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Use of allelic loss to predict malignant risk for low-grade oral epithelial lesions Cheng, Xing 2000

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Use of Allelic Loss to Predict Malignant Risk for Low-Grade Oral Epithelial Lesions by Xing Cheng M.Sc.,Xian Medical University, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Oral Biological and Medical Sciences, Faculty of Dentistry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November 1999 ©Xing Cheng, 1999  In presenting this thesis in partial fulfillment o f the requirements for an advanced degree at the University o f British Columbia, I agree that the library shall make it freely available for reference and study. I further agree that permission for extensive copying o f this thesis for scholarly purposes may be granted by the head o f m y department  or b y his or her  representatives. It is understood that copying or publication o f this thesis for financial gain shall not be allowed without m y written permission.  Department o f Oral Biological and Medical Sciences The University o f British Columbia Vancouver Canada  ABSTRACT Oral squamous cell carcinomas (SCC) are believed to develop through progressing stages of oral premalignant lesions (histologically divided into hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia and carcinoma in situ, CIS) before finally become invasive. Prognosis is poor once invasion occurs and S C C is formed. The key to improve this gloomy prognosis may lie in early diagnosis and proper management of oral premalignant lesions. However, the majority o f oral premalignant lesions, particularly low-grade lesions (hyperplasia, mild and moderate dysplasia) do not progress into cancer. Since carcinogenesis is underlain by changes to critical control genes, it is hypothesized that the small percentage of progressing premalignancies differs genetically from the majority of non-progressing lesions. One of the best approaches to identifying genetic changes critical to oral cancer progression is to compare progressing and non-progressing oral premalignant lesions. However, such samples are rare, and little information is available on genetic changes in progressing and non-progressing lesions. This thesis, for the first time, compared genetic changes in 116 cases of progressing and non-progressing low-grade oral premalignant lesions by microsatellite analysis for loss o f heterozygosity (LOH) using 19 probes for 7 chromosome arms. The progressing and non-progressing cases showed dramatically different L O H patterns of multiple allelic losses. A n essential step for progression seems to involve L O H at 3p and/or 9p as virtually all progressing cases showed such loss. However, L O H at 3p and/or 9p also occurred in non-progressing cases. Individuals with L O H at 3p and/or 9p but no other arms exhibit only a  ii  slight increase of 3.8-fold in relative risk for developing cancer.  In contrast, individuals with  additional losses (on 4q, 8p, l l q , 13q or 17p), which appeared uncommon in non-progressing cases, showed 3 3-fold increases in relative cancer risk. The results suggest that L O H patterns will facilitate the prediction of the malignant potential of low-grade premalignancies. They also demonstrate the predictive value of assaying allelic loss at arms other than 3p and/or 9p. We may more precisely predict the malignant potential of low-grade premalignancies i f we combine molecular analysis with the clinical assessment to premalignent lesions. Clinically, the data also support the belief that the molecular tools such as microsatellite analysis for L O H may be used in differentiating between progressing and non-progressing lesions. The identification of molecular changes that can be used to predict the likely behavior of lowgrade lesions would allow the clinician to identify which patients with low-grade lesions should be managed more aggressively and thus, should improve prognosis.  iii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENT  iv  LIST OF T A B L E S  vii  LIST OF FIGURES  viii  ABBREVIATIONS  ix  ACKNOWLEDGMENTS  :  xi  DEDICATION  1.  xii  INTRODUCTION  1  1.1. Importance of studying oral premalignant lesions  1  1.2.Oral mucosa  2  1.3.Oral premalignant lesions and their relation to oral S C C  2  1.4. Problems with the histological progression model  5  1.5. Molecular biology o f carcinogenesis  6  1.5.1. Genetic pathway of carcinogenesis  6  1.5.2. The major genes involved in tumorigenesis  9  1.6. Loss o f heterozygosity (LOH) and microsatellite analysis  11  1.7. L O H in oral and head and neck malignant lesions  13  1.8. L O H in oral and head and neck premalignant lesions  18  1.9. A molecular progressing model for oral S C C  21  2.  S T A T E M E N T OF T H E P R O B L E M S  23  3.  OBJECTIVES  25  4.  HYPOTHESIS  26  iv  5.  E X P E R I M E N T A LDESIGN A N D STATISTICAL A N A L Y S I S  26  5.1. Experimental design  27  5.2. Statistical analysis 6.  7.  8.  28  MATERIALS AND METHODS  29  6.1. Sample collection  29  6.2. Sample sets  29  6.3. Histological diagnostic criteria for the samples  31  6.4. Slide preparation  32  6.5. Microdissection  33  6.6. Sample digestion and D N A extraction  33  6.7. D N A quantitation  34  6.8. Primer-extension preamplification (PEP)  34  6.9. Coding samples  35  6.10. End-labeling  35  6.11. P C R amplification  35  6.12. Scoring of L O H  36  RESULTS  38  7.1. Frequency o f allelic loss  38  7.2. Pattern o f allelic loss  39  7.3. Progression risk  41  7.4. Comparison of L O H pattern in matching premalignant and malignant lesions  44  7.5. Clinical history o f the lesions  46  DISCUSSION  53  8.1. High frequency of allelic loss characterized dysplastic lesions  53  8.2 Increased frequency o f allelic loss in progressing lesions  54  v  8.3. The pattern of allelic loss characterized progressing lesions  55  8.4. L O H as markers to predict cancer risk - further statistic analysis  57  8.5. Most progressed lesions may be derived by clonal outgrowth from the earlier lesions.. 60 8.6. Does evidernce of recurrence serve as a further indicator of cancer risk in premalignant lesions?  61  8.7. Summary  63  9.  REFERENCES  65  10.  APPENDIX  76  vi  LIST OF TABLES  Table 1. L O H frequencies (%) i n Head and Neck and Oral Cancers  76  Table 2. L O H and oral premalignant lesions  20  Table 3. Samples sets and groups  30  Table 4. Accumulation of allelic loss in progressing and non-progressing lesions  38  Table 5. Patterns of allelic loss in progressing and non-progressing lesions  40  Table 6. The time between initial biopsy and current date /the date when lesion has progressed to S C C Table 7. Probability o f lesions not progressing to cancer after 5 years follow-up  78 43  Table 8. Comparisons of L O H patterns in initial biopsies with that seen in CIS/SCC that later developed at the same anatomical site Table 9. Characteristics of patients with dysplasia Table 10. L O H frequencies and clinical history in progressing lesions Table 11. Allelic loss in recurrent premalignant lesions without a history of progression  44 46 rAl 52  vii  LIST OF FIGURES Figure 1. Histological progression model of oral premalignant and malignant lesions  4  Figure 2. Genetic pathway of carcinogenesis  8  Figure 3. Tumor Progression model for head and neck cancers as proposed by Califano et al 1996 Figure 4. Probability of having no progression to cancer, according to LOH pattern Figure 5. LOH analysis of 2 patients (a, b)  22 42 45  ABBREVIATIONS APC  adenomatous polyposis coli gene  BCCA  British Columbia Cancer Agency  bcl-1,2  B-cell lymphoma  CDK  Cyclin-dependent kinase  CIS  Carcinoma in situ  DNA  Deoxyribonucleic acid  docl V H L  The gene responsible for von Hippel-Lidadu syndrome  erbB  Erythroblastosis  FHIT  Fragile histidine triad  H&E  Hematoxylin and eosin  HHSCC  Head and neck S C C  LOH  Loss of heterozygosity  MEN1  Multiple endocrine neoplasia type 1  NBCCS  Nevoid basal cell carcinoma syndrome  NF1,2  neurofibromatosis type I and II  PEP  Primer-extension preamplification  PC  Phenol-chloroform  PCR  Polymerase chain reaction  p 1 6 / I N K 4 A / M T S - 1/CDK2A A tumor suppressor gene, encodes a cell cycle protein that halt cell-cycle progression ras  Rat sarcoma  Rb  Retinoblastoma gene  RFLP  Restriction fragment length polymorphism  ix  SCC  Squamous cell carcinoma  SDS  Sodium dodecyl sulfate  TSG  Tumor suppressor gene  T0R-II  Transforming growth factor type II receptor  UBC  University of British Columbia  WHO  World Health Organization  x  ACKNOWLEDGMENTS I would like to express m y deepest thanks to m y supervisors, D r . Lewei Zhang and D r . M i r i a m P. Rosin, for giving me the opportunity to learn and earn an M . S c . degree under their professional guidance, as well as their support throughout m y degree. I am also grateful to D r . Robert Priddy and D r . Joel Epstein for their valuable contributions to clinical issues in m y thesis. Thanks also to D r . Douglas Waterfield for chairing m y final oral examination. I appreciate very much the assistance o f all the people i n the laboratory. Special thanks go to my dearest friend, X i a o L e i Zhang for her special support and understanding.  xi  DEDICATION  To my Dear Mom and Dad To my Dearest Daughter  1. INTRODUCTION  .1.  Importance of studying oral premalignant lesions  Head and neck cancers, including those o f the mouth and upper air and food passages (oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx), the salivary glands, and thyroid, account for approximately 6% o f all human malignancies in the western world. The incidence is much higher in the Far East and India i n particular with up to 40% o f malignancies occurring in the head and neck region. The prognosis o f oral cancer has not significantly improved during the past two decades: the 5-year-survival rate is still less than 50% and is one o f the lowest among the major types o f cancers despite recent advances in surgery, radiotherapy, and chemotherapy (Raybaud-Diogene et al 1996; Todd et al 1995). A s oral cancer is believed to progress from oral premalignant lesions, the key to improve the gloomy prognosis o f oral cancer may lie i n early diagnosis and proper management o f oral premalignant lesions. The research o f our lab focuses on oral premalignant and malignant lesions. This thesis represents one aspect o f the focus the investigation o f molecular changes in early oral premalignancies o f oral mucosa.  1  1.2.  Oral mucosa  The oral cavity is lined by oral mucosa, which consists o f overlying epithelium and underlying lamina propria. The overlying epithelium o f oral mucosa is stratified squamous epithelium. The connective tissue lamina propria underneath the epithelium contains blood and lymphatic vessels, small nerves, fibroblasts, collagen, and elastic fibers.  The stratified  squamous epithelium could be divided largely into basal and prickle cells i n addition to there are other types: merkel, langerhans and melanocyte cells.  The one-layered basal cells  separate the overlying epithelium from the underlying connective tissue.  They are the only  cells that have the capacity to divide in the epithelium and subsequently give rise to either more new basal cells or differentiate into prickle cells located above them. The majorities o f oral premalignant and malignant lesions arise from this stratified squamous epithelium o f oral mucosa and the malignant tumors are called squamous cell carcinoma ( S C C ) .  1.3.  Oral premalignant lesions and their relation to oral SCC  Oral S C C is believed to be a result o f a multistage carcinogenesis process over a long period o f time.  This multistage process involves progression from normal to premalignant  lesions and finally invasive S C C . A premalignant or precancerous lesion has been defined by the W o r l d Health Organization ( W H O , 1978) as a morphologically altered tissue i n which cancer is more likely to occur than i n its apparently normal counterpart. In the oral cavity, most premalignant lesions present clinically as leukoplakias.  Other types o f clinical forms  exist, such as erythroplakia and possibly lichen planus ( W H O 1978). Leukoplakia means a "white patch" and occurs on mucous membranes such as the mucosa o f the oropharynx,  2  larynx, esophagus, and genital tract.  Not necessarily white, leukoplakias also may appear  yellow to light brown, especially in smokers. The W o r l d Health Organization ( W H O , 1978) defines leukoplakia in the oral cavity as a white patch or plaque o f oral mucosa that cannot be characterized clinically or pathologically as any other diagnosable disease and is not removed by rubbing. It is only a clinical term. When a biopsy is taken, a leukoplakia w i l l show microscopically hyperkeratosis and/or epithelial hyperplasia (acanthosis) with or without epithelial dysplasia. Dysplastic lesions are further divided into mild, moderate, and severe forms depending upon how much o f the tissue is dysplastic. M i l d dysplasia is lesions i n which the dysplastic cells are confined to the basal layer and the cells exhibit the smallest degree o f the above changes. W i t h moderate and severe dysplasia, the epithelial layers involved and the severity of the cellular changes are progressively increased. In carcinoma in situ, the dysplastic cells occupy the entire thickness o f the epithelium (bottom to top changes) although the basement membrane is still intact.  