between cellular C M N and C F S , prompted us to screen the cohort of C M N cases for CFS-associated ETV6-NTRK3  5.2.2  gene fusions [404].  R T - P C R Analysis of C M N Cases  We performed R T - P C R to detect ETV6-NTRK3  fusion transcripts using a  previously described assay [356]. A s shown i n Fig. 16, 8/9 cellular C M N s and 2/2 mixed C M N s were positive for the expected 731 bp ETV6-NTRK3  fusion transcript,  while all 4 classical C M N s were negative. Sequencing of the amplification products demonstrated identical fusion sequences as those described for C F S ([356]; data not shown). We also screened primary tumor tissue from 12 cases of C C S K as w e l l as one case of predominantly spindle cell monomorphic W T i n a 16 m o n t h These cases were uniformly negative for identical ETV6-NTRK3 (data not shown).  child.  fusion transcripts  109  £-1 u 2«  •5? v 1 N o fi QJ  ro  g  QJ  LO  ^  eft OJ  gI •  1"( S1—I  >H  PH  '  cn  feu c4  .£ u PH 3I  H  .* g  CN CN  c  ra CJ  <N  .g  OJ  _ fi  fi  O  u  QJ -*-»  0)  U  CO  S*  i  BH  E-H 0 u I  vo  cn m  m  CN  oo  m o  W VO  01  JH  VO  ro  ON  CO  g  QJ >  ro O fi b QJ QJ  3 6 60 ro  fi  110 5.2.3  Northern Blot A n a l y s i s  To confirm our results, we performed N o r t h e r n blot analysis of a cellular and classical C M N using ETV6 and NTRK3  probes.  Both samples demonstrated  6.2-, 4.3-, and 2.4-kb ETV6 transcripts (data not shown), as expected for this ubiquitously expressed gene [316]. However, only the cellular C M N expressed a 4.3kb transcript also hybridizing either a full length NTRK3 c D N A probe or a probe for the NTRK3 P T K region (see Fig. 17), as is observed for CFS [356]. These data indicate that cellular C M N , but not classical C M N , C C S K , or W T , expresses identical ETV6NTRK3 fusion transcripts as those detected i n CFS.  5.2.4  F I S H Analysis  We next wanted to determine whether there was a correlation i n C M N between the expression of the ETV6-NTRK3  gene fusion and trisomy 11 as we had  previously observed for CFS. W e therefore prepared touch preparations of each C M N case and probed them w i t h an a-centromeric chromosome 11 probe.  As  shown i n a representative example i n Fig. 18, trisomy for chromosome 11 was observed i n every case w h i c h expressed ETV6-NTRK3  fusion transcripts. T r i s o m y  11 was never observed i n C M N cases lacking this gene fusion  (see Table 8),  including the cellular C M N case w h i c h was R T - P C R negative.  5.3  DISCUSSION Congenital mesoblastic nephroma ( C M N ) is a renal, spindle cell tumor of  infancy w h i c h is subdivided into a cellular, mixed, and classical forms based on  Ill  28S - |  18S - • (3-Actin  Figure 17. N o r t h e r n analysis of C M N cases. Blots were probed w i t h a 3' NTRK3 partial c D N A probe encoding the P T K domain (NTRK3-PTK; top panel), and a (3actin c D N A probe (bottom panel) to test for equal loading. The ETV6 c D N A probe detected previously described 6.2, 4.3, and 2.4 kb transcripts i n multiple samples (data not shown), including C F S and C M N cases, while NTRK3-PTK detected a 4.3 kb species only i n C F S and cellular C M N cases (lanes 1 and 3; arrow). Lane 2, classical C M N ; 4, Ewing's TC71; 5, S A N - 2 ; 6, human brain R N A .  112  Figure 18. FISH analysis for trisomy 11. The presence of an extra copy of chromosome 11 was determined by FISH analysis on touch preparations made from primary tissue specimens. A n a-centromeric 11 probe was used to probe touch preparations of all C M N cases. Shown above is a cellular C M N case w i t h three copies of chromosome 11.  113 mitotic activity and degree of cellularity. Histologic and cytogenetic evidence suggested that C M N and CFS are histogenetically related. screen C M N cases for the t(12;15)(pl3;q25)-associated  has  This prompted us to  ETV6-NTRK3  gene f u s i o n  previously reported i n CFS. T w o of two mixed and 8 of 9 cellular C M N s were positive for the ETV6-NTRK3  gene fusion while all 4 classical C M N cases tested  were negative for this alteration.  We also found a striking correlation  between  trisomy 11 and fusion gene expression, w i t h all C M N cases harboring the NTRK3  gene fusion displaying an extra copy of chromosome  11 by FISH.  ETV6This  included two cases (cases 1 and 2, Table 8) w i t h cytogenetically proven extra copies of chromosome 11. Our findings strongly support the notion that cellular C M N and CFS are histogenetically related. The data do not support a relationship w i t h C C S K or W T as has been previously proposed [224,394,395]. Molecular testing for  ETV6-NTRK3  gene fusions therefore provides a potential modality for the diagnosis of cellular C M N . O u r data also suggest that classical and cellular C M N are genetically distinct entities, as no cases w i t h classical morphology displayed either ETV6-NTRK3  gene  fusions or trisomy 11. It is tempting to speculate, as have others [403], that cellular and classical C M N represent the renal counterparts of C F S and IFB, respectively, particularly given the overlapping age ranges of these lesions.  