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Allelic imbalance at 11q in oral cancer and premalignant lesions An, Ding 2002

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A L L E L I C I M B A L A N C E AT 11Q IN O R A L C A N C E R A N D PREM ALIGN ANT LESIONS by  DING A N B.Sc, Lanzhou University, China, 1998  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R Of SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biological and Medical Sciences; The Faculty of Dentistry)  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A May 2002 © D i n g A n , 2002  A L L E L I C I M B A L A N C E AT 11Q IN O R A L C A N C E R A N D PREMALIGNANT LESIONS by  DING A N B.Sc, Lanzhou University, China, 1998  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R Of SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biological and Medical Sciences; The Faculty of Dentistry)  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A  May 2002 © D i n g A n , 2002  In  presenting  degree  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  freely available for copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department The University of British Vancouver, Canada  DE-6 (2/88)  Columbia  purposes  gain shall  requirements that  agree  may  representatives.  financial  permission.  I further  the  It not  that  the  Library  permission  be  granted  is  understood be  for  allowed  by  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Oral squamous carcinomas ( S C C ) remain a significant public health problem worldwide despite advances i n therapy and local disease control. N e w innovative strategies must be developed for the prevention, early detection and treatment o f oral carcinoma. Such approaches w i l l be heavily dependent on a better understanding o f the molecular mechanisms underlying carcinogenesis at this site.  This thesis describes a series o f studies done on oral cancers and premalignant lesions to better define the role o f alterations on chromosome 11 i n the development o f oral cancer at this site. Although numerous studies have reported the presence o f alterations on this chromosome arm in oral cancers, few studies have examined premalignant lesions i n order to determine whether such alterations play a role i n the development o f the disease. The objectives o f this thesis were: 1) to use microsatellite analysis to examine D N A extracted from severe dysplasia, carcinoma in situ (CIS) and S C C for novel alterations on 1 l q ; 2) to determine at what stage o f oral cancer development the alteration occurred by performing microsatellite analysis on a spectrum o f stages o f oral premalignant lesions (hyperplasia, m i l d dysplasia, moderate dysplasia, severe dysplasia, CIS) as w e l l as invasive S C C . The data obtained was compared to 2 previously studied hotspots on 1 l q : the int2 (1 l q l 3 ) and D11S1778 (1 lq22-23); and 3) to determine the significance o f allelic imbalance ( A l ) at int2 (1 l q l 3 ) and D11S1778 (1 lq22-23) to the progression o f  ii  oral premalignant lesions by comparing frequencies o f loss for different locus i n lowgrade dysplasia with_known outcome, i.e. low-grade lesions that did not progress into cancer with morphologically similar lesions that did develop into S C C .  In summary, the data suggest that at least 3 regions o f alteration are present on 1 l q i n both oral premalignant and malignant lesions and that 1 o f these regions, identified as containing the novel marker D11S4207, might play a significant role i n the early development o f the disease. The data further support the use o f these markers to identify progression risk for early oral premalignant lesions.  iii  T A B L E OF C O N T E N T S  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST O F T A B L E S  ix  LIST O F F I G U R E S  xi  LIST O F A B B R E V I A T I O N S  xii  ACKNOWLEDGEMENTS  xiv  DEDICATION  I.  xv  INTRODUCTION  1  1.1.  Overview  1  1.2.  Clinical, histological and molecular alterations during oral carcinogenesis  4  1.2.1.  Clinical alterations during oral carcinogenesis  4  1.2.2.  Histological alterations during oral carcinogenesis  7  1.2.2.a.  Normal oral mucosa  7  1.2.2.b.  Oral premalignant lesions and squamous cell carcinoma (SCC)  8  iv  1.2.3.  Molecular alterations during oral carcinogenesis  12  1.2.3.a.  The importance of studying genetic changes in cancer progression  12  I.2.3.b.  Oncogenes and tumor suppressor genes (TSGs)  14  1.2.3.c.  Oncogenes and TSGs in oral premalignant and malignant lesions  18  1.2.3.d.  Microsatellite analysis amd loss of heterozygosity (LOH) studies  19  1.2.3 .e.  Microsatellite analysis of oral cancer and premalignant lesions  24  1.2.3.f.  Genetic progression model for head and neck cancer  25  1.2.3 .g.  Prediction of risk of progression for oral premalignant lesions (OPL)  29  1.3.1.  Genetic changes on 11 q in a variety o f cancers  1.3.2.  Genetic alterations at 1 l q in head and neck squamous cell carcinoma (HNSCC)  31  34  1.3.3.  