Invasion o f dysplastic cells through the basement membrane into  the underlying stroma and/or the dissemination o f these cells to other sites through lymphoid and circulatory systems are events associated with development o f invasive S C C . The presence and absence o f dysplasia and the degree o f dysplasia is believed to have a huge impact on the malignant risk o f the premalignant lesions.  A l l studies to date have  shown that leukoplakia with dysplasia has a higher malignant risk than leukoplakia without dysplasia. A large clinical study by Silverman et al (1984) found that during a mean onset o f 7.2 years after presentation, more than 36% o f leukoplakia lesions with microscopic epithelial dysplastic features eventually underwent malignant transformation whereas  leukoplakia  without dysplasias only demonstrated a malignancy rate o f 15%. The risk o f dysplasia and degree o f dysplasia is further demonstrated by studies from the uterine cervix and other  3  systems and organs including skin and respiratory system. As a result, currently the gold standard for judging the malignant potential of premalignant lesions in these organs and systems, including the oral cavity, is the presence and degree of dysplasia. Using these criteria, a histological progression model has been established for the oral cavity (Fig. 1). Premalignant lesions are classified histologically into categories with progressively increased risk of becoming invasive SCC: epithelial hyperplasia (without dysplasia), mild, moderate and severe dysplasias, and carcinoma in situ (CIS).  Figure 1: Histological progression model of oral premalignant and malignant lesions  HISTOM-1 JPC  Other factors also affect the malignant potential of oral premalignant lesions. These include location and duration of the lesion, gender of the patient, appearance of the lesion (homogenous vs non-homogenous) and presence of C. albicans (Waal, 1997). Most studies on the malignant transformation of oral premalignant lesions have been done on leukoplakias (clinical presentation of oral premalignant lesions), frequently without knowledge of dysplasia  4  for all the study cases.  The reported malignant risk for leukoplakia varies from study to  study, ranging from as low as 0.13% to as high as 24% depending upon the patient population and follow-up time (Papadimitrakopoulou et al, 1997; Lumerman et al, 1995; Silverman et al, 1984).  1.4.  Problems with the histological progression model  The histological progression model has a better predictive value for high-grade preinvasive lesions (severe dysplasia and CIS) which are believed by many to have a much higher possibility o f progression into invasive lesions than low-grade lesions including hyperplasia without dysplasia and those with low-grade dysplasia (mild and moderate dysplasias) i f left untreated. Some even, believe that oral C I S w i l l inevitably become cancer i f left untreated (Regezi et al, 1989). A s a result, high-grade preinvasive lesions are generally treated aggressively, and the histological progression model has served as a good guidance for the aggressive treatment. While the histological progression model has a good or reasonable predictive value for high-grade preinvasive lesions, it is poor at predicting the malignant potential o f low-grade lesions. This model is more problematic i n guiding the treatment o f the low-grade lesions. The majority o f these low-grade lesions do not progress into oral cancer, either remaining static or regressing, with only a small percentage progressing. O n the other hand, these lowgrade lesions constitute the bulk o f leukoplakias and account for more than 90% o f leukoplakias (hyperplasia without dysplasia, 80.1%; early dysplasia, 12.2%; late dysplasia, 4.5% and S C C , 3.1%) (Waldron Lumerman et al, 1975). This poses a management dilemma for clinicians. Aggressive treatment does not seem to be justified for the majority o f these  5  lesions, both in terms o f side effects and cost.  N e w methods that could identify that small  percentage o f progressing low-grade lesions from the majority o f non-progressing lesions are highly desired. The significance o f establishing these new methods lies i n two aspects. First, this w i l l facilitate the understanding o f the mechanisms o f early carcinogenesis; and second, this w i l l have direct impact on the clinical management o f these lesions. If we could understand the critical events occurring during early carcinogenesis, we may not only be able to predict the malignant potential at a very early stage, but can also plan management o f this small percentage  of  chemoprevention).  progressing  lesions  accordingly  (e.g.,  aggressive  treatment  or  Successful treatment o f these early lesions and prevention o f their  progression w i l l decrease the mortality and morbidity o f oral S C C drastically. A central dogma o f carcinogenesis is that alteration to critical control genes underlies malignant transformation.  The investigation o f these critical changes in genes has been  greatly facilitated recently with the rapid development o f molecular biology techniques. This thesis has investigated some o f the molecular changes in early oral premalignant lesions.  1.5.  Molecular biology of carcinogenesis  1.5.1. Genetic pathway of carcinogenesis  In 1976, N o w e l proposed that neoplastic transformation occurred in a single cell that had a critical genetic alteration giving it a growth advantage over its neighboring cells. The tissue would then go on to accumulate multiple mutations with these subsequent mutations being random i n nature; however, the determination o f which mutated cells would expand  6  into genetic clones in the tissue would be dependent on a variety o f intracellular and environmental factors, including earlier mutations. Thus, all daughter cells i n a tumor would share early initiating events but during tumorigenesis subclones would arise with additional genetic changes (that give them a selective advantage) and lead to heterogeneity.  The non-  random nature o f this mutation selection is responsible for the preferred molecular pathways that are envisaged for many cancers.  Ilyas Lumerman et al (1996) summarized that for a  normal cell to become malignant, it must acquire the stepwise accumulation o f genetic changes and a minimum number o f necessary mutations which help it to overcome growth controls. These mutations and the order o f development o f the mutation profile comprise the "genetic pathway" o f carcinogenesis.  7  Figure 2. Genetic pathway of carcinogenesis  *Mutation profile: the group o f mutations which are essential for tumour development. * * A cell, gets greatest survival advantage over its surrounding cells, w i l l undergo colonial expansion and outgrowth, becomes predominant. ***Not selected, become apoptosis.  The understanding o f the genetic pathways is essential in identifying those lesions that are following the pathways to cancer from those lesions with less critical genetic changes and with less cancer risk.  Since the alteration to critical control genes underlies malignant  transformation, it may be logical to assume that progressing premalignant lesions are genetically  different  from  morphologically  similar  non-progressing  lesions.  The  identification o f molecular changes that can be used to predict the likely behavior o f low-  8  grade lesions would allow the clinician to identify which patients with low-grade lesions should be managed more aggressively  (either by more frequent screening or by early  treatment, using traditional approaches such as surgery, or newer techniques such as chemopreventive regimes), and to better control and prevent the progression of premalignant lesions to cancer.  7.5.2. The major genes involved in tumorigenesis  It is now well established that clonal evolution of cancer is due to a progressive accumulation of critical genetic alterations, including at least two large groups: (1) the protooncogenes, which can be activated in the tumorigenic process to increase cell proliferation and induce malignant transformation, and (2) the tumor suppressor genes (TSGs), which maintain several growth control checkpoints and control the ability of cells to invade or metastasize. Proto-oncogenes code for proteins that regulate the many functions of the normal cell, including the control of cell division, the production of enzymes that alter cellular activity, and the production of intercellular adhesion molecules and cell surface molecules that are bound by extracellular molecules.  They include genes for growth factors, growth factor  receptors, protein kinases, signal transducers, factors.  nuclear phosphoproteins and transcription  Proto-oncogenes act in a dominant fashion to positively regulate cell growth and  differentiation.  Mutation of these proto-oncogenes to oncogenes can occur in the coding  region of gene, resulting in alteration of structure and activity in coded proteins.  Many  oncogenes have been identified in the literature; however, few of them have been reported to occur in HNSCC.  Some of the oncogenes that have been found altered or expressed at  9  abnormal levels i n head and neck cancers are ras, cyclin-Dl,  myc, erbB, bcl-1, bcl2, int-2  CK8 and CK19 (Staibano et al, 1998; Kannan et al 1996; Bartkova et al 1995; X u et al 1995; Gaffey et al 1995; Michalides et al 1995; Anderson et al 1994; Clark et al 1993; Eversole et al 1993; W o n g et al 1993; Riviere et al 1990; Yokota et al 1986). In  contrast,  TSGs  function  antagonistically  with cellular proto-oncogenes  to  negatively regulate cell growth and differentiation. The functions o f T S G s must be lost in order for tumorigenesis to occur. According to Knudson's hypothesis (1985), both copies o f a tumor suppressor gene have to be inactivated for its protective function to be lost i n a cell. Experience with known suppressor genes, such as the retinoblastoma gene, suggests that this process involves two separate events, the first quite often involving a point mutation in one allele, followed b y loss o f loci containing the w i l d type gene i n the remaining allele. Some o f the T S G s involved in head and neck cancers include p53, Rb (retinoblastoma),  andpl6INK4A  (Gallo et al 1999; Jares et al, 1999; Liggett et al, 1999; Papadimitrakopoulou et al, 1999; Sartor et al,  1999; Partridge et al, 1999a and 1998; Pavelic et al, 1997; Reed et al 1996;  Gleich et al 1996; Largey et al 1994). Other potential candidates are FHIT (fragile histidine triad), APC (adenomatous polyposis coli), doc-1 VHL (the gene responsible for von HippelLidau syndrome) and TfiR-II  (the gene coding for transforming growth factor type II  receptor). (Croce et al, 1999; U z a w a et al 1999 and 1994; M a o et al 1998 and 1996; Waber et al 1996;Todd et al 1995). This meticulous balance between growth inducers (coded by proto-oncogenes) and suppressors (coded by tumour suppressor genes) controls the rate o f division i n normal cells. These genes are altered through a multistep process i n which a cell accumulates many genetic changes, breaking the balance o f normal cell growth and leading to the malignant phenotype. Recent advancement i n the techniques o f molecular analysis has rapidly revolutionized our 10  ability to look at these genetic alterations. M y research w i l l focus on loss o f tumor suppressor genes (TSGs). Functional loss o f T S G s is one o f the most common genetic alterations during carcinogenesis (Leis et al, 1996).  Therefore, defining chromosomal regions harboring  biologically important suppressor genes may have broad practical implications not only on our comprehension o f progression o f tumors but also on the clinical management o f cancers and premalignant lesions. This thesis has studied regions o f chromosome loss that contain presumptive T S G s by employing a polymerase chain-based microsatellite heterozygosity  1.6.  analysis for loss of  (LOH).  Loss of heterozygosity (LOH) and microsatellite analysis  L O H is defined as a loss o f genomic material (as small as a few thousand nucleotides to as large as a whole chromosome) in one o f a pair o f chromosomes.  The L O H assay is  designed to assess polymorphic chromosomal regions that map close to or within putative or known recessive cancer-related genes.  The concept o f L O H is consistent with Knudson's  two-hit hypothesis, which states that inactivation o f one o f the two alleles by either a germline or somatic mutation w i l l provide a growth advantage to the tumor cell because only one more inactivation o f the remaining allele is needed.  L O H analysis has been employed as a means  of identifying critical loci containing T S G s and has subsequently led to the discovery o f several important genes o f this class, including the retinoblastoma (Rb) gene and the genes responsible for multiple endocrine neoplasia type 1 {MEN1), the nevoid basal cell carcinoma syndrome (NBCCS), adenomatous polyposis coli (APC), and neurofibromatosis type I and II (NF1 andNFII,  respectively) (Fearon et al, 1997 and Ah-See et al, 1994).  11  Two methods have been available for the study o f L O H or allelic loss: the more classical approach o f restriction frequent length polymorphism ( R F L P ) analysis, and the newer method o f microsatellite analysis. This thesis employed microsatellite analysis for at least two reasons.  