The fact that both  mixed C M N cases tested i n this study expressed ETV6-NTRK3  fusion  lends support to the intriguing possibility that the  transcripts  mixed form represents a  transitional stage i n w h i c h distinct regions w i t h i n classical C M N have acquired the  114 chromosomal  aberrations  found  in  cellular  CMN.  Tissue  microdissection  experiments may be useful to address this question. It remains unclear as to how ETV6-NTRK3  expression confers a proliferative  advantage to tumor cells. The gene fusion links the H L H dimerization d o m a i n of the E T V 6 ETS family transcription factor to the P T K domain of N T R K 3  [356].  NTRK3 is a member of the N T R K family of receptor PTKs and binds n e u r o t r o p h i n 3 (NT-3) w i t h h i g h affinity [358, 359]. N T - 3 binding induces receptor d i m e r i z a t i o n and autophosphorylation of P T K tyrosine residues.  These  residues  serve  as  anchors for downstream signal transduction molecules such as S H C , phospholipase C y l (PLCyl), and PI-3K [358, 359].  We have hypothesized that the E T V 6 H L H  domain induces ligand-independent dimerization and constitutive activation of N T R K 3 signaling. The finding that all fusion positive C M N and CFS cases demonstrate trisomy 11 suggests that this alteration also contributes to tumorigenesis. paternally  expressed  member  of a cluster  of imprinted  The IGFII gene, a  genes  localized  to  chromosome H p l 5 . 5 , encodes an insulin-like growth factor expressed i n certain h u m a n tumors and overgrowth syndromes [406]. It is therefore possible that some form of complementarity or synergism occurs between E T V 6 - N T R K 3 and IGFII signaling pathways that is required for CFS or C M N tumor cells to proliferate, as has been observed for other oncogenes [407]. Further studies w i l l be necessary to elucidate  the  comparative  roles of these alterations  i n oncogenesis  determine if this relationship is unique to tumors of very young children.  and  to  115  CHAPTER VI M O L E C U L A R STUDIES OF THE E T V 6 - N T R K 3 FUSION PROTEIN  6.1  INTRODUCTION Tyrosine kinase receptors are activated through a process k n o w n as l i g a n d  mediated receptor dimerization. Briefly, after a ligand, such as a growth factor, has attached itself to the extracellular ligand binding domain of a tyrosine kinase receptor, the receptor undergoes a conformational change favoring its interaction with  another  similar  receptor  [37].  This  interaction  induces  the  cross  phosphorylation of certain tyrosine moieties w i t h i n the intracellular domain, thus activating  the  receptor-dimer  complex  leading to  further  interactions  with  cytoplasmic substrate proteins. The deactivation of the receptor-dimer complex by specific protein tyrosine phosphatases (PTPs) is equally important i n the regulation of signal transduction [408]. W h e n a cell acquires a chromosomal translocation, a rearrangement may be generated w h i c h produces a fusion gene (discussed i n the previous chapters). T h i s fusion gene consists of part of one gene fused to a part of another gene. In the case of ETV6 gene fusions, we saw how a translocation can result i n the fusion of the H L H d o m a i n from ETV6 to the protein tyrosine kinase d o m a i n from PDGF0R, A B L or J A K 2 [316, 318-320, 409].  These  fusion  proteins  either  lack  the  extracellular ligand binding (regulatory) domains for the tyrosine kinase receptors; instead, these domains are replaced by the H L H domain encoded by the ETV6 gene.  116 The H L H domain is k n o w n to induce dimerization, and therefore, acts to ablate the necessity for ligand induced activation resulting i n the constitutive activation of the tyrosine kinase d o m a i n [339, 410, 411]. The downstream pathways affected by the activated tyrosine kinase domain are, therefore, constantly activated. Other studies have shown fusion genes i n v o l v i n g the ETV6 gene are able to heterodimerize w i t h normal ETV6 as well as the ability to h o m o d i m e r i z e g i v i n g rise to the concept that the introduction of constitutively activated signaling pathways or the disruption of normal ETV6 function, or a combination of both may be the source of oncogenesis [321,412,413]. W e therefore wanted to determine the specific characteristics of the E T V 6 NTRK3  gene product, including downstream interactions (similar to the ones that  interact w i t h N T R K 3 ) , dimerization status (homo- and heterodimerization)  and  phosphorylation status.  6.2  RESULTS 6.2.1  Expression and Phosphorylation Status of ETV6-NTRK3 and ETV6NTRK3 Mutant Proteins in NIH3T3 Cells  Analysis  of  the  ETV6-NTRK3  nucleotide  sequence  using  Lasergene  Navigator software ( D N A S T A R ) estimated the molecular weight of the f u s i o n protein to be approximately 74,300 Da. Immunoprecipitation of lysates derived from primary C F S tumor cells grown i n culture, in vitro translated E T V 6 - N T R K 3 as w e l l as N I H 3 T 3 cells infected w i t h an  ETV6-NTRK3  retroviral  construct  (supplied b y D . W a i i n our lab) w i t h either the a - E T V 6 : H L H or a - N T R K antibodies  117 followed by i m m u n o b l o t t i n g analysis using the opposite antibody detected a doublet (due to the presence of two initiation sites w i t h i n ETV6) i n the 70-80 k D a range, confirming the presence of E T V 6 - N T R K 3 proteins (see Fig. 19). We  were interested i n determining w h i c h domains of the  ETV6-NTRK3  protein ( H L H domain, P T K domain, A T P binding domain, etc.) were important for transformation  and what these domains were responsible for.  