Mechanism o f alteration at 1 l q l 3 and 1 lq22-23  1.3.4.  11 q alterations i n head and neck premalignant lesions  1.3.5.  M a i n genes on chromosome 1 l q  36  43  45  I.3.5.a.  CCND1 {cyclin Dl, PRAD1) gene at 1 l q l 3  48  I.3.5.b.  int2 (FGF3) gene at 1 l q l 3  I.3.5.C.  HST1 gene at 11 ql3  50  I.3.5.d.  EMS1 gene at l l q l 3  51  49  v  1.3.5.e.  MENI gene at 1 l q l 3  I.3.5.f.  RIN1 gene at l l q l 3  52  I.3.5.g.  y47Mgeneat llq23.1  53  II.  52  STATEMENT OF PROBLEMS  56  II. 1.  Where are the additional tumor genes at 1 l q l 3 located?  56  II.2.  A t what stages o f oral cancer development does A I occur for int2 (1 l q l 3 ) , D11S1778 (1 lq22.3) and any novel loci identified i n this study?  57  III.  OBJECTIVES  59  IV.  HYPOTHESES  60  V.  MATERIALS AND METHODS  61  V . 1.  S ample collection  61  V.2.  Sample sets  61  V.3.  Diagnostic criteria for the samples  V.4.  Clinical information  64  V.5.  Slide preparation  65  V.6.  Microdissection  ...66  V.7.  Sample digestion and D N A extraction  ,  vi  64  66  V.8.  D N A quantification  67  V.9.  Primer extension preamplification (PEP)  67  V.10.  Coding samples  68  V.ll.  End-Labeling  68  V.12.  Microsatellite analysis: P C R amplification  70  V . 13.  Scoring o f allelic imbalance  71  RESULTS  72  V I . 1.  Choice o f microsatellite markers for this study  72  VI.2.  A l at D11S4207 i n oral S C C s and premalignant lesions  75  VI.3.  The timing o f induction o f A l at D11S4207, int2 and D11S1778 during  VI.  histological progression  VI.4.  81  Further evidence in support o f the A l at D11S4207 being an independent event. 85  VI.5.  Fine-mapping at D11S4207  VI.6.  A l l e l i c imbalance at chromosome 1 l q and malignant progression risk  88  DISCUSSION  93  VII.  87  V I I . l . A l l e l i c imbalance o f genes at D11S4207, D11S1778 and int2-cycline Dl during  vii  multistage oral carcinogenesis  93  VII. 1.1.  Temporal changes o f the 3 loci at 11 q  94  V I I . 1.2.  Significance o f the changes o f the 3 loci at 1 l q  96  VII. 1.2.1.  Significance of AI at Dl 1S4207  VII.1.2.2.  Significance of AI at DUS1778 (1 lq22-23) and int2 (M2-cyclin Dl region)  VII.2.  A l l e l i c imbalance at D11S1778 and int2 is associated with cancer risk  VII.3. D11S4207, a new hot spot at 1 l q l 3  VII.4. Ending M a r k  96  99  100  102  104  BIBIOGRAPHY  105  viii  LIST OF T A B L E S  Table 1.  A l i n oral lesions  26  Table 2.  A l on 1 l q i n various cancers as detected by microsatellite analysis  33  Table 3.  A l at loci on 1 l q i n H N S C C s  35  Table 4.  Amplification at 1 l q l 3 as identified with F I S H i n H N S C C  41  Table 5.  Genetic alterations at 1 l q o f head and neck premalignant lesions  44  Table 6.  List o f genes located at 1 l q l 3  46  Table 7.  Histological groups i n sample set 1  62  Table 8.  Histological groups i n sample set 2: the progression test series  63  Table 9.  A l l e l i c imbalance at D11S4207 i n a spectrum o f primary lesions with different  .  histological diagnoses  77  Table 10. A l l e l i c imbalance at D11S4207, int2 and D11S1778 oral premalignant lesions and S C C  84  Table 11. Patterns o f alteration at Dl 1S4207 and int2: frequencies at which these alterations occur together or independent o f each other  86  Table 12. Patterns o f alteration at D11S4207 and D11S1778: frequencies at which these alterations occur together or independent o f each other  ix  87  Table 13. Demographic information o f patients with low-grade dysplasia  Table 14. A l l e l i c imbalance o f DllSI778  90  (ATM) and int2 (intl-cyclin  Dl) i n progressing  and non-progressing hyperplasia and low-grade dysplasia  91  Table 15. Association o f allelic imbalance at D11S4207 and 3p &/or 9p i n low-grade dysplasias  99  LIST OF FIGURES  Figure 1. Clinical presentation o f oral premalignant lesions  5  Figure 2. Histological progression model for oral premalignant and malignant lesions. 11  Figure 3. Schematic illustration o f microsatellite analysis and A I  23  Figure 4. Molecular progression model o f oral cancer proposed by R o s i n and Zhang  (2001)  28  Figure 5. The procedures o f B F B cycle model of 1 l q l 3 amplification  38  Figure 6. Microsatellite results for cases with A I at D11S4207 and retention at int2  74  Figure 7. Microsatellite analysis o f S C C cases at D11S4207 and int2  76  Figure 8. Microsatellite analysis o f hyperplasia cases at D11S4207  79  Figure 9. Comparison o f A I frequencies observed at D11S4207 with those at 3 p l 4 , 9p21 and 8p  80  Figure 10. Comparison o f A I frequencies observed at D11S4207 with those seen with microsatellite markers at D11S1778 and mt2  83  Figure 11. Probability o f having no progression to cancer, according to A I at Dl 1S1778 or at int2  92  xi  LIST O F A B B R E V I A T I O N S AI  allelic imbalance  ATM  ataxia telangiectasia mutated  BAC  bacterial artificial chromosome  BFB  Breakage-fusion-bridge  CCND1  cyclin D l  CIS  carcinoma in situ  dmin  double-minute