First, microsatellite repeat markers are highly polymorphic and well-  distributed throughout the human genome. They show levels o f heterozygosity between 3080%, significantly above the level observed with the R F L P substitutions at endonuclease recognition sites.  analysis based on base  Second, this PCR-based approach is much  more sensitive than the R F L P analysis and requires only small quantities o f D N A (5 nanograms or less per reaction). For these reasons, the microsatellite analysis procedure has become the major tool for the majority o f current L O H studies. Microsatellites contain runs o f short and tandemly iterated sequences o f di, tri, or tetranucleotides, such as G T G T G T . . . or G T A G T A G T A . . . or G T A C G T A C G T A . . . . These short repetitive D N A sequences are called microsatellites.  The number o f such tandem  repeats is found to be highly polymorphic in the population, with each individual typically containing a different number o f copies (generally 4 to 40) o f the repeat at each particular locus. In addition, they are well interspersed throughout the human genome (e.g., estimated every 30-60 K B for C A repeats) and are highly conserved through successive generations (Ah-See et al 1994). Testing o f highly polymorphic microsatellite markers from a specific chromosomal region allows rapid assessment o f allelic loss by comparing the alleles in tumor D N A to normal D N A .  Therefore microsatellites are good way to research the T S G s either  close to or within these chromosome spots. Loss o f heterozygosity suggests that a putative tumor suppressor gene nearby may be also lost.  12  1.7.  L O H in oral and head & neck malignant lesions  Recent studies including those from this lab have shown that the loss o f specific regions o f chromosomes that contain tumor suppressor genes is a common event i n oral SCCs.  In this thesis, microsatellite markers on chromosome arms 3p, 4q, 8p, 9p, l l q , 13q  and 17p were used, since they have been reported to lie within regions most frequently lost i n oral S C C s . Each o f these regions w i l l be discussed briefly.  Chromosome  3:  H i g h frequency o f L O H at chromosome 3p has been reported i n head  and neck cancers (Table 1 i n the appendix). The losses appear to center around 3p 13-21.1, 3p21.3-23, and 3p24-25 (Partridge et al, 1999b, 1998; Scully et al, 1996; Partridge et al,\996; R o z et al, 1996; Maestro et al, 1993). The number o f regions showing allele loss at 3p (3p 12.1-14.2, 21.3-22.1 and 24-26) is consistent with the progressive accumulation o f genetic errors during the development o f oral S C C (Partridge et al, 1996). Each o f the three regions is presumed to contain at least one putative T S G . W i t h i n the region o f 3 p l 4 . 2 exists one o f the most common fragile site locus, called F R A 3 B , in the human genome. Fragile sites are portions o f chromosomes that are extremely weak and break easily. Consequently, these weak areas may be easy targets for carcinogens such as those found in tobacco.  The gene, FHIT (Fragile histidine triad), was recently identified at this  fragile site and appears to be involved in various cancers such as esophageal, gastric, colonic, breast, cervical, small cell lung, and head and neck carcinomas (Pennisi et al, 1996; Sozzi et al, 1996; Ohta et al, 1996; W i l k e et al, 1996; M a o et al 1996a and W u et al ,1994). encodes a protein with 69% similarity to a Schizosaccharomyces 5', 5 " ' - P l , P4-tetraphosphate  It  pombe enzyme, diadenosine  ( A p 4 A ) asymmetrical hydrolase which cleaves the  AP4A  13  substrate into 5' - A D P and A M P . Current theories suggest that diadenosine tetraphosphate may accumulate in the cells in the absence o f the normal expression o f the gene and may eventually lead to D N A synthesis and cell replication (Mao etal, 1996). Several recent studies have shown that FHIT may be significantly involved i n oral S C C development (Croce et al, 1999; M a o et al, 1998a and 1998b ) and suggest that alteration to this gene may play an important role in early stage i n the development o f this cancer (Mao et al, 1996b). It was recently suggested in some tissues and organs, particularly those associated with exposure to environmental carcinogens, alterations i n FHIT occur quite early i n the development o f human cancer (Croce et al, 1999). Croce concluded that FHIT loss in bronchial tissue indicates the occurrence o f genetic alterations associated with the early steps o f carcinogenesis. L O H at 3 p l 4 has been shown to be involved i n oral premalignant lesions (Mao et al, 1996a). U n t i l now there is sufficient evidence for only one gene, FHIT, to be responsible for the L O H at the region 3 p l 4 . 3 , although the evidence in support o f it being a T S G is still considered to be controversial (Mao et al, 1998b). T S G s that are responsible for L O H at the other two regions (3p24-pter, and 3p21.3) are still not clear. For example, the region o f 3p2425 contains the V H L gene, which is thought to be a member o f a novel class o f glycananchored membrane proteins that function i n signal transduction and cell adhesion (Waber et al, 1996), and its alteration has been reported especially in VHL-associated cancers (van den Berg, et al, 1997; K o k et al, 1997 and Decker et al, 1997).  U z a w a also mentioned the  possibility that the V H L gene may in involved in oral S C C development (1998). However, mutations o f the V H L gene could not be identified and the V L H gene was not inactivated by hypermethylation i n H N S C C . Hypermethylation is an alternation method o f inactivity o f a gene that does not require direct mutation to the gene.  It is possible that allelic loss o f  14  chromosome arm 3p i n H N S C C involves regions surrounding the V H L locus but does not include the V H L gene. Another T S G i n H N S C C may exist i n the regions surrounding D 3 S 1110 at 3p 25 (Uzawa et al, 1998; Waber et al, 1996). Chromosome 9:  L O H on 9p is by far the most commonly reported chromosomal defect  in head and neck cancers, with L O H reported i n 72% o f malignant lesions.  The most  commonly affected region is chromosome 9p21-22. In addition, L O H at 9p22-q23.3 is also common (> 70% o f head and neck cancers) (Scully et al 1996 and Nawroz et al, 1994). The putative T S G s are near the interferon locus and are not clearly identified. A t 9p21, the prime T S G candidate involved i n the head and neck cancers is pi6 know as MTS-1 kinase4a,  and  for major tumor-suppressor CDKN2A  (INK4A/MTS-1/CDKN2A)  1, INK4a  for cyclin-dependent  kinase  (also  for inhibitor o f cyclin-dependent inhibitor 2 A ) .  The T S G  pi6  encodes a cell cycle protein that inhibits cyclin-dependent kinases  ( C D K ) 4 and 6, preventing phosphorylation o f R b protein and consequently inhibiting the cell cycle transition o f the G l - S phase (Reed et al, 1996). The major biochemical effect o f p i 6 is to halt cell-cycle progression at the G l / S boundary.  The loss o f p i 6 function may lead to  cancer progression b y allowing unregulated cellular proliferation ( W i l l i a m et al, 1998). Although mutations o f this gene are not apparently frequent for oral cancer, this might suggest that either this gene is inactivated b y an alternative mechanism such as homozygous deletion or by methylation o f the 5 ' C p G - r i c h region, which results i n a complete blook o f gene transcription (Papadimitrakopoulou et al, 1997; Rawnsley et al, 1997; Matsuda et al, 1996 and M e r l o et al, 1995). Reed and Papadimitrakopoulou found that - 8 0 % o f the head and neck cancers and premalignant lesions were p i 6 inactivated at the protein and/or D N A level and suggest that inactivation o f p i 6 may play an important role in early head and neck cancer development (Papadimitrakopoulou et al, 1997 and Reed et al, 1996). Alternatively, another  15  tumor suppressor gene may exist i n this region (Waber et al, 1997; Dawson et al, 1996; Reed etal, 1996).  Chromosome 17:  L O H on 17p has been reported i n 50% o f head and neck cancers, most  frequently involving 17pl3 and 17p 11.1-12 (Scully et al, 1996; Field et al, 1996; Adamson et al, 1994; Nawroz et al, 1994). The region 17pl3 harbors the gene p53 (17pl3.1), which has been reported to have the highest frequency (~50%) o f mutations i n human  cancers.  Mutation at p53 is also one o f the most common events i n H N S C C ( Lazarys et al, 1995). Its protein functions as mediator i n several activities, including transcription activation, D N A repair, apoptosis, senescence, and G I and G 2 cell cycle inhibition.  In addition, increasing  evidence also shows that a region, defined by the cholinergenic receptor B l ( C H R N B 1 ) locus at 17pl 1.1-12, that is tightly linked to thep53 regions may contain a novel T S G .  Chromosome 4:  L O H on chromosome 4 has been studied i n cancers o f many systems  and organs including hepatocellular, bladder, ovarian and cervical cancers.  The putative  tumor suppressor locus was localized to a region near the epidermal growth factor gene on 4q25 and 4q24-26. Loss at 4q25 occurs i n 70% o f head and neck cancers (Pershouse et al, 1997) and loss at 4q26-28 occurs i n 47% (Califano et al, 1996; B o c k m i h l et al, 1996). The combination o f allelic deletions and chromosomal transfer studies strongly suggests the presence o f a T S G within 4q24-26.  This region was involved i n >80% o f the tumors  examined, suggesting that a putative chromosome 4q T S G may play an important role i n the evolution o f H N S C C (Pershouse et al, 1997).  16  Chromosome 8:  Investigation o f 8p regions in head and neck squamous carcinoma has  shown a relatively high incidence o f alterations (31%-67%) ( W u et al, 1997; Califano et al, 1996; Bockmuhl et al, 1996; Scholnick et al, 1996; EI-Naggar et al, 1995; Field et al, 1995; Ah-aee et al, 1994; L i et al, 1994). Deletion mapping o f oral and oropharyngeal S C C defines three discrete areas on chromosome arm 8p: 8p23, 8p22, and 8pl2-p21 ( W u et al, 1997 and EI-Naggar et al, 1995). Several studies have linked allelic loss at 8p to a higher stage ( W u et al, 1997) and poor prognosis (Scholnick etal, 1996 and L i et al, 1994).  Chromosome 11:  L O H on human chromosome 11 has also been commonly reported i n a  variety o f human cancers, including H N S C C (39%-61%) (Lazar et al, 1998; Venugopalam et al, 1998; Bockmuhl et al, 1996; Califano et al, 1996; U z a w a et al, 1996; El-Nagger et al, 1995; Nawroz et al, 1994). The common region o f loss at this chromosome seems to be near the INT-2 locus at 1 l q l 3 (Nawroz et al, 1994). It is possible that some o f this region's allelic imbalance may be due to amplification rather than L O H (Nawroz et al, 1994). Amplification o f this region associated with poor prognosis was also reported (Papadimitrakopoulou et al, 1997).  Chromosome 13:  More than half o f H N S C C s shows L O H o f 13q i n regions close to the  R B (retinoblastoma) locus, but not R B gene (52-67%) (Ogawara et al, 1998; Ishwad et al, 1996; Maestro et al, 1996; Bockmuhl et al, 1996; Califano et al, 1996; Nawroz et al, 1994). A hot spot o f D 1 3 s l 3 3 at 13ql4.3, which lies just telomeric to the R B gene, was reported (Yoo et al, 1994). A recent study done by Ogawara showed L O H on 13ql4.3 correlated with lymph node metastasis o f oral cancer (p<0.0024). H i s results also suggest that L O H on 13q is a common event in oncogensis and/or progression o f oral S C C and the existence o f a new 17  suppressor gene near D13S273-D13S176 loci which may play a role i n these events since no significant variation in R B protein expression was detected (Ogawara et al, 1998). The study of Harada et al, (1999) confirmed  that L O H in chromosome  13 showed a significant  correlation with lymph node metastasis in esophageal squamous cell carcinoma, included in H N S C C . They reported that unidentified TSG(s) in region 13ql2-13 might be involved.  1.8.  L O H in oral and head & neck premalignant lesions Since tumorigenesis is a sequential accumulation o f genetic alterations, analysis o f  early and late stage lesions may define the genetic changes associated with the development and progression o f H N S C C .  