We  therefore  generated a series of constructs (see Table 5 i n chapter 2) w h i c h were used to infect N I H 3 T 3 cells including:  E T V 6 - N T R K 3 (as the positive control), Vector (NIH3T3  cells transfected w i t h M S C V p a c vector alone containing no insert, as the negative control), A H L H (we deleted the H L H dimerization d o m a i n i n E T V 6 - N T R K 3 to test for its significance i n oncogenesis), PLGyQ, PLCyT, PLCyE (the tyrosine residue specific for P L C y l binding was replaced w i t h a glutamine (Q), threonine (T), or a glutamate (E) residue, respectively, to ablate the ability of P L C y l to bind to E T V 6 N T R K 3 and test for its significance i n oncogenesis), A c t i v a t i o n Loop Dead ( A L D ) (the three tyrosines k n o w n to be essential for autophosphorylation of N T R K 3 were mutated i n order to determine their importance i n the oncogenic process), and KinaseDead  (KD) (the  phosphorylation  A T P binding  of the E T V 6 - N T R K 3  site  was  mutated  so  that  tyrosine  protein w o u l d be ablated i n order  to  determine the significance of tyrosine phosphorylation i n the oncogenic process) [414, 415]. O f these constructs, only E T V 6 - N T R K 3 and the PLCyQ, PLCyT, PLCyE mutants were able to transform NIH3T3 cells while cells infected w i t h Vector,  118  co ai  ^ S  CO  2 Q  CN  LO  fi  CU  2 fi (3  g3 CS  fi£ i  Q  V  ^SH  O 'fi  rc a> ON  g  PH  -  > 3 < f— 1  .5 U QJ  CN  * fi  P_CN  $ a; co fi M 4S 3 "a3 2d ^  v  v  r  -1  < !> 1 — 1  Z fi  pq *H  MH <U CO CO ^  ro  i OJ  fi M  c ro  -!->  OH  CU  CO  I rofi -fibp • ON HH rH OJ I X  W  t—1  D  a l-H PH  la  u  QJ  119 A H L H , A L D and K D constructs appeared morphologically normal. These cells were subsequently analyzed by immunoprecipitation and i m m u n o b l o t t i n g , as described above for cells expressing E T V 6 - N T R K 3 (see Fig. 19). To determine  the tyrosine phosphorylation status of the  ETV6-NTRK3  protein and the various mutants, we immunoprecipitated lysates from v a r i o u s N I H 3 T 3 cells expressing either E T V 6 - N T R K 3 or one of the mutant constructs w i t h anti-NTRK3  antibody  and  subsequently  immunoblotted  with  the  anti-  phosphotyrosine antibody, RC-20. W e were able to show tyrosine p h o s p h o r y l a t i o n for E T V 6 - N T R K 3 , A L D , and PLCyQ,  PLCYT,  PLCyE, while Vector, A H L H , and K D  failed to show any signs of tyrosine phosphorylation (see Fig. 20).  6.2.2 To  E T V 6 - N T R K 3 Homodimerizes and Heterodimerizes w i t h E T V 6 examine  the  possibility  that  ETV6-NTRK3  is  capable  of  homodimerization, we took advantage of the h i g h affinity between glutathione-Stransferase (GST) and glutathione-agarose beads [416]. A vector coding for a 5'-GST protein was used to generate an G S T - E T V 6 - N T R K 3 construct, w h i c h was used to transfect SF9 insect cells for the production and subsequent purification of G S T E T V 6 - N T R K 3 protein. We checked for homodimerization by m i x i n g purified G S T - E T V 6 - N T R K 3 fusion  protein w i t h in vitro  translated  Methionine and glutathione beads. ETV6-NTRK3  protein by the  ETV6-NTRK3  radiolabeled w i t h  3 5  S-  E T V 6 - N T R K 3 was p u l l e d d o w n w i t h the G S T -  addition of glutathione  beads,  but  not  with  glutathione beads alone suggesting that E T V 6 - N T R K 3 is able to homodimerize (see  00  01  in  c re > '53 'Z,  re >  5 CQ  co  TO  <  PQ  CO  121 Fig. 21a). Since dimerization is thought to act through the H L H d o m a i n we tested the ability of the A H L H mutant  (ETV6-NTRK3  dimerize w i t h either ETV6 or E T V 6 - N T R K 3 .  lacking the H L H domain)  to  The A H L H mutant d i d not d i m e r i z e  w i t h E T V 6 nor the E T V 6 - N T R K 3 protein (see Fig. 21b). To determine if E T V 6 N T R K 3 was able to heterodimerize w i t h ETV6, we co-m vitro NTRK3  and  ETV6,  immunoblotted  with  immunoprecipitated anti-ETV6:HLH  coimmunoprecipitating  with  anti-NTRK3  antibody.  along w i t h E T V 6 - N T R K 3  translated E T V 6 -  Figure  antibody  21c  shows  and ETV6  protein suggesting that  the  E T V 6 - N T R K 3 is able to heterodimerize w i t h ETV6.  6.2.3  Downstream Interactors Affected b y the E T V 6 - N T R K 3 M o l e c u l e  NTRK3  is k n o w n to interact w i t h specific cytoplasmic tyrosine kinases  including S H C , GRB2, SH2Bp, r A P S , PI3K, and P L C y l [310, 311, 417, 418]. W e were interested i n determining if these molecules were able to interact w i t h the E T V 6 N T R K 3 fusion protein. W e were interested i n determining w h i c h regions of the ETV6-NTRK3  protein were important i n downstream interactions.  