Few studies (Table 2) have investigated the premalignant stages  of the lesions while there are many studies o f L O H on oral S C C . The main difficulties lie i n the fact that: 1) premalignant lesions are small and therefore it is extremely hard to obtain sufficient amount o f D N A for molecular analysis, 2) big hospitals or research centers typically have better access to cancers rather than premalignant lesions, and 3) it is much harder to microdissect premalignant lesions compared to carcinomas. The limited number o f studies on premalignant lesions either used only a small number o f cases or primers, or did not correlate L O H with degree o f dysplasia and mostly form high-grade dysplasias. Nonetheless, results from these studies clearly show that L O H is a frequent event in premalignant lesions (Califano et al, 1996; M a o et al, 1996a; E m i l i o n et al, 1996; R o z et al, 1996; El-Naggar et al, 1995). For example, a similar frequency o f L O H at 9p was reported i n preinvasive lesions (71%) as in S C C (72%) ( V a n der Riet et al, 1994). This suggests that loss o f 9p is an early event i n the progression  o f oral  cancer  (Papadimitrakopoulou et al, 1997 and V a n der Riet et al, 1994). Similarly L O H at 3p have been found to occur very early during oral carcinogenesis and i n a significant number o f oral  18  m i l d dysplasia or even hyperplasias (Zhang et al, 1997). O n the other hand, data from this lab showed that L O H at 17p was not found i n reactive hyperplastic lesions and mild dysplasia o f oral mucosa, indicating loss at 17p occurs later than L O H at 3p and 9p (Zhang et al, 1997). EI-Naggar and his colleges (1998) recently found L O H at 8p in 27% o f the dysplastic lesions and i n 67% o f the invasive oral and laryngeal S C C s . The highest frequency o f allele losses i n dysplasia and cancer were detected i n the same loci: 8p21 and 8p22. In addition, allelic losses in both dysplastic and corresponding invasive specimens were noted at the same loci, suggesting their emergence from a common preneoplastic clone. The studies suggested that inactivation o f TSG(s) within these loci may constitute an early event i n the evolution o f oral and laryngeal S C C s . Moreover, a study b y M a o and co-workers (Mao et al, 1996a) showed that L O H i n oral premalignant lesions could be used to predict risk o f cancer progression o f these premalignant lesions. They reported that the presence o f L O H at 9p21 & / o r 3 p l 4 in oral leukoplakia was associated with a greater probability o f progression o f this premalignant lesion into S C C : 7 o f 19 (37%) cases with such L O H progressed to S C C i n their study, as compared to only 1 o f 18 (6%) cases without L O H .  19  T a b l e 2. L O H and oral premalignant lesions  Authors  Chromosome arm  Degree of oral dysplasias  studied  L O H and risk of malignant transformation  VanderRiet, 1994  9p21-22  Severe dys/ CIS lesions  EI-Naggar, 1995  3p, 5q,8p, 9, 11 q, 17  Severe dysplasia adjacent to cancer  LOH at 8p loci has higher risk of tumor's aggressive feature  Califano, 1996  3p, 4q, 6p, 8, 9p, 1 lq, 13q, 14q, 17p  Dysplasia with no indication of severity  Not evaluated  Mao, 1996a  3pl4, 9p21  Reported as leukoplakias some of which were dysplastic  Patients with LOH at 9p and/or 3p have higher risk of cancer development  Mao, 1996  5q (APC)  Emilion, 1996  3p, 17p(p53), 18q(DCC)  Primary dysplasias: Mild: 8 cases Moderate: 4 cases Severe: 5 cases Dysplasia adjacent to cancer: Mild: 4 cases Moderate: 6 cases Severe: 3 cases  LOH was not associated with the degree of dysplasia, but the number of allelic loss increased while tumor developed.  Roz, 1996  3p  Severe/CIS lesions only  Not evaluated  Zhang, 1997  3p, 9p, 17p  Reactive lesions: 29 cases Mild dysplasias: 10 cases Moderate dysplasias: 11 cases Severe dys/CIS: 16 cases Cancer: 22 cases  Loss of more than one chromosome arm is associated with degree of  Dysplasia: 5 cases CIS: 3 cases  Not evaluated  Not evaluated  *"""• 20  El-Naggar, 1998  Partridge, 1998  1.9.  8p  3p21 8p21-23 9p21 13ql4.2 (Rb) 17p 11.2 (tp53) 18q21.1 (DCC)  Eight paired severe dysplasia and corresponding invasive lesions 31 Dysplasia with no indication of severity  Both lesions emergent from a common preneoplastic clone The probability of progressing to SCC was much greater for cases showing AI at two or more relevant loci  A molecular progressing model for oral S C C  In the late 80s, Fearon and Volgelstein, among the first people to describe molecular progression, suggested that a) tumors progress via the activation of oncogenes and the inactivation of TSGs, each generating a growth advantage for a clonal population of cells; b) specific genetic events generally occur in a distinct order of progression; but c) the order of progression is not necessarily the same for each individual tumor, and therefore it is the accumulation of genetic events that determines tumor progression. It has been estimated that at least 6-10 independent genetic events are required in order for head and neck cancers to occur (Emilion et al 1996). It is now accepted that the histologic progression of oral cancer (from hyperplasia  mild dysplasia -> moderate dysplasia -> severe dysplasia -> CIS ->  SCC) is underlain by the accumulation of such changes to critical genes. In a landmark study by Califano and his colleagues (1996), LOH was investigated in a whole spectrum of oral premalignant lesions including hyperplasia, dysplasia, CIS and SCC. The study proposed a 21  genetic progression model for oral carcinogenesis (Fig. 3). The model proposes that LOH at 9p is the earliest event associated with transition from normal to benign hyperplasia; LOH at 3p and 17p is associated with dysplasia, whereas CIS and SCC were characterized by additional deletions on 4q, 6p, 8, 1 lq, 13q, and 14q (Fig 3).  Figure 3  Tumor Progression Model for Head and Neck cancers as proposed by Califano etal 1996  Alternate Precursor Lesion Normal Mucosa  —  9p loss  \  3p,17p loss  \ i  Dysplasia  N  1 lq, 13q 14q loss  Carcinoma in situ  6p, 8, 4q loss  Invasive cancer  _ J Benign Squamous Hyperplasia est  22  2.  S T A T E M E N T O F T H E PROBLEMS  Oral S C C is believed to progress through stages o f oral premalignant lesions (hyperplasia, mild dysplasia, moderate dysplasia, and severe dysplasia, CIS) before finally become invasive.  While the prognosis o f oral premalignant lesions are excellent, once  invasion occurs and oral S C C is formed the prognosis is poor and about half o f the patients die within 5 years o f diagnosis despite recent advancement in treatment. Those who survive still face severe cosmetic and functional morbidity. The understanding and intervention o f oral premalignant lesions w i l l be critical in the reduction o f the mortality and morbidity o f oral S C C . However, the majority o f oral premalignant lesions do not progress into oral S C C and aggressive treatment o f these lesions is not justified. A t present, we can not identify those progressing oral low-grade premalignant lesions from the majority o f non-progressing lowgrade oral premalignant lesions. Since cancer is underlain by changes to the critical control genes, the understanding o f molecular changes during early oral cancer development may be critical i n the establishment o f molecular markers for the identification o f high-risk oral premalignant lesions. It is likely that the progressing low-grade oral premalignant lesions are molecularly  different  from  the  morphologically similar  non-progressing  low-grade  premalignant lesions. W i t h the rapid development o f molecular biology techniques, there have been numerous studies on the molecular changes i n human cancers, including oral S C C s . However, few studies are done in oral premalignant lesions because these lesions are not readily available and are small i n size. A number o f studies done on premalignant lesions are all limited either i n the number o f cases used or the number o f probes used.  Furthermore,  most o f these studies were either done on high-grade oral premalignant lesions or the degree o f dysplasia was not mentioned.  There is a marked lack o f information on the molecular  23  changes o f low-grade oral premalignant lesions, which are the majority o f oral premalignant lesions, and are the hardest to predict i n terms o f their malignant risk. A molecular model has been proposed by Califano et al, (1995) based on studies on oral premalignant and malignant lesions.  However, the study merged all dysplasias together.  It is well known that the  prognosis o f low-grade oral premalignant lesions (e.g. m i l d dysplasia) differs drastically from that o f high-grade oral premalignant lesions (e.g., severe dysplasia). Furthermore, there is no data available on the characteristics o f molecular changes o f those oral premalignant lesions that have known to have progress into cancer. The main problem is that such lesions are hard • to find.  This thesis w i l l investigate the molecular changes o f low-grade oral premalignant  lesions, and compare the characteristics o f genetic changes o f those premalignant lesions that have progressed into CIS or S C C with those with no known progressing history.  24  OBJECTIVES  To characterize the pattern o f genetic changes in premalignant lesions by means o f L O H analysis using microsatellite markers for the 7 chromosomal regions known to be frequently lost i n oral tumors: 3p, 4q, 8p, 9p, 1 l q , 13q and 17p.  To determine whether the L O H profile was significantly altered i n cases that have progressed to CIS or S C C as compared to those that have not.  25  4.  HYPOTHESIS  1)  Molecular changes  as assayed by the L O H analysis occur early during oral  carcinogenesis, including low-grade oral premalignant lesions.  2)  Progressing early lesions (low-grade dysplastic and hyperplasia without dysplasia) are genetically different from morphologically similar non-progressing lesions.  If the data support the first hypothesis, it would suggest that loss o f regions o f chromosomes that contain presumptive tumor suppressor genes is critical for early oral carcinogenesis. If the data support the second hypothesis, it would suggest that early lesions progressing to cancer, though morphologically indistinguishable from those without a history o f progression, are in fact different genetically. It is this genetic difference that underlines the behavior o f these lesions. It would also support the belief that the molecular tools such as microsatellite analysis for L O H may be used i n differentiating between progressing and nonprogressing lesions. The identification o f molecular changes that can be used to predict the likely behavior o f low-grade lesions would allow the clinician to identify which patients with low-grade lesions should be managed more aggressively, either b y more frequent screening or by early treatment, using traditional approaches such as surgery, or newer techniques such as chemopreventive regimes.  26  5.  5.1.  EXPERIMENTAL DESIGN A N D STATISTICAL ANALYSIS  E x p e r i m e n t a l design  This is a case — control study (Chap, 1997, p2 and K i n g , 1996, p38).  The study  subjects were those who already had a certain condition (premalignant lesions known to have progressed to C I S or S C C ) : These subjects are the cases and this thesis investigated the characteristics shared b y the cases. The comparison group (those lesions without progression history) — the controls — was selected so that it resembled the cases as closely as possible (Brunette, 1996).  W e tried to avoid the usual problems usually i n a case control design:  information bias or data collected i n the past under uncertain conditions. First, there was no information bias in our study because there were no significant differences i n quality or availability o f data between the cases and controls. Second, the experimental data ( L O H data) were collected under the same experimental conditions. Although some data were collected in the past under uncertain conditions, like gender, age, site and smoking history, there was no significant difference between cases and controls i n terms o f gender distribution, age distribution, site distribution, and smoking history ( Table 9). These matched factors can no longer be evaluated as etiologic agents since they w i l l be equalized in the cases and the controls (Brunette, 1996). moderate  Moreover, the histological diagnosis o f hyperplasia or m i l d or  dysplasia was reconfirmed b y two pathologists ( L Z and R P ) using criteria  established by the W o r l d Health Organization ( W H O collaborating Reference centre 1978). This study was a retrospective design.  W e obtained some clinical information by  tracking recurrent lesions as they appeared in our database and by following the case histories o f treatment in hospital charts.  However, there was no evidence that treatment was less  27  aggressive among progressing cases compared with those without a history o f progression (see details in discussion part).  5.2.  Statistical analysis  Associations between L O H and progression were examined using Fisher's exact test (two-tailed). Clinical differences between progressing and non-progressing groups were examined using either Fisher's exact test (gender distribution and smoking habit) or unpaired t-test (age and follow up time).  A l l p values were two-sided. A P value o f 0.05 or less was  considered significant. Time-to-progression curves  were  estimated  by the  Kaplan-Meier method  and  comparisons were performed using log-rank test (Chap, 1997 and Armitage et al, 1987). Relative risks were determined using C o x regression analysis (Chap, 1997 and Armitage et al, 1987).  28  6.  M A T E R I A L SA N D M E T H O D S  6.1.  Sample collection.  This study used paraffin-embedded archival samples from the provincial Oral Biopsy Service o f British Columbia. This centralized Oral Biopsy Service provides service to dentists and E N T surgeons throughout the province, at no cost to the provider or patient, with more than 3,500 biopsies o f oral lesions received per year (19 years archived). This provides a large collection o f early lesions that can be followed over time. Cases that progressed into cancer were identified by linking the database o f this Service to the British Columbia Cancer Registry, which receives notification o f all histologically confirmed cases o f cancer and C I S diagnosed i n the Province.  6.2.  Sample sets  T w o sample (archival paraffin blocks) sets were used (Table 3): Set 1: Oral lesions from patients with no subsequent history o f head and neck cancer. W e refer to these cases as non-progressing. Set 2: Oral lesions from patients that later progressed to CIS or S C C .  Both sets o f samples included hyperplasia (without dysplasia) group and low-grade dysplasia (mild or moderate dysplasia) group.  29  Table 3. Samples sets a n d groups Number of Cases Lesion Type  Lesions with no history o f head a n d neck cancer  Epithelial Hyperplasia  33  (without dysplasia) M i l d Dysplasia  31  Lesions that later progressed to CIS o r SCC  _  (low-grade dysplasia) Moderate Dysplasia  (low-grade dysplasia)  23  14  The criteria for choosing Set 1 samples included:  1)  A histological diagnosis of a case confirmed by two pathologists using criteria established by the World Health Organization (WHO collaborating Reference centre 1978).  2)  The provision that the sample was large enough to yield sufficient DNA from both the epithelium and from the connective tissue for multiple LOH analyses.  3)  Confirmation that these patients had no prior history of head and neck cancer and that they had not developed such cancer so far was obtained both from hospital records and by using a computer linkage with the British Columbia Cancer Registry. All but three of these cases had at least 3 years of follow up time.  The inclusion criteria for selection of the set 2 lesions in addition to those described for set 1 included:  1)  Both the primary hyperplastic or dysplastic lesions and their matching CIS or SCC had to be from the same anatomical site as recorded on pathology reports and patients charts;  30  2)  The interval between the primary lesions and later CIS or S C C had to be longer than 6 months.  This criterion was used to exclude cases that might be due to inadequate  biopsy, with the time interval chosen for exclusion being arbitrary.  6.3.  Histological diagnostic criteria for the samples  The generally accepted histological diagnostic criteria for dysplasia includes loss o f basal cell polarity, more than 1 layer o f basaloid cells, increased nuclear to cytoplasmic ratio, drop-shaped rete ridges, irregular stratification, increased and/or abnormal numbers o f mitosis in the basal compartment as well as increased mitotic figures in the superficial half o f the epithelium, cellular pleomorphism, nuclear hyperchromatism, enlarged nucleoli, reduction o f cellular cohesion, and keratinization o f single cells or cell groups in the spinous cell layer ( W H O collaborating Reference centre 1978). Based on the above-mentioned criteria as well as the extent o f the epithelial tissue that is dysplastic, dysplasias are divided into mild, moderate, and severe forms. Those classified as mild indicate that the dysplastic cells are confined to the basal layer and that they exhibit the smallest degree o f changes.  W i t h moderate and severe dysplasias, the epithelial layers  involved and the severity o f the cellular changes increase progressively. In carcinoma in situ, the dysplastic cells occupy the entire thickness o f the epithelium (from bottom to top), although the basement membrane is still intact.  Invasion o f dysplastic cells through the  basement membrane into the underlying connective tissue and the dissemination o f these cells to other sites through the circulatory systems are characteristics o f malignancy (i.e., cancer) (Zhangetal, 1997) ( F i g 1).  31  The histological diagnoses o f the lesions were performed independently by D r . R . Priddy and D r . L . Zhang, oral pathologists at University o f British Columbia.  Only those  cases i n which the two pathologists agreed on the diagnosis were used for the study.  6.4.  Slide preparation  A case was chosen after the pathologists agreed on the diagnosis, and after the determination that the block was b i g enough and contained sufficient epithelial and connective tissues for molecular analysis. The tissue block for the case was removed from the archive, one 5 micron thick slide was cut and stained with H & E (hematoxylin and eosin) and coverslipped for reference. The actual samples for microdissection were 12 microns thick and approximately 15 slides, each with multiple thick sections, were cut for each tissue block. They were also stained with H & E but left uncoverslipped. The H & E procedure for the slides is described below: Baked at 3 7 ° C i n an oven overnight, then at 6 0 - 6 5 C for 1 hour, and left at room temperature 0  to cool; Deparaffmized b y two changes o f xylene for 15 m i n each; Dehydrated in graded ethanol (100%, 95% and 70%), 5 m i n each; Hydrated by rinsing in tap water, 2 min; Placed in G i l l ' s Hematoxylin for 5 min; Rinsed in tap water, 2 m i n ; Blued with 1.5 % (w/v) sodium bicarbonate, 1 m i n ; Rinsed in water, 2 min; Counterstained with eosin for 10 sec; 32  Dehydrated in graded alchohol (75%, 9 5 % and 100%), 3 m i n each; Cleared in xylene, 3 min twice for coverslipping (for the H & E slide) or submitted for microdissection.  6.5.  Microdissection  Microdissection o f the specimens was either performed or supervised b y D r . L . Zhang. Areas o f dysplasia were identified microscopically. Epithelial cells i n these areas were then meticulously dissected from adjacent non-epithelium tissue under an inverted microscope using a 2 3 - G needle. The non-epithelial underlying stroma dissected out from the same tissue block was used as controls (Zhang et al, 1997).  6.6.  Sample digestion and DNA extraction  The microdissected tissue was placed in a 1.5 m l eppendorf tube and digested i n 300 ul o f 50 m M T r i s - H C L ( p H 8.0) containing 1% sodium dodecyl sulfate (SDS) proteinase K (0.5 mg/ml) at 48C° for 72 or more hours. During incubation, samples were spiked with 10 or 20 ul o f fresh proteinase K (20 mg/ml) twice daily. The D N A was then extracted 2 times with P C - 9 , a phenol-chloroform mixture, precipitated with 70% ethanol i n the presence o f glycogen, and washed with 70% ethanol. The samples were then re-suspended in L O T E , a low ionic strength Tris buffer, and submitted for D N A quantitation (Zhang et al, 1997).  33  6.7.  DNA quantitation  The fluorescence analysis using the Picogreen kit (Molecular Probes) was used to quantitate D N A . Absorbance was read from an S L M 4800C spectrofluorometer ( S L M Instruments Inc. Urbana, I L ) . The sample D N A was then determined from the standard curves. A series o f dilutions were done subsequently to adjust the concentration o f D N A to five ng/pl with L O T E buffer (Zhang et al, 1997).  6.8.  Primer-extension preamplification (PEP)  If the concentration o f D N A was less than 100 ng total, a procedure called P E P was first done. P E P involves amplification o f multiple sites o f the genome using random primers and low stringency conditions and hence  increases  the  amount  o f total D N A for  microsatellite analysis. It was carried out i n a 60 p i reaction volume containing 20 ng o f the D N A sample, 900 m M o f T r i s - H C L o f p H 8.3, 2 m M o f d N T P where N is A , C , G and T, 400 n M o f random 15-mers (Operon Techologies, SP 182-2, Poly N , 15 mer), and 1 pi o f Taq D N A polymerase ( G i b c o B R L , 5U/pl). reaction.  T w o drops o f mineral o i l were added prior to the  P E P using the automated thermal cycler (Omigene H B T R 3 C M , Hybaid Ltd)  involved 1 cycle o f pre-heat at 95 C° for 2 min, 50 cycles o f 1) denaturation at 92 C° for 1 min, 2) annealing at 37 C° for 2 m i n , and 3), ramping from 37 C° to 55 C° at 10 sec/degree, polymerization at 55 C° for 4min.  34  6.9.  Coding samples  A l l samples were coded i n such a way that the analysis o f L O H would be performed without the knowledge o f the sample diagnosis.  6.10.  End-Labeling  One more step before P C R was end-labeling o f one o f the primer pair. The reaction contained a 50ul mixture o f P C R water 38ul, 10 x buffer for T4 polynucleotide kinase 5 u l , 10 x B S A l u l , one o f the primer pair l u l , T4 polynucleotide kinase 3 u l , and [y- P] A T P (20 32  u C i , Amersham) 2 u l . It was then run for 1 cycle at 37 C° for 60 m i n using the thermal cycler (Zhang etal, 1997).  6.11.  PCR amplification  Microsatellite L O H analysis i n this study was done on chromosome arms 3p, 4q, 8p, 9p, l l q , 13q and 17p.  The pairs o f  P end-labeled polymorphic probes (primer pairs,  chromosome markers, Research Genetics - Huntsville, A L ) that flank the area o f tandem repeats in the chromosomal region o f interest mapped to the following regions: 3pl4.2 (D3S1234, D3S1228, D3S1300); 4q26 (FABP2); (D8S262); 8p23.3 (D8S264); l l q l 3 . 3 (INT2); (CHRNB1)  9p21-22 (IFNA,  l l q 2 2 . 3 (D11S1778);  4q31.1 (D4S243); 8p21.3 (D8S261); 8p23.3 D9S171,  13q32 (D13S170);  and 17p 13.1 {tp53 and D17S786).  D9S736,  D9S1748,  13ql4.3 (D13S133);  D9S1751); 17pll.2  These markers are localized to regions 35  previously shown to be frequently lost i n head and neck tumors (Lazar et al, 1998; EI-Naggar et al, 1998; Zhang et al, 1997; Maestro et al, 1996; U z a w a et al, 1996; Califano et al, 1996; M a o et al, 1996a; E m i l i o n et al, 1996; R o z et al, 1996; EI-Naggar et al, 1995; Ah-See et al, 1994; Nawroz et al, 1994; Adamson et al, 1994; W u et al, 1994). Some o f the markers, like the regions 3 p l 4 (instead o f 3p21.3-23 and 3p24-25) and 9p21 (instead o f 9p22-23), are preferentially chosen for analysis, because high frequencies o f L O H at these regions have not only been reported i n head and neck cancers but also have been shown to be associated with the risk o f malignant transformation o f oral premalignant lesions (Mao et al, 1996a). P C R amplification using the thermal cycler was carried out in a 5 pi reaction volume containing 5 ng o f genomic D N A , 1 ng o f labeled primer, 10 ng o f each unlabeled primer, 1.5 m M each o f d A T P , d G T P , d C T P , and d T T P , 0.5 units o f Taq D N A polymerase ( G I B C O , B R L ) , P C R buffer [16.6 m M ammonium sulfate, 67 m M Tris (pH8.8), 6.7 m M magnesium chloride, 10 m M (mercaptoethanol, 6.7 m M E D T A , and 0.9% dimethyl sulfoxide], and 2 drops o f mineral oil. Amplification involved 1 cycle o f pre-heat at 95C° for 2 min; 40 cycles o f 1) denaturation at 95 C° for 30 sec, 2) annealing at 50-60 C° (depending on the primer used) for 60s, and 3) polymerization at 70 C° for 1 min; and 1 cycle o f final polymerization at 70 C° for 5 min. The P C R products were then diluted 1:2 i n loading buffer and separated on an 7% urea-formamide-polyacrylamide gels, and visualized by autoradiography.  The films  were then coded and scored for L O H (Zhang et al, 1997).  6.12.  Scoring o f L O H  For each case, two samples, the epithelial cells i n the lesion and the connective tissue underneath (which serves as a control), were subjected to amplification at the same time. A n 36  informative  case w i l l  yield two alleles, one maternal  electrophoretic migration dependent  and the  on the different sizes.  other paternal,  with  Samples were scored b y  comparison o f the intensity o f the autoradiographic bands (which represent the P C R product) o f the lesion with that o f the normal connective tissue control. A l l e l i c imbalance detected as loss or marked reduction (>50%) o f one o f these allelic bands is termed L O H . A case is noninformative i f both paternal and maternal alleles are o f the same size. In this situation, an alternative information probe was replaced. A l l samples showing L O H would be subjected to repeat analysis after a second independent amplification and re-scored whenever the quantity o f D N A was sufficient.  37  7.  7.1.  RESULTS  Frequency o f allelic loss  LOH was present in 68 of 116 (59%) pre-malignant lesions studied, occurring more frequently among dysplastic (55 of 77, 71%) than hyperplastic (13 of 39, 33%) lesions (P < 0.001). LOH frequencies were dramatically elevated in lesions that later progressed to cancer (Table 4). All progressing lesions (both hyperplastic and dysplastic) showed LOH at one or more of the 19 microsatellite loci tested. LOH was detected in only 21% of the nonprogressing hyperplasias and 59% of dysplasias. Multiple chromosomal arm loss was characteristic of progressing lesions (50% of hyperplasia and 91% of dysplasia, see Table 4). It was absent in non-progressing hyperplasia and occurred in only 31% of the non-progressing dysplasias.  T a b l e 4. A c c u m u l a t i o n of allelic loss i n progressing a n d non-progressing lesions  Hyperplasia  •  L o w - g r a d e dysplasia  Without progression  With progression  33  6  7(21)  6 (100)  >1 a r m lost  0  >2 arms lost  ~0~  # o f lesions # with L O H  a  Without progression  With progression  54  23  0.001  32 (59)  23 (100)  0.0001  3 (50)  0.002  17(31)  21(91)  <0.0001  Y(50y  0.002  11 (20)  13 (57)  0.003  P  P  a  A total of 7 chromosomal loci were tested. Values in parentheses are percentages  38  7.2.  Pattern o f allelic loss  The most common losses for both progressing and non-progressing cases were on 3p and 9p with a higher frequency in the progressing cases (Table 5).  A m o n g non-progressing  cases, 4 o f 30 (13%) hyperplasias and 13 o f 53 (25%) dysplasias had a loss at 3p.  L O H at 9p  was rare i n non-progressing hyperplasias (3% o f cases), but present i n 24 o f 52 (46%) dysplasias.  