analysis of the  ETV6-NTRK3  chimera  determined  that the  Sequence  S H C and  PI3K  interaction site were lost as a result of the position of the breakpoint, but the P L C y l site was retained  [356].  W e therefore  wished to elucidate the  downstream  interactors w i t h E T V 6 - N T R K 3 by testing S H C , SH2Bp\ GRB2, PI3K, and P L C y l (rAPS was not included due to the lack of antibody). W e lysed cells expressing the ETV6-NTRK3  protein, immunoprecipitated  with  anti-NTRK3  antibody,  subsequently immunoblotted w i t h antibodies toward either S H C , G R B 2 ,  and  122  II ro J3  m  CN  ro ro  ro cu 3 ro  cn ro ro T3  rosi-sax s .a ;  PQ  ro  « 5 a  ro a;  gre jo ro  CO  CN  ro  ro  ills  123 SH2BP (data not shown), PI3K or P L C y l (see Fig. 22).  Of these molecules, o n l y  P L C y l coimmunoprecipitated w i t h E T V 6 - N T R K 3 as w e l l as w i t h the A L D mutant. Similar analysis of the other E T V 6 - N T R K 3 mutants,  i n c l u d i n g Vector, PLCyQ,  PLCyT, PLCyE, A H L H , and K D failed to coimmunoprecipitate P L C y l (see Fig. 23). None of the constructs, including E T V 6 - N T R K 3 , were able to coimmunoprecipitate S H C , GRB2, PI3K or SH2Bf3.  6.2.4  Subcellular Localization  We were interested i n the subcellular localization of the E T V 6 - N T R K 3 f u s i o n protein as this w o u l d help explain its mechanism  of action as an oncogenic  molecule w i t h i n the cell. Cells infected w i t h E T V 6 - N T R K 3 , A H L H , K D , or Vector constructs were fixed i n paraformaldehyde, incubated w i t h a n t i - N T R K 3 antibody followed by another  incubation w i t h  analyzed by confocal microscopy.  rhodamine-anti-rabbit  and  subsequently  Our preliminary results suggest that  ETV6-  N T R K 3 is mainly localized w i t h i n the cytoplasm w i t h l o w amounts i n the nucleus. Similar results were obtained for A H L H and K D , while Vector showed relatively little fluorescence and was used as the negative control (see Fig. 24).  6.3  DISCUSSION The ETV6 gene has been shown to be i n v o l v e d i n numerous  translocations  giving rise to various gene fusions i n human leukemias (see Table 7, Chapter 3).  12  3 4  GRB2>-  F I G U R E 22. E T V 6 - N T R K 3 interacts w i t h P L C y b u t not w i t h S H C , GRB2, or PI3K p85 subunit. Whole cell lysates were prepared from a human medullary thyroid carcinoma cell line overexpressing wild-type N T R K 3 (lane 1), as well as from NIH3T3 cells expressing E T V 6 - N T R K 3 (lane 2), Vector (lane 3), or K D (lane 4). Immunoprecipitation was performed with antibodies against the N T R K 3 P T K domain followed by immunoblotting with antibodies directed against S H C , GRB2, PI-3K, or P L C y l as indicated. Only wild-type N T R K 3 was found to associate w i t h S H C , GRB2, and PI-3K (Lanes 1 of top three panels), while both N T R K 3 and E T V 6 - N T R K 3 bound P L C y (lanes 1 and 2 of bottom panel).  125  ON  OO  fN  co M  CN  5 2 3 ^  •2 T3  CO  CO  p $  QJ  126  F I G U R E 24. Confocal microscopy of E T V 6 - N T R K 3 and A H L H expressing NIH3T3 cells. NIH3T3 cells expressing either E T V 6 - N T R K 3 (panels A and B) and A H L H (panels C and D) were probed with either a - N R T K 3 antibody (panels A and C) or w i t h a - E T V 6 : H L H antibody (panels B and D). W h e n probed with a - N T R K 3 antibody, confocal microscopy showed localization of E T V 6 - N T R K 3 and A H L H to the cytoplasm with relatively low amounts in the nucleus. Probing w i t h a - E T V 6 : H L H antibody detected w i l d type E T V 6 i n addition to E T V 6 - N T R K 3 and as a result showed more intense nuclear staining.  127 Most of the gene fusions encoding the H L H d o m a i n of  ETV6  as the 5' end, possess  sequences encoding a tyrosine kinase domain as the 3' end [320, 409, 419]. This is expected to result i n constitutive activation of the P T K d o m a i n due to the l i g a n d independent dimerizing capabilities of the H L H domain. The recently characterized  gene fusion i n C F S and cellular  ETV6-NTRK3  C M N was identified i n our studies predominantly as a protein doublet i n lysates derived from N I H 3 T 3 cells infected w i t h either an E T V 6 - N T R K 3 construct or one of the mutants.  This can, i n part, be explained by the fact that the  two transcription initiation sites.  ETV6  gene has  Following the translation of these two species,  post-translational modifications (primarily tyrosine phosphorylation of the N T R K 3 portion) can further divide each of the two bands producing a total of four bands (two unphosphorylated and two phosphorylated). A study analyzing the E T V 6 and E T V 6 - C B F A 2 fusion protein identified this fusion protein as several species due to alternative initiation sites i n the  ETV6  different  gene i n addition to the post-  translational modification of these proteins [338, 344].  