In contrast, 67% o f the progressing hyperplasias and 64% dysplasias showed  L O H at 3p; 50% o f the progressing hyperplasias and 83% dysplasias showed L O H at 9p. The frequency o f loss at other arms (4q, 8p, 1 l q , 13q, 17p) was low for nonprogressing cases. O n l y 2 o f 33 hyperplasias (6%) had loss on any o f these arms (1 at 1 l q , 1 at 13q). Nineteen o f 54 (35%) o f non-progressing dysplasias had loss on these arms, most frequently at 17p (20% o f cases) and 8p (15%) followed by 1 l q (12%), 4q (8%), and 13q (4%). Further increases i n L O H frequencies at 4q, 8p, 1 l q , 13q and 17p occurred i n lesions that progressed to tumors. For dysplasias, this increase was significant for 8p, 1 l q and 13q, with the increase for 4q approaching significance (P = 0.057). There was also a doubling i n the frequency o f L O H on 17p (from 20% to 4 1 % o f cases), although this increase was not significant (P = 0.087).  For hyperplasias, increases were significant in comparisons o f  progressing versus non-progressing lesions for 4q, 8p, and 17p, with 1 l q approaching significance (P = 0.062).  39  Table 5  Patterns of allelic loss i n progressing a n d non-progressing lesions  Hyperplasia  L o w - g r a d e dysplasia  Without progression  With progression  P  3p  4/30 (13)  4/6 (67)  0.014  9p  1/32 (3)  3/6(50)  -j-jjjjjj—  4q  0/31 (0)  2/6 (33)  0.023  8p  0/31 (0)  llq  Without progression  With progression  P  13/53 (25)  14/22 (64)  0.003  24/52 (46)  19/23 (83)  0.005  4/48 (8)  6/21 (29)  0.057  2/6(33) ' " 0.023  8/51 (15)  11/21 (52)  0.003  1/31 (3)  3/6 (33)  0.062  6/52(12)  9/23 (39)  0.011  13q  1/32 (3)  0/4 (0)  1  2/53 (4)  7/21 (33)  0.002  17  0/33 (0)  2/6 (33)  0.020  11/54 (20)  9/22 (41)  0.087  5/29(17)  6/6 (100)  0.004  30/54 (56)  22/23 (96)  0.004  0/32 (0)  3/6 (50)  0.002  15/54 (28)  18/23 (78)  0.0001  Arm  P  3p & / o r 9p 3p & / o r 9p plus  a  b  any other a r m a  Loss/informative cases (% loss) b  Bold means p< 0.05, was considered significant.  40  7.3.  Progression r i s k  Specific L O H patterns in premalignant lesions were examined for association with disease progression b y using the Kaplan-Meier method (Armitage et al, 1987). Almost all the progressing lesions (28 o f 29, 97%) had L O H on 3p & / o r 9p. Time-to-progression curves (see table 6 i n appendix o f time between initial biopsy and current date or the date on which a lesion has progressed to S C C , with time o f first biopsy set at 0) were plotted as a function o f L O H at 9p (Fig. 4 A ) , 3p (Fig. 4 B ) , or a combination o f 3p & / o r 9p (Fig. 4 C ) . A l l were significant.  A further comparison was made o f cases with loss on these 2 arms in the  presence and absence o f L O H at any o f the other 5 chromosomes (4q, 8p, l l q , 13q and 17p; Fig.4D). A significant difference was again observed. Finally, we separately compared timeto-progression for cases in which 3p & / o r 9p L O H was restricted to these 2 arms alone with additional losses on each o f the chromosome arms (Fig.4E — 41). Significant P values were observed for combinations that included 8p, 1 l q or 13q. Additional analyses included an assessment o f relative risk o f progression for each o f the L O H patterns presented  and a determination o f the proportion o f cases without  progression at 5 years follow-up. These results are tabulated in Table 7 with an assessment o f their significance respectively.  41  Figure 4 . Probability of having no progression to cancer, according to L O H pattern  B t i i  t  —  . . . .  3p  3p Het  and  r  9p Het  Is ip'LdH p value  3 p &/or  <.01  p_value 10  12  9pLtiH '  '  1  14  <.01  16  6  8  10  12  Years  ' d p a n d 9 p Het  3 f l &/or 9 p L O H  Is  only  3 p &/pr 9 p L O H  only  3p'&/or9p I^OHonly  dl  3 p &/br 9 p  §s  LdHplusothers 3 p &/or 9 p L O H  p_value  <.Q1  p_value 10  12  14  plus 4 q  3 p &/or 9 p L O H  =.09  p_value a  16  10  12  plus 8p  =.03  14  10  12  14  16  Years  3 p &/pr 9 p L O H  \  s &  only  8  1  „  3 p &/or 9 p L O H ' p l u s l l q  p_yalue  «  i  3 p &/or 9 p L O H . o n l y  3p&/or 9 p L O H plus  12  14  only  3p&/or 9p LOH  plus  17p  8  8 p value  10  2  13q  §  =.05 8  &/or 9 p L O H  Is  9  a » j?  0.  ^ 1  6  8  10  <.01 12  p_value 14  8  =.08  10  Years  A, progression as a function of LOH at 9p (No LOH = 69; LOH = 47). B, progression as a function of LOH at 3p (No LOH = 80; LOH = 36). C, progression as a function of LOH at 3p &/or 9p (No LOH = 56, LOH = 60). D, progression as a function of LOH at 3p &/or 9p when this loss occurred in the absence or presence of LOH at any other arm (No additional arms lost = 26, LOH on at least 1 of the following arms: 4q, 8p, 1 lq, 13q or 17p = 34). E, F, G, H, and I, progression as a function of LOH at 3p &/or 9p when this loss occurred with no additional arms lost (n = 26) or with LOH at 4q (n = 10), 8p (n = 18), 1 lq (n = 15), 13q (n = 8) or 17p (n = 21) respectively.  42  12  14  Table 7. Probability of lesions not progressing to cancer after 5 years follow-up  Proportion (%) L O H pattern  #of cases  R R (95% CI)  of non-progressing cases (95% CI)  48  100  -  9p Het  69  93 (87-99)  1.0  9p L O H  47  61 (48-78)  3.97(1.68-9.15)  3p Het  80  85(76-95)  1.0  3p L O H  36  63 (48-83)  3.74(1.76-7.93)  a  56  98 (95-100)  1.0  A l l cases with 3p & / o r 9p L O H  60  63 (50-77)  24.1 (3.3-176)  3p & / o r 9p L O H (but no other arms)  26  74 (57-95)  3.75 (1.32-10.7)  3p & / o r 9p L O H (+ L O H at any other arm)  34  53 (38-74)  33.4 (4.48-249)  3p & / o r 9p L O H (but no other arms)  26  74 (57-95)  1.0  3p & / o r 9p plus 4q L O H  10  60 (36-99)  2.3(0.86-6.16)  3p & / o r 9p plus 8p L O H  18  52 (32-84)  2.59(1.05-6.37)  3p&/or9pplus l l q L O H  15  50 (29-86)  2.49 (0.98-6.31)  3p&/or9pplus 13qLOH  8  0  7.08 (1.93-25.9)  3 p & / o r 9 p p l u s 17p L O H  21  54 (35-83)  No L O H 9p*':  3p* : 2  3p & / o r 9p* : 3  3p & / o r 9p H e t  3p & / o r 9p + others* : 4  b  2.2 (0.18-5.5)  "Includes 8 cases with L O H at other arms Calculation does not include 2 non-progressing cases that have less than 5 years follow-up *'The R R estimates the risk of 9p L O H group relative to group of 9pHet. * The R R estimates the risk of 3p L O H group relative to group of 3pHet. * The R R estimates the risk of each group relative to group of 3p&/or 9p Het * The R R estimates the risk of each group relative to group of 3p&/or 9p L O H (but no other arm loss).  b  2  3  4  43  7.4.  C o m p a r i s o n o f L O H pattern i n matching p r e m a l i g n a n t a n d malignant lesions  In the Table 8, a comparison was made of the regions of loss in biopsy pairs of progressing cases. Twenty-five of 29 progressing cases had later CIS/SCC biopsies available for LOH analysis. In 17 cases (68%), allelic losses in the premalignant lesions (upper versus lower allele) were found in the later lesion (see Fig 5. b), although 14 of these cases showed additional losses on other chromosome arms. The remaining 8 cases showed an alteration in the pattern of loss in the tumor for some of the loci that had LOH in the early biopsy. T a b l e 8: C o m p a r i s o n s o f L O H patterns i n initial biopsies w i t h that seen i n C I S / S C C that later developed at the same anatomical site # o f cases Cases i n w h i c h later biopsy  Case ID #  25  was available for analysis  Those with same LOH  3/25(12%)  #281 (6 losses), #470 (3 losses); #365 (1 loss)  14/25 (56%)  #385, #405, #416, #271, #293, #399, #185,  pattern in both biopsies Those with LOH in first biopsy also present in later  #377, #245, #286, #464, #121,#223, #460  biopsy, but with additional losses in the later lesions in the following arms: 3p  4/14 (29%)  #405, #121, #399, #185  9p  1/14 (7%)  #416  17p  3/14(21%)  #385,#405, #223  4q  5/14 (36%)  #385,#271, #293, #245, #286  8/14 (57%)  #405, #385, #223, #293,#185,#377,#245, #460  llq  6/14 (43%)  #385, #405, #416, #185, #286, #464  13q  3/14(21%)  #245, #377, #121  8/25 (32%)  #173, #446, #472, #401, #95, #406, #122, #473  4  #471, #363, #197, #79  Different LOH patterns L a t e r biopsy not available  44  Figure 5. LOH analysis of 2 patients (a, b)  a  Patient 1 D8S264 (Bp)  IFNA  D13S170  (9p)  (13p) •  D17S786  (17p) f t  f CD  CD  b  CD  • CD  Patient 2 IFNA  (9p)  D11S1778  (11q)  tp53  (17p)  ill C  D T  C  D  DNA was isolated from control stroma microdissected from lesion biopsies.  T  C D T  (C), dysplasia (D) or tumor (T)  Microsatellite markers, the chromosomal arm  being assayed and patient numbers are indicated above each block,  a: A rare mild  dysplasia with multiple allelic loss: loss of lower allele at D8S264; upper allele at IFNA;  upper allele at D13S170; and lower allele at D17S786.  b:  Patient with a mild  dysplasia (D) that later progressed to a SCC (T). The mild dysplasia shows the same pattern of multiple allelic loss as the tumor: loss of the lower allele at IFNA and D11S1778;  and upper allele at tp53  7.5.  Clinical history of the lesions.  There was no significant difference between the progressing low-grade dysplasias and those without a history of progressing in terms of gender distribution (56% male in progressing cases vs. 57% of those without a history), age distribution (mean age 58 years in progressing cases vs 55 in those without a history), site distribution, and smoking history (of those with known habits, 78% of progressing cases vs. 85% of those without a history were smokers). However, on average non-progressing cases were monitored for over twice the duration (96 versus 37 months) to ensure that progression did not occur. (Table 9). Table 9 Characteristics of patients with dysplasia  Features  Nonprogressing  Progressing  P  Age (mean, years) Sex (% male) % with smoking history  " 55  ~~58  0T4T6  57 85  56 -^g  1  Follow-up (mean, months)  96  -  37  ^ 0.0001  A history of recurrence was found for 2 of 6 progressive hyperplasias (compared with none of the 33 cases without a history of progression, p = 0.0192) and for 13 of 25 (52%) progressing low-grade dysplasia (compared to 4 of 55, or 7%, of dysplasias without a history of progression, p < 0.0001, Table 10 and 11). Furthermore, despite the fact that low-grade dysplasias in British Columbia are generally treated conservatively, 6 of the 25 progressive dysplasias had a further wide removal of their lesion (Table 10) probably because of their recurrence and progression.  46  Table 10  Id# 405  385  406  416  471  363  173  Age/ sex  Site  43/M  Tongue  66/F  68/M  57/M  70/M  70/F  74/M  L O H frequencies and clinical history in progressing lesions  Diagnosis  Time (mo.)  Chromosomal arms showing L O H  Hyperplasia  0  9p  SCC  7  9p, 3p, 17p, 8 p , l l q  Hyperplasia  0  3p  SCC  16  3p, 17p,llq, 8p,4q  Hyperplasia  0  9p, 17p, l l q  Hyperplasia  8  NA  SCC  12  9p, 17p, 3p, 4q  Hyperplasia  0  3p  SCC  72  3p, 9p, l l q  Hyperplasia  0  3p, 4q, 8p  CIS  105  NA  Hyperplasia  0  3p,9p, 17p,4q,8p, l l q  Hyperplasia  8  NA  Moderate dysplasia  31  NA  Hyperplasia  46  NA  SCC  76  NA  M i l d dysplasia  0  3p, 9p, 8p, l l q , 13q  a  Tongue  Retromolar  Retromolar  Gum  Tongue  Tongue  M i l d dysplasia  122  56/F  Tongue  b  NA  SCC  16  3p, 9p, 8p, l l q , 17p  M i l d dysplasia  0  3p, 9p, 8p, l l q , 17p  M i l d dysplasia  15  NA  Severe dysplasia,  32  NA  99  3p, 9p, 8p, 4q, 13q  C  removed with wide margin SCC  47  223  29/  Tongue  M i l d dysplasia,  0  9p  Persistently recurring  NA  NA  SCC  51  9p, 1 7 p , 8 p  M i l d dysplasia  0  3p,9p, 17p, l l q , 13q  Hyperplasia  4  NA  CIS  10  3p, 9p, 17p, l l q , 13q, 4 q  M i l d dysplasia  0  3p, 9p, 17p  SCC  26  3p, 9p, 1 7 p , 4 q , 8 p  M i l d dysplasia  0  9p,17p  Clinical recurrence and  8  NA  SCC  10  9p, 17p, 3p  M i l d dysplasia  0  NA  M i l d dysplasia  11  9p  M i l d dysplasia  23  9p  S C C (probably from V C )  26  9p  M i l d dysplasia  0  8p, 9p  S C C (very inflamed)  60  8p  M i l d dysplasia  0  3p,9p,4q,8p, l l q  Hyperplasia  2  NA  SCC  85  3p,9p,4q,8p, l l q  M  leukoplakia treated b y multiple wide electrocauterization  271  69/F  Floor o f mouth  293  47/  Tongue  M  399  44/F  Tongue  treated with bleomycin  365  446  807F  47/F  Gum  Floor o f mouth  472  60/  Tongue  M  48  185  50/  Tongue  Moderate dysplasia  0  9p, 4q  Clinical recurrence o f  48  NA  SCC  83  9p, 4q, 3p, 8p, l l q  Moderate dysplasia  0  3p,9p  Moderate dysplasia  6  NA  SCC  7  NA  Moderate dysplasia  0  3p, 4q  Moderate dysplasia  *7  NA  Moderate dysplasia  35  NA  SCC  70  3p, 4q, 8p, 13q  Moderate dysplasia  0  3p, 8p, l l q  Moderate dysplasia  5  3p, 8p, l l q , 9p  SCC  11  8p, l l q , 9p, 3p, 13q  Moderate dysplasia  0  3p, 9p  SCC  56  3p, 9p, 8p, 4q, 13q  Moderate dysplasia  0  3p, 9p, 17p, 4q, 8p, l l q  SCC  52  3p, 9p, 17p, 4q, 8p, l l q  M  leukoplakia and all grossly visible lesion removed with margin (no bx submitted)  197  39/  Tongue  M  377  52/  Cheek  M  followed by wide laser excision  473  73/  Gum  M  removed with wide margin  245  281  70/F  53/  Cheek  Tongue  M  49  401  42/  Tongue  Moderate dysplasia  0  9p, 17p, 4q, l l q , 13q, 8p,  SCC  15  9p, 17p, 4q, l l q , 13q  Moderate dysplasia*  0  3p, 17p, 8p, 13q  CIS  11  3p, 17p, 8p, 13q, 9p, 4q, l l q  Moderate dysplasia  0  3p, 9p, 4q, l l q , 13q  Recurrence of  NA  NA  SCC  46  3p,9p,4q, l l q , 13q, 8p  Moderate dysplasia  0  3p,9p,17p,4q  CIS  29  NA  Moderate dysplasia  0  3p, 17p, 8p  SCC  27  3p, 17p, 8p, l l q  Moderate dysplasia  0  9p, 8p  Moderate dysplasia  4  NA  SCC  29  9p, 8p, 3p, 13q  Moderate dysplasia  0  8p, l l q , 13q  Moderate dysplasia  5  NA  CIS  6  8p, l l q , 13q  Moderate dysplasia  0  9p, 13q  CIS  32  9p  M  286  65/  Cheek  M  460  46/  Floor of  M  mouth leukoplakia but no further biopsy or treatment were given  79  464  121  470  60/F  71/F  80/F  46/  Tongue  Tongue  Gum  Lower lip  M  95  b  76/F  Tongue  Time between biopsies, with time of first biopsy set at 0. Chromosome arms that are bold are those showing LOH in 1 biopsy but not in the other; italicized  arms have different alleles lost in consecutive biopsies *Note: This patient had history of cancer in multiple organs prior to buccal mucosa dysplasia: (1) 11 years ago: cervical lymphoma treated with chemotherapy and prednisolone; (2) 6 years ago: Bladder 50  tumor treated with surgery; (3) 3 years ago: lung SCC treated with surgery; and (4) 1 year ago: prostate cancer treated with surgery and estrogen  51  Table 11  Id#  Allelic loss in recurrent premalignant lesions without a history of progression  Age/  Site  Diagnosis  Time  Chromosomal arms  (mo.)