Further studies are still  needed to clarify the role of each species i n the oncogenic process. The A H L H and K D mutants were unable to transform N I H 3 T 3 cells and d i d not show any sign of being tyrosine phosphorylated. The only difference between K D and E T V 6 - N T R K 3 is a single amino acid (lysine 380 to an arginine), that blocks A T P b i n d i n g w h i c h is crucial for P T K activity. Previous attempts to inactivate the A T P b i n d i n g site i n N T R K  molecules  showed similar  results  as the  NTRK  molecule was no longer able to tyrosine phosphorylate (even after ligand-induced dimerization) and interact w i t h downstream molecules [420]. This indicates that a n  128 intact P T K d o m a i n is essential for transformation. The A H L H mutant lacks most of the helix loop helix domain from exons 3 and 4, w h i c h has been s h o w n to be important i n dimerization.  Dimerization is essential for bringing the  tyrosine  kinase domains i n close proximity so that they can phosphorylate each  other,  suggesting that the H L H domain is critical i n the process of activation of the tyrosine kinase d o m a i n i n the E T V 6 - N T R K 3 fusion. The A H L H and K D mutants, therefore, provide evidence that dimerization and tyrosine phosphorylation are both required for transformation of NIH3T3 cells. We were able to show that the E T V 6 - N T R K 3 can h o m o d i m e r i z e as w e l l as heterodimerize w i t h ETV6.  E T V 6 - N T R K 3 heterodimerization w i t h E T V 6 m i g h t  ablate the normal function of ETV6 by interfering w i t h its D N A binding potential. There has been some evidence suggesting that ETV6 may actually be a t u m o r suppressor gene [421-423].  Furthermore, we were able to show that the  ETV6-  N T R K 3 fusion protein was predominantly localized w i t h i n the cytoplasm w i t h lower amounts i n the nucleus. Further studies are necessary to determine the role of dimerization and oncogenesis i n these tumors. Our  results suggest that most of the k n o w n signaling interactors  with  N T R K 3 (namely, S H C , GRB2, PI3-K, and SH2BB) do not associate w i t h the E T V 6 N T R K 3 molecule. Phospholipase C - y l (PLCyl) was, however, able to interact w i t h the E T V 6 - N T R K 3 fusion protein. p h o s p h o l i p i d phosphodiesterases  P L C y l is a member of the family of i n o s i t o l [424]. There are a total of three members to this  family i n c l u d i n g B, y, and 8. PLCy is activated by the intracellular tyrosine kinase  129 domains of its respective receptors [425]. Activation of P L C y leads to the p r o d u c t i o n of intracellular second messengers inositol (1, 4, 5)-triphosphate (InsP ) and sn 1, 23  diacylglycerol (DAG) [424]. A t the amino terminus of the P L C molecules is a structure k n o w n as the pleckstrin homology d o m a i n (PH) responsible for facilitating b i n d i n g of P L C y to PtdInsP and other inositol phosphates [426]. The P L C molecules also contain an EF 2  H a n d domain w h i c h is thought to b i n d C a , as these molecules are C a + +  enzymes [427]. Deletion of the EF H a n d abrogates activity [428].  ++  dependant  The two most  highly conserved structures i n m a m m a l i a n P L C molecules are the X and Y boxes. These structures are essential for activity and may also determine w h i c h substrates and subsequent reactions the molecule can support [429].  O n the tertiary l e v e l ,  these boxes form a structure k n o w n as a T I M barrel. W i t h i n the T I M barrel are P H , SH2 and S H 3 domains responsible for the specific interactions w i t h  substrate  molecules such as PtdInsP (SH2 domains were first discussed i n Chapter 1 as 2  structures essential for the interaction w i t h phosphorylated tyrosines) [430].  The  last d o m a i n i n P L C y (conserved and present i n PLC|3 and 8) is the C2 d o m a i n , w h i c h is C a  + +  dependant and seems to act as an interface between the EF H a n d and  the T I M barrel catalytic domain [431, 432]. P L C y l is a ubiquitously expressed tyrosine kinase substrate responsible for the control  of  intracellular  levels  of  Ca  ++  and  D A G [433].  After  tyrosine  phosphorylation of P L C y l , the molecule translocates to the membrane where the P H domains recognize PI 4, 5-P and Ins 1, 4, 5-P as ligands. The deactivation by 2  3  130 phosphatases is evident, but unclear. In addition to tyrosine phosphorylation, the P L C y l molecule undergoes serine-threonine phosphorylation, but the kinases and phosphatases of this system have not been characterized yet. The m a i n reaction catalyzed by P L C y l is the conversion of Ptdlns 4,5-P to D A G and Ins 1,4,5-P . Ins 1, 2  3  4, 5-P is responsible for stimulating the secretion of C a 3  ++  from the endoplasmic  reticulum [433]. Ins 1, 4, 5-P is responsible for tethering protein kinase C (PKC) to 3  the membrane while D A G acts as a potent activator of P K C [433]. There has been some evidence suggesting that P K C then activates the M A P K pathway t h r o u g h RAF  and can ultimately  result  i n an increase  i n cellular proliferation  (in  undifferentiated cells) and other cellular responses such as contraction, secretion and membrane conductance (in differentiated cells) [434, 435]. Our studies showed that there is no loss i n the transformation capabilities of n o n - P L C y l binding E T V 6 - N T R K 3 mutants, suggesting that P L C y l signaling may not contribute to E T V 6 - N T R K 3 transformation.  O u r results failed to show  an  association between E T V 6 - N T R K 3 and k n o w n N T R K 3 interactors such as S H C , GRB2, PI3-K, and SH2BB.  These results, however, do not rule out that these  molecules are i n v o l v e d indirectly i n E T V 6 - N T R K 3 transformation activity. Other adaptor molecules may link E T V 6 - N T R K 3 to k n o w n N T R K 3 signaling pathways. These molecules must be assayed directly to assess their possible roles i n E T V 6 N T R K 3 signaling. Alternatively, a completely novel pathway may be i n v o l v e d . The increase i n proliferation and transformation  seen i n E T V 6 - N T R K 3  infected  NIH3T3 cells can be explained by the fact that some other molecule is able to  131 associate w i t h E T V 6 - N T R K 3 and activate proliferative a n d / o r cell s u r v i v a l signal transduction pathways. The possibility that the E T V 6 - N T R K 3 fusion is interacting w i t h normal ETV6 and dysregulating it is unlikely, since the K D mutant is capable of d i m e r i z i n g w i t h normal ETV6 (data not shown), but is non-transforming. view of this, the A H L H mutant  In  is non-transforming because of its inability to  dimerize and activate the tyrosine kinase domain and not because of its inability to dimerize and interfere w i t h normal ETV6 function. Further studies such as yeast two hybrid screening w i l l be necessary (and are currently being performed i n o u r laboratory) for characterizing new and essential downstream interactors w i t h the N T R K 3 portion (and possibly the ETV6 portion) of the E T V 6 - N T R K 3 chimeric fusion.  132  CHAPTER VII SUMMARY AND CONCLUSIONS  The dilemma i n finding a cure for cancer is that there w i l l not be one cure for all cancers, but rather a specific conglomerate of approaches for each i n d i v i d u a l cancer.  Developing treatment protocols for a specific cancer w o u l d require a  number of factors. The first requirement, w h i c h w o u l d be crucial i n d e t e r m i n i n g the course of action for a particular tumor, is the ability to accurately differentiate and diagnose that tumor from other tumors.  Secondly, an in-depth knowledge of  the molecular basis for tumorigenesis of the specific tumor is essential.  Once the  tumor has been characterized at the molecular level, the knowledge o n h o w it has evolved and maintains itself needs to be integrated i n order to develop a treatment strategy. The treatment protocol w o u l d need to target tumor cells while l e a v i n g normal cells unharmed. We  have  accomplished i n  part  the  first  two  tasks  fibrosarcoma (CFS), a soft tissue pediatric spindle cell lesion.  with  congenital  C F S is difficult to  diagnose because of its histologic similarity w i t h adult type fibrosarcoma, aggressive fibromatosis and infantile fibromatosis.  Cytogenetic analysis on a series of C F S ,  ATFS,  a recurring  A F B and  IFB cases revealed  rearrangement  involving  chromosomes 12 and 15 only i n CFS. Whole chromosome F I S H of a C F S case u s i n g a chromosome  12 painting probe revealed a portion of chromosome  smaller acrocentric chromosome.  This rearrangement  12 on a  was not seen i n n o r m a l  133 fibroblastic tissues. This warranted the characterization of the rearrangement o n a molecular level, i n hopes of finding a recurring molecular marker of C F S w h i c h w o u l d be an invaluable tool for the pathologist.  7.1  IDENTIFICATION OF  A  RECURRING  t(12:15)  in  CONGENITAL  FIBROSARCOMA Cytogenetic analysis revealed 12pl3 and 15q25-q26. chromosome  FISH  specific probes  a rearrangement  analysis and  using  region  involving  a combination  specific  chromosomes  of  a-centromeric  Y A C probes  translocation of material from chromosome 12pl3 to chromosome  identified  a  15q26. One of  the Y A C s , namely 817_H_1, was found to be split, indicating that it spanned  the  breakpoint and contained the gene i n v o l v e d . This Y A C was k n o w n to harbor the ETV6 gene, w h i c h has been rearranged i n numerous hematopoietic  malignancies.  H i g h resolution FISH mapping using cosmids specific for certain exons of the ETV6 gene placed the breakpoint w i t h i n the ETV6 gene thus confirming its i n v o l v e m e n t . E T V 6 belongs to the ETS family of transcription factors and has been s h o w n to be i n v o l v e d i n gene fusions i n hematopoietic malignancies. gene i n both  hematopoietic  malignancies  and  The i n v o l v e m e n t of a  sarcomas  has  been  described  previously, as the ERG and FLU genes (other ETS family members) are found to be fused w i t h two similar genes, EWS and TLS/FUS, translocations i n h u m a n solid tumors and leukemias.  as a result of  chromosome  134 7.2  THE  ETV6-NTRK3  GENE  FUSION  CHARACTERIZES  CONGENITAL  FIBROSARCOMA W e hypothesized that E T V 6 was i n v o l v e d i n a gene fusion i n C F S . ETV6 gene is k n o w n to be oriented i n a telomere chromosome  12pl3.  Therefore  The  to centromere fashion  the t(12;15) i n C F S is expected to result i n the  translocation of 5' ETV6 material to chromosome  15. We therefore performed 3'  rapid amplification of c D N A (3'RACE) using k n o w n ETV6 sequence primers amplify and clone the breakpoint.  