*  showing L O H  Moderate dysplasia  0  No L O H  M i l d dysplasia  10  NA  M i l d dysplasia  0  3p, 9p, 17p, 8p, 13q, 14q,  sex 26  65/M  Left anterior gingiva  88  68/F  Right floor o f mouth  8q M i l d to moderate  9  NA  M i l d dysplasia  15  NA  Hyperplasia  0  17p, 4q (non-informative  dysplasia  277  47/?  Floor o f mouth  for 3p)  189  43/F  Soft palate  M i l d dysplasia  13  Moderate dysplasia  0  9p, 4 q , 8 p  Moderate dysplasia  32  NA  Time between biopsies, with time o f first biopsy set at 0.  52  8.  DISCUSSION  Over the last decade, genetic analysis o f tumors has led to significant breakthroughs i n our understanding o f alterations i n tissues that underlie tumorigenesis.  One o f the current  research challenges is to begin to apply such technology to the earliest clinical lesions, both to improve our understanding o f the genetic alterations that underlie early cancer development and, hopefully, to provide potential indicators o f risk for such lesions.  8.1.  High frequency of allelic loss characterized dysplastic lesions  This study showed that loss o f regions o f chromosomes containing presumptive tumor suppressor genes occurred early during oral carcinogenesis. A significant percentage o f lowgrade oral dysplasias showed allelic loss suggesting that loss o f these regions may play an important role in the evolution and growth advantage o f these premalignant clones. The presence o f histological evidence o f dysplasia is presently the gold standard for judging malignant potential o f oral premalignant lesions. This study showed that advent o f dysplasia was accompanied b y significantly increased frequency o f L O H when compared to hyperplasias. When the patterns o f loss among hyperplastic and low-grade dysplastic lesions were compared (Table 4), L O H was found i n only 13 o f 39 (33%) cases showing loss on any o f the 7 arms studied i n hyperplastic lesions. In contrast, such loss occurred i n the majority o f dysplastic lesions, 55 o f 77 (71%) samples (p < 0.001).  This increase i n allelic loss i n  dysplastic lesions also was evident when the percentage o f cases showing multiple arm loss was determined.  While multiple losses among hyperplastic lesions was limited to 3 o f 39  (7.7%) cases, 38 o f 77 (49%) o f low-grade dysplasias showed > 1 arm lost (pO.OOOl). Our  53  results are consistent with M a o ' s : Lesions with dysplastic alteration had a higher rate o f microsatellite alterations (3pl4 and 9p21), 50% vs 3 1 % (P = 0.08). It is well accepted that presence o f dysplasia has been associated with an increased risk o f malignant transformation (van der W a a l et al, 1997). The fact that the frequency o f L O H was associated with the presence o f dysplasia would suggest a cancer predictive value for L O H and also suggest that molecular changes may underline the dysplastic phenotypes.  8.2  Increased frequency of allelic loss in progressing lesions  Linkage o f specific patterns o f genetic alteration to disease progression is often limited by the difficulty o f obtaining clinical specimens from the same lesion over time. In this study, microdissected early oral premalignant lesions from 116 patients with or without a history o f progression into CIS or invasive S C C were analyzed for L O H at on 7 chromosomes i n order to identify genetic differences between progressing and non-progressing lesions, and to identify genetic profiles that have predictive value for early premalignant lesions. One o f the more striking observations made i n this study is markedly increased frequency o f allelic loss i n progressing lesions as compared to the non-progressing lesions. A l l such progressing lesions, both hyperplasias and dysplasias, showed loss on at least 1 arm. In contrast, hyperplasias and low-grade dysplasias without a history o f progression had loss i n 2 1 % and 59% o f cases respectively (Table 4). Furthermore, multiple arm loss (>1 arms lost) was absent i n hyperplasias without a history o f progression but present i n 3 o f 6 progressing hyperplasias.  A l s o , the majority o f progressing dysplasias had >1 arms lost (91% o f cases  compared to 3 1 % o f dysplasias without a history o f progression) with 57% o f cases having loss at >2 arms (compared with 20% o f dysplasias without a history o f progression).  54  These data support the hypothesis that progressing early lesions and those without a history o f progression can appear morphologically or phenotypically similar, yet differ significantly at the genetic level. Furthermore, the data suggest that assaying hyperplasias and low-grade dysplasias for L O H frequencies might be o f clinical use i n differentiating between low-risk and high-risk lesions.  8.3.  The pattern of allelic loss characterized progressing lesions  The earliest loss was at 3p and occurred before the advent o f dysplasia.  Even  hyperplasia without a history o f progression demonstrated 3p loss i n 13% o f the cases. This may suggest that T S G s at 3p, such as FHIT, may play an important role i n early oral carcinogenesis. The most common loss i n low-grade dysplasia was at 9p (46%), supporting the hypothesis that p i 6 gene dysfunction plays important role i n early oral carcinogenesis. The fact that the most common losses for both sets o f cases were on 3p and 9p was consistent with results from previous studies that have shown that L O H at 3 p l 4 and 9p21 (the regions studied in this paper) are frequent occurrences in oral premalignant lesions and likely to occur early i n oral carcinogenesis (Zhang et al, 1996; Califano et al, 1996; M a o et al, 1996a; R o z et al, 1996). The loss at 3p & / o r 9p was not only the earliest and most common event during oral carcinogenesis, it was also significantly higher i n the progressing lesions (Table 5), with this trend apparent when 3p and 9p are considered separately, even more apparent when they are considered together. Virtually all progressing lesions (22/23, 96% i n dysplasia and 6/6, 100% in hyperplasia) had loss on 3p & / o r 9p, compared to 5 o f 29 (17%) hyperplasias without a 55  history o f progression and 30 o f 54 (56%) o f low-grade dysplasias without a history o f progression. These data suggest that L O H at 3p & / o r 9p is not simply one o f those random genetic alterations but rather a prerequisite for progression o f oral premalignant lesions. From a clinical point o f view, this would suggest that analysis o f L O H at 3p and 9p may be used as an important initial screen for the malignant risk o f leukoplakia.  However,  because o f the high frequency o f L O H at 3p and 9p i n early lesions without a history o f progression, especially i n dysplasia (56%), these markers may not be the best markers for prediction o f prognosis, at least not by themselves. L O H on the other 5 chromosome arms (4q, 8p, l l q , 13q and 17p) occurred rarely or at low frequency i n non-progressing lesions. In contrast, a significant number o f progressing lesions  including both  hyperplasia and  low-grade  dysplasia showed  L O H at  these  chromosome arms. L O H i n these chromosome arms have also been reported to occur in oral premalignant lesions; however, such losses (especially for chromosome arm 4q, 8p, l l q and 13q) were generally seen i n high-grade preinvasive lesions (severe dysplasia/CIS) or S C C (Califano et al, 1996; unpublished data from this lab). This would suggest that L O H s on these chromosome arms are relatively Tate' events i n the preinvasive stages and should be rare or absent in hyperplastic or low-grade dysplastic lesions. This was indeed the case for the lowgrade dysplasias without a history o f progression in this study. In contrast, progressing l o w grade dysplasias had increased frequencies o f loss for these chromosome arms. Progressing low-grade dysplasias had significant increases for 8p (p = 0.003), 1 l q (p = 0.01) and 13q (p = 0.002) with the increase for 4q approaching significance (p = 0.057).  There was also a  doubling in the frequency o f L O H on 17p (from 20% to 4 1 % o f cases); although this increase was not significant or only marginally significant.  56  The high frequency o f L O H on these arms i n high-risk advanced oral preinvasive lesions and in lesions with proven progression would suggest L O H on these arms indicates a higher cancer risk (than those with only 3p & / o r 9p). In fact, when the percentage o f cases that showed a combination o f 3p & / o r 9p loss with additional losses at any o f the remaining arms was determined, this occurrence was shown to be present i n 0% o f hyperplasias without a history o f progression compared to 50% o f progressing hyperplasias and in 28% o f dysplasias without a history o f progression and 78% o f progressing dysplasias, both are significantly different (Table 5). These results would suggest that additional losses on these chromosome arms indicate cancer risk.  8.4.  L O H as markers to predict cancer risk - further statistic analysis  The present study  showed that progressing  and non-progressing  lesions  had  significantly different L O H profiles. H o w could we use the study data for the evaluation o f the cancer risk o f oral premalignant lesions? Since loss at 3p & / o r 9p was the most common and earliest event and seemed to a prerequisite for progression, it may be used as an initial screening for assessing cancer risk o f oral premalignancies. If L O H at 3p & / o r 9p had been used as an initial screening for our study set, without knowledge o f L O H at other arms, those cases with 3p & / o r 9p L O H (with or without L O H at other arms) would have had a 24-fold increase i n the relative risk o f cancer progression as compared to those without L O H at either 3p or 9p (Fig.4C, Table 7). This is consistent with the results o f M a o et al, (1996a) who found L O H at 3p & / o r 9p predicts cancer risk o f oral leukoplakias. However, since there was a high frequency o f allelic loss on these arms in non-progressors and the relative cancer risk for those with L O H at 3p & / o r 9p but no other arms was only increased by 3.8-fold i n the  57  relative risk o f cancer progression as compared to those without L O H at either 3p & / o r 9p (Fig.4D, Table 7), additional markers are essential for better prediction o f prognosis. The study results suggest that loss at any o f the other 5 chromosomes (4q, 8p, l l q , 13q, and 17p) in addition to L O H at 3p & / o r 9p seem to provide a better predictive value. Those cases with such losses had a 3 3-fold increased risk o f progressing to cancer compared to cases that retained both o f these arms (Fig 4 D , Table 7). Furthermore, time-to-progression curves showed that lesions that had 3p & / o r 9p loss with additional loss on at least one o f the indicated arms had a significantly shorter progression time than those with 3p & / o r 9p loss only (p<0.01,Fig. 4 D ) . To determine which o f the additional losses (on 4q, 8p, l l q , 13q or 17p) would most significantly increase progression risk, the study separately compared those cases with 3p &/or 9p loss alone against those cases with 3p & / o r 9p loss plus each o f the additional losses (Figure 4 E to I). A significantly shorter time-to-progression was observed when either 8p (p<0.03, by the logrank test), l l q (p<0.05) or 13q (p<0.01), L O H was present i n addition to 3p & / o r 9p L O H .  Comparisons with 4q (P = 0.09) or 17p (P = 0.08) were not statistically  significant, although a trend was observed. For each premalignant L O H pattern, the probability o f having no subsequent progression is summarized in Table 7. Forty to sixty percent o f individuals with additional losses at 4q, 8p, l l q or 17p developed cancer within 5 years, corresponding to a 2.2-2.6-fold increase i n relative risk o f cancer progression as compared to those with L O H at 3p&/or 9p only. Moreover, cases with additional 13q loss had a 7-fold increase in risk o f progression. Six o f the 8 cases with loss on this arm had 5 years o f follow-up and all showed progression within this timeframe.  