on  to  Sequence analysis of R A C E products revealed 5'  ETV6 sequence until nucleotide 1033, w h i c h corresponds to the last nucleotide i n ETV6 exon 5. The remaining sequence was compared to public databases and was 100% homologous  to a portion of the NTRK3  gene.  The rearrangement  confirmed by Southern analysis and the presence of an ETV6-NTRK3  was  gene fusion  was confirmed by N o r t h e r n analysis and RT-PCR. A s this is one of two recurring chromosomal abnormalities along w i t h trisomy 11, it was hypothesized that this fusion gene is etiologic i n CFS oncogenesis.  7.3  TRISOMY 11 A N D THE  ETV6-NTRK3  GENE FUSION LINK CONGENITAL  FIBROSARCOMA TO CONGENITAL MESOBLASTIC N E P H R O M A Congenital mesoblastic nephroma ( C M N ) is an infantile spindle cell t u m o r of the kidney w h i c h has an excellent prognosis similar to that of C F S . C M N is subdivided into classic and cellular forms depending o n the degree of cellularity and mitotic activity of the spindle cells. The cellular variant is virtually identical histologically and cytogenetically to CFS, and this morphologic overlap has led to  135 the hypothesis that these tumors are histogenetically related. have  reported  common  trisomies  Cytogenetic studies  i n C F S and cellular C M N , particularly of  chromosome 11. We analyzed C M N cases and found that the t(12;15)(pl3;q25) rearrangement i n C F S is also present i n cellular C M N and may underlie the distinctive biological properties of these two tumors. ETV6-NTRK3  Analysis of m R N A  revealed the expression of  chimeric transcripts i n 8 of 9 cellular C M N cases as w e l l as i n 2 of 2  mixed C M N cases. Four of four classical C M N cases were negative for the NTRK3.  In addition, we found trisomy 11 to be strongly correlated w i t h the  presence of the ETV6-NTRK3 Our  ETV6-  gene fusion.  studies therefore indicate that congenital fibrosarcoma  and cellular  congenital mesoblastic nephroma are histogenetically related. Table 9 s u m m a r i z e s the results of R T - P C R analysis on a series of C F S , C M N , A T F S , IFB, and A F B primary tumor samples to date. Briefly, the E T V 6 - N T R K 3 fusion was found i n 100% of CFS cases (n=15), 90% of cellular C M N cases (n=10) and 100% of m i x e d C M N cases (n=2) analyzed. Fusion transcripts were not detected i n A T F S (n=10), IFB (n=12), and A F B (n=5) cases, nor i n classical C M N cases analyzed (n=4).  Only  one of ten cellular C M N cases was negative for the fusion transcript.  7.4  M O L E C U L A R STUDIES O F T H E E T V 6 - N T R K 3 F U S I O N P R O T E I N Understanding the oncogenic process requires the familiarization w i t h the  complex biochemical interactions w i t h i n the tumor cell.  W e generated a f u l l  length E T V 6 - N T R K 3 construct encoding the helix-loop-helix ( H L H ) dimerization  136  T A B L E 9. Summary of ETV6-NTRK3  TUMOR  (TEL-TRKC)  Analysis.  ETV6-NTRK3  ETV6-NTRK3  NUMBER ANALYZED  FUSION POSITIVE  CFS  12  12  0  ATFS  10  0  10  IFB  12  0  12  AFB  5  0  5  1. Classical  4  0  4  2. Cellular  10  9  1  3. Mixed  2  2  0  FUSION NEGATIVE  CMN  Abbreviations. CFS, congenital fibrosarcoma; A T F S , adult-type fibrosarcoma; IFB, infantile fibrosarcoma; A F B , aggressive fibrosarcoma; C M N , congenital mesoblastic nephroma.  137 domain of ETV6 fused to the protein tyrosine kinase (PTK) d o m a i n of N T R K 3 . NIH3T3 cells were infected w i t h recombinant retroviral viruses carrying either the full-length ETV6-NTRK3  c D N A or one of the mutants (kindly p r o v i d e d by D a n i e l  W a i i n our laboratory). Cells expressing the ETV6-NTRK3 transformed  phenotype  construct exhibited a  and formed macroscopic colonies i n soft  hypothesized that chimeric proteins mediate  transformation  agar.  We  by dysregulating  N T R K 3 signal transduction pathways v i a ligand-independent dimerization and PTK-autophosphorylation.  To test this hypothesis, a series of different  were generated to help determine protein  were  necessary  for  mutants  w h i c h regions of the E T V 6 - N T R K 3  oncogenesis.  We  showed  that  fusion  ETV6-NTRK3  homodimerizes and is capable of forming heterodimers w i t h wild-type E T V 6 in vitro.  The H L H domain of ETV6 i n the E T V 6 - N T R K 3 fusion was deleted i n order  to investigate the role of protein dimerization i n transformation ( A H L H mutant). The A H L H mutant was not able to associate w i t h ETV6, nor w i t h E T V 6 - N T R K 3 . Cells expressing this mutant protein were morphologically non-transformed failed to grow i n soft agar.  and  To investigate the role of the N T R K 3 P T K d o m a i n ,  N I H 3 T 3 cells were transfected w i t h a variety of E T V 6 - N T R K 3  mutants  with  activation loop amino acid substitutions ( A L D ) as well as a kinase inactive m u t a n t unable to b i n d A T P (KD). The three P T K activation-loop tyrosines mutated ( A L D ) to phenylalanines  still  became  tyrosine phosphorylated  but were  unable  to  transform NIH3T3 cells. The K D mutant failed to autophosphorylate and lacked transformation ability. Of a series of signaling molecules well k n o w n to b i n d to wild-type N T R K 3 , only P L C y l was found to associate w i t h and become tyrosine  138 phosphorylated by E T V 6 - N T R K 3 .  Interestingly, several P L C y l b i n d i n g mutants  were unable to b i n d P L C y l , but were still capable of transforming N I H 3 T 3 cells suggesting that another transforming  pathway is being activated and is responsible for  abilities of E T V 6 - N T R K 3 .  the  In addition, preliminary subcellular  localization studies showed that the E T V 6 - N T R K 3 fusion protein localizes m a i n l y i n the cytoplasm, w i t h limited presence i n the nucleus.  O u r studies confirm that  E T V 6 - N T R K 3 is a transforming protein that requires both an intact d i m e r i z a t i o n domain and a functional P T K domain for transformation activity.  7.5  GENERAL COMMENTS The discovery of the ETV6-NTRK3  gene fusion i n C F S and cellular C M N  lead us to screen other cancers for the same gene fusion. Interestingly, we were able to detect the ETV6-NTRK3 patient.  gene fusion i n a breast carcinoma from a 6 year o l d  Cytogenetic analysis on  this  case  confirmed  the  presence  of  the  t(12;15)(pl3;q25) but failed to show further evidence of any other c h r o m o s o m a l abnormalities including trisomy chromosome 11. Our transfection studies h a v e shown that the ETV6-NTRK3  gene fusion product has transformation  ability,  w h i c h supports the notion that the breast carcinoma may have arisen solely due to the gene fusion.  Another group has recently identified the ETV6-NTRK3  fusion i n an adult acute myeloid leukemia [436].  The ETV6  gene has  gene been  implicated i n numerous hematopoietic rearrangements, but this is the first report of NTRK3  involvement w i t h a leukemia.  This makes the ETV6-NTRK3  gene  fusion the only documented gene fusion to date w h i c h has been involved i n both a  139 solid tumor as w e l l as a leukemia. The ETV6-NTRK3  gene fusion may therefore  have a wider spectrum of involvement i n h u m a n malignancies.  Further studies  are required to test this possibility i n detail. Recent evidence suggests that the ETV6 central d o m a i n (previously thought to contain no k n o w n domains) mediates transcriptional repression by associating with  SMRT  and  mSin3A,  while  the  ETV6  H L H domain  transcription through a mechanism that is independent [437]. This d o m a i n is a part of the ETV6-NTRK3  represses  of k n o w n  gene  corepressors  gene fusion. Further studies are  therefore needed to determine if there are other molecules interacting w i t h E T V 6 N T R K 3 w h i c h are responsible for oncogenesis.  This is currently being explored i n  our laboratory by the use of yeast-2-hybrid screening. Future studies, some of w h i c h are being explored currently i n our laboratory, include elucidating the signal transduction pathways w h i c h are being utilized by ETV6-NTRK3.  To look for other interactors w i t h E T V 6 - N T R K 3 , yeast-2-hybrid  screening is currently being used to identify novel interactors. To help increase the » resolution of the yeast two hybrid approach, the E T V 6 - N T R K 3 has been d i v i d e d into two so that interactors w i t h the ETV6 portion and the N T R K 3 portion can be studied independently. Specific inhibitors of signaling pathways (e.g., w o r t m a n n i n specifically inhibits PI-3K; PD98059 inhibits M E K 1 , and molecules) should be tested to determine  U73122 inhibits P L C  their impact o n tumor  growth and  whether or not they can be used to treat CFS and cellular C M N i n patients. Similarly further research is needed to see if N T R K specific inhibitors could be used to treat CFS and cellular C M N (e.g., K252a and CEP-751 are effective N T R K tyrosine  140 kinase inhibitors domain  [438, 439]).  To explore the possibility that the  of ETV6 is dimerizing w i t h other  molecules  or  functions to the oncogenic process, the 3'-portion of NTRK3  dimerization  contributing  other  i n v o l v e d i n the C F S  translocation has recently been fused to an inducible dimerization domain, F K B P [440, 441], i n our laboratory.  This w i l l allow us to control dimerization  3 6 v  and  therefore the oncogenic activity of the N T R K 3 portion of E T V 6 - N T R K 3 i n N I H 3 T 3 cells  and  will  transformation  provide process.  valuable  information  For example,  if the  on  the  proliferative  introduction  dimerization domain results i n the failure of transformation, explanation  might be that the ETV6 portion  of  artificial  then a possible  of E T V 6 - N T R K 3  important domain that is required for proper transformation  the  and  contributes  an  ability. Finally, some  of the most useful information can be derived from in vivo models.  Currently o u r  laboratory is exploring the oncogenic activity of E T V 6 - N T R K 3 i n transgenic mice.  141  REFERENCES 1.  Enzinger, F . M . and Weiss, S.W. Mosby. 989.  Soft Tissue Tumors. 1988, St. Louis: C . V .  2.  Soule, E . H . and Pritchard, J. Cancer, 40 , 1711-1721.  3.  Neifeld, J.P., Berg, J.W., G o d w i n , D . , and Salzberg, A . M . 1978. 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