58  In conclusion, although prospective studies involving large numbers o f subjects over time are necessary  to fully  understand  the  relation between  chromosomal loss and  tumorigenesis, the study data do suggest that L O H patterns w i l l facilitate the prediction o f the malignant potential o f low-grade premalignancies. Can we use this information clinically? In addition to being used for deletion mapping o f tumor suppressor genes, the L O H assay has begun to be used to assist pathologists i n screening for patients with premalignant lesions and cancer. M a o (1996c) analyzed L O H i n urothelial cells obtained from urine sediments o f cancer patients and correctly identified 9 5 % o f the patients with cancer. There have also been prognostic studies that attempt to correlate the degree o f L O H and survival rate (Partridge et al, 1996; Scholnick et al, 1996; Nawroz et al, 1996; Field et al 1995). For example, Nawroz (1996) found that patients with L O H at any o f their 12 markers all had advanced disease (stage III or I V ) , most had nodal metastases, and half o f them died o f disease. Recently, Califano (1999) tried to use detection o f genetic alterations for molecular identification o f the site o f origin o f the primary tumor; Harda (1999) and Ogawara (1998) both found L O H on 13q was significantly correlated with lymph node metastasis i n oral S C C and H N S C C ; Partridge (1999c) found that patients with allelic imbalance at 3p24-26, 3 p l 3 and 9p21 have an approximately 25 times increase i n their mortality rate relative to patients retaining heterozygosity at these loci. She suggested that it would be possible to develop a molecular staging system which w i l l be a better predictor o f outcome than conventional clinicopathological features,  as the molecular events represent fundamental  biological  characteristics o f each tumour; Matsuura (1998) found L O H o f chromosome 9p21 and 7p31 is correlated with high incidence o f recurrent in H N S C C . Finally, M a o (1998) has suggested that clinical and histologic assessments o f the response to chemporeventive agents may be  59  insufficient to determine their efficacy and that critical genetic alterations could be used as independent biomarkers to augment the ability to evaluate the efficacy o f such agents. A l l these studies positively support our inquiry. Our data suggest that L O H at 3p & / o r 9p would place the patient into an at risk category since nearly all progressing cases i n our cohort showed such alteration. Their relative risk increases with additional loss on other arms. Although additional losses on any o f these arms is an indicator o f probable progression, the loss o f 13q might signal the need for active intervention with either traditional or novel forms o f therapy such as chemoprevention.  8.5.  Most progressed lesions may be derived by clonal outgrowth from the earlier lesions.  Comparing the L O H pattern o f premalignant and malignant i n patients that later progressed to C I S or S C C , we have found that most o f L O H patterns in the premalignant lesions  were found i n their matched later lesions, although 14 o f these cases showed  additional losses on other chromosome arms and the remaining 8 cases showed an alteration in the pattern o f loss i n the tumor for some o f loci that had L O H i n the early biopsy (Fig. 5 b and Table 8).  However, in 3 o f the latter cases this difference was restricted to a single arm  out o f 5 or 6 arms lost. For example, i n case #173 the early lesion contained a L O H at 13q that was not found in the later lesion; however, the pair showed loss o f the same alleles on 3p, 9p, 8p and l l q . Our analysis o f lesions with progressed cases suggests that for most progressing lesions, the later cancer was derived by clonal outgrowth from the earlier lesions. In other words, the later cancer progressed from the early lesion.  60  8.6.  Does evidernce of recurrence serve as a further indicator of cancer risk in premalignant lesions?  W e obtained some clinical information for our subjects b y tracking recurrent lesions as they appeared i n our database and by following the case histories o f treatment i n hospital charts.  W i t h the information available, the data showed a significantly higher number o f  recurrences among progressing lesions (61% o f progressing dysplasias vs. 7% dysplasias without a history o f progression) (Table 10 and 11). Although there are some studies that correlated L O H and recurrence in head and neck cancer (Matsuura et al, 1998; Lydiatt et al, 1994 and 1998), no study showed the relationship between recurrence and cancer risk i n premalignant lesions. There are at least two possible explanations for our results: 1) the recurrence resulted from failure to totally remove these lesions and this failure was more prevalent among progressing cases; and 2) the recurrence reflected a more aggressive lesion with an elevated rate o f genetic change. The study does suggest that a large percentage o f progressing lesions were not totally removed.  In 17 o f 25 (68%) progressing lesions, loci lost in the early lesions showed the  same pattern o f loss (upper versus lower allele) i n the later lesion, suggesting that they were derived by clonal outgrowth from the earlier lesions.  Interestingly, many o f these lesions  were considered to be totally eliminated using histological and clinical criteria. However, there is no evidence that treatment was less aggressive among progressing cases compared with those without a history o f progression. In fact, i n 6 o f the 25 progressing dysplasias the decision was made to treat aggressively again, based on the recurrence and/or progression o f the lesions (Table 10: #473, #122, #223, #399, #185 and #377). Despite wide removal o f the  61  lesion or wide laser excision or multiple wide electrocauterization or treatment with bleomycin, the same site later still progressed into cancer. Our data support the second probability that these progressing lesions were indeed more aggressive in nature, possibly due to the specific genetic alterations that they had undergone. The recurrence reflected a more aggressive lesion with an elevated rate o f genetic change. It is well known that many oral leukoplakias, which histologically consist o f hyperplasia & / o r dysplasia, w i l l not progress into cancer even when left untreated.  This  would imply that incomplete removal is only important clinically when the premalignant lesion is a high-risk lesion (genetic profile more important).  In a rare, long-term. study,  Banoczy (1977) followed 670 patients with oral leukoplakia for up to 30 years (average 10 years). The treatment for many o f these lesions was described as 'removal o f local irritants and conservative treatment'. A large percentage o f these lesions either regressed (29.7%) or remained static (25.8%). It is possible that we did not see as many recurrent lesions without a history o f progression as progressing lesions because the clones o f genetically altered cells regressed or remained static i n lesions without a history o f progression, even i f they were not totally removed during the incisional biopsy. The result that 3 o f the 4 recurrent premalignant lesions without a history o f progression had multiple losses (Table 11) supports  the  hypothesis that the recurrent lesions were biologically more aggressive and that this behavior was associated either directly or indirectly with the amount o f genetic damage in the lesion. Moreover, whether or not the failure o f these recurrent lesions to progress was a result o f their complete removal or insufficient time for follow-up is still not clear although a significantly greater length o f follow-up was observed for non-progressing lesions (progressing cases, 37  62  months vs. non-progressing, 96 months, P = 0.0001) (Table 9). W e may pay more attention to the recurrent lesions clinically.  8.7.  Summary  The results from this study suggest that L O H patterns w i l l facilitate the prediction o f the malignant potential o f low-grade premalignancies. In this study, we have demonstrated the predictive value o f allelic loss subsequent to 3p & / o r 9p loss, especially with 13q loss which increases the relative risk i n progression by 7 fold.  W e may more precisely predict the  malignant potential o f low-grade premalignancies i f we combine L O H patterns with the predictive value o f dysplasia and clinical attributes such as recurrence after treatment. W e are only beginning to understand the processes that control the malignant transformation o f oral premalignancies.  For example, despite the significant association  between multiple L O H and cancer risk, the speed o f the malignant transformation was not affected by the number o f the L O H i n a lesion (Table 10).  Some premalignancies had a  relatively low number o f regions showing L O H and yet progressed into cancer rapidly while others, with numerous losses, seemed to 'sit' for a long time before S C C occurred.  This  suggests that other changes, either genetic or epigenetic, must control or influence the malignant transformation.  Such changes could include not only allelic losses at other  chromosomal regions but also mutations to proto-oncogenes and T S G s or even epigenetic changes such as D N A methylation or histone deacetylation (Bakin et al, 1999). Alternatively, the immune system or homeostatic regulation o f premalignant cells by surrounding normal cells such as epithelial-mesenchymal interaction could also play a role i n controlling outgrowth o f such lesions.  63  Furthermore, the study also suggests that not all cells in the high-risk premalignancies were removed even though the margins appeared clinically and histologically clean. Previous studies have shown that the presence of genetically damaged cells at the margins of surgical resection of oral SCC could account for tumor recurrence, despite gross and histologically free surgical margins (Westra et al, 1998). The same principle might be applied to oral premalignant lesions. 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L O H Frequencies (%) in Head and Neck and Oral Cancers Chromo  Oral Cancer  Head and Neck Cancers  some arm Wu  Uzawa  Roz  Ishwad  Ah-  Nawroz  Adamso  Field  1994  1996  1996  1996  See  1994  n 1994  1995  1994  ElMaestr  Naggar  oe  1995  1996 *  14  30  27  lq  0  23  28  2p  0  19  28  2q  8  15  13  44  67  52  3q  0  50  13  4p  12  38  8  4q  11  37  13  5p  11  19  17  5q  43  25  29  0  38  21  6q  5  23  25  7p  8  23  8  0  29  7  10  40  35  7  38  21  24  72  62  72  35  13  20  35  IP  3P  6p  7q 8  52  50  58  7  11  P  8q 9p 9q  48  47  53  76  iop  0  23  9  lOq  0  21  13  lip  5  17  13  45  61  23  12p  7  18  14  12q  12  25  15  0  54  27  5  39  11  15q  6  5  12  16p  0  10  13  16q  0  10  13  17p  31  52  17q  19  31  30  18p  0  27  16  18q  0  23  49  19p  0  32  0  19q  0  40  29  20p  0  30  0  20q  7  9  9  5  26  8  16  29  0  llq  56  6  33  13p 13q  67  14p 14q 15p  50  50  21p 21q 22p 22q  *Blanks indicate information not available.  77  Table 6 The time between initial biopsy and current date /the date on which a lesion has progressed to SCC  Groups  Case# Arms with L O H  Arms with Nl  # arms Time between initial lost  biopsy and current date/SCC (mo.)  Without progression set Mild dysplasia  108  4q  0  65  180  0  122  184  0  161  186  3p,9p,17p,llq  4  164  187  9p,4q  2  183  188  9p  1  123  191  3p,9p,llq  3  155  201  9p  1  145  206  0  130  207  0  112  1  159  233  0  103  239  0  28  1  145  209  9p  240  9p  241  9p  4q  1  127  242  llq  9p  1  122  246  3p,Hq  135 mmmm  248  0  112  256  0  112  258  3p,9p,17p  3  103  270  8p  1  110  78  277  17p,4q  3p,llq  2  147  12  1  65  21  0  66  26  0  65  51  0  52  1  52  0  54  0  41  55  3p  60  8p  64  Moderate  88  9p,17p,8p,13q  3p  4  42  92  9p  8p,13q  1  40  143  3p,9p,17p,8p,13q  5  37  163  3p,9p,17p,llq  4  37  167  9p  1  141  168  3p,9p,17p,8p  4  62  169  3p,9p  2  142  0  164  dysplasia  llq  176 177  3p,9p,17p,8p  4  32  189  9p,4q,8p  3  170  190  3p,9p,17p,8p  4  145  214  3p,9p  2  105  232  9p  1  147  234  HHHHH  1  109  243 257  4q  .  8p  274  0  104  0  110  0  Zo  22  9p,17p  2  65  25  4q  1  64  . . .................  79  32  3p,9p,17p,llq  34  8p  4q  4  44  1  47  0  47  0  59  76  0  66  101  0  41  0  41  0  41  66 71  Hyperplasia  4q,9p  102  4q  103 111  8p,Hq  0  41  127  3p  0  35  129  3p  1  37  130  3p  1  37  131  13q  1  36  132  0  36  133  0  37  0  35  135  0  38  136  0  35  137  0  36  134  138  3p  41  3p  139  0  41  140  0  37  141  0  36  142  0  42  144  0  36  146  9p  1  37  148  llq  1  36  80  149  13q  0  36  150  4q  0  37  151  mmm  37  152  0  36  171  3p,8p,llq  0  34  172  9p  0  34  179  0  34  183  0  34  11  0  43  23  0  42  28  0  43  With progression set Mild and  173  3p, 9p, 8p,llq,13q  5  16  122  3p,9p,17p,8p,llq  5  99  1  51  5  10  moderate dysplasia  223 8p  271  3p,9p,17p,llq,13q  293  3p,9p,17p  3  26  399  9p,17p  2  10  365  9p  1  26  446  9p,8p  2  60  472  3p,9p, 4q, 8p, l l q  5  85  185  9p,4q  2  83  197  3p,9p  2  7  377  3p,9p  2  70  473  3p,8p,llq  3  11  4q,13q  81  Hyperplasia  2  56  6  52  6  15  3p,17p,8p,13q  4  11  460  3p,9p,4q,llq,13q  5  46  79  3p,9p,17p,4q  4  29  464  3p,17p,8p  3  27  121  9p,8p  o L  29  470  8p,llq,13q  3  6  95  9p,13q  4q  2  32  405  9p  13q  1  7  1  16  3  12  1  72  3p,4q,8p  3  105  3p,9p,17p,4q,8p,llq  6  76  245  3p,9p  281  3p,9p,17p,4q,8p,llq  401  9p,17p,4q,8p,llq,13q  286  17p  3p  13q  385 406  9p,17p,llq  416  HHHHHi  471 363  13q  82  

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