chromosomes  DNA  deoxyribonucleic acid  FGF  fibroblast growth factor  FISH  fluorescence in situ hybridization  H & E  hematoxylin and eosin  hsrs  homogeneously staining regions  HNSCC  head and neck squamous cell carcinoma  HST1  heparin secretory transforming factor 1  LOH  loss o f heterozygosity  Mb  megabase pair  MEN1  multiple endocrine neoplasia type 1  OPL  oral premalignant lesion  PCR  polymerase chain reaction  SCC  squamous cell carcinoma  TSG  tumor suppressor gene  WHO  W o r l d Health Organization  xiii  ACKNOWLEDGEMENTS  I would like to take this opportunity to thank m y supervisor Dr. L e w e i Zhang for her support and guidance throughout m y degree; and to extend m y warmest gratitude to m y co-supervisor Dr. M i r i a m P. Rosin for her academic help and constant moral support. I am also grateful to Dr. Douglas Waterfield for being m y committee member and for his valuable comments. I really appreciate technicians and other students i n D r . Rosin and Dr. Zhang's lab for their support.  I also would like to take this opportunity to thank m y grandma, m y uncle Y u and his wife Huijun, m y brother Fan and m y cousin Ruobing for their continuous support and understanding.  xiv  DEDICATION  To My Mom and My Dad Who Love Me and Whom I Love Forever  I.  INTRODUCTION  1.1.  Overview  Oral cancer is the sixth most common cancer in the world, accounting for about 3% o f all new cancers i n Western countries (Harras et al, 1996; Greenlee et al, 2001) and up to 40% o f all cancers in places such as India (Saranath et al, 1993). Despite refinement o f surgical techniques and adjuvant therapies, the prognosis o f oral cancer has remained unchanged for decades with 5-year survival rates o f 40-50% i n the Western world and even lower in undeveloped countries (20-43%) (Rao and Krishnamurthy, 1998; Greenlee et al, 2001). The major cause o f this high mortality rate is the late-stage at which most cancers are identified, resulting i n a high local recurrence and formation o f second primary malignancies even i n successfully treated cases (Cooper et al, 1989; D a y and Blot, 1992; Lippman and Hong, 1989; Shikhani et al, 1986). The development o f novel approaches for the prevention, early detection, and effective treatment o f this disease are critical to improving outcome. Such approaches are dependent on a better understanding o f the molecular and cellular mechanisms underlying the tumorigenesis process.  Molecular analyses o f oral cavity tumors have uncovered a number o f recurrent genetic events that appear to underlie the development o f the disease. A m o n g these changes are frequent alterations to loci (and genes) on chromosome 1 l q . Microsatellite analysis o f  1  oral tumors suggests the presence o f at least 2 regions on this chromosomal arm containing putative T S G s or oncogenes. These regions include 1 l q l 3 and 1 lq22-23, each o f which contain loci that show frequent allelic imbalance i n tumors, with such alteration occurring in 30-80% o f cases (Dwight et al, 2000; Ah-See et al, 1994; Venugopalan et al, 1998; D ' A d d a et al, 1999; U z a w a et al, 1996; Lazar et al, 1998; H u i et al, 1996). The region around 1 l q l 3 is o f particular interest i n that amplification o f this region (as identified with fluorescent in situ hybridization or F I S H ) has been reported in 30-50% o f oral cancers (Fujii et al, 2001; Ott et al, 2002; Bayerllein et al, 2000; A l a v i et al, 1999; Williams et al, 1993; M u l l e r et al, 1997; Meredith et al, 1995; Fortin et al, 1997). Several genes potentially important for creating the malignant phenotype are located at l l q l 3 or l l q 2 2 - 2 3 including int2, FGF4, EMS1, RIN1, MEN1, PAK1, ATM and the most commonly reported gene, cyclin Dl.  However, the evidence in  support o f the involvement o f the majority o f these genes is still limited.  There is little information on the involvement o f alterations on 1 l q i n oral premalignant lesions. Such alterations have been reported as being present i n head and neck premalignant lesions (El-Naggar et al, 1995; Califano et al., 1996; P o h et al, 2001). However, the numbers o f cases used i n these studies is small and the association with the degree o f dysplasia is reported i n only one o f these studies (Poh et al, 2001). That study suggested that 1 l q alterations may be occurring late i n carcinogenesis, between severe dysplasia and S C C (Poh et al, 2001). There is also some preliminary evidence from this laboratory that suggests that allelic imbalance at 1 l q is associated with an increased risk for progression (Rosin et al, 2000). These studies need to be confirmed using a larger  2  number o f cases and expanding the analysis to other loci on this arm i n order to better identify both the sites containing genes o f importance to progression o f oral premalignant lesions and to determine whether or not such markers can be used as indicators o f risk o f progression for early lesions.  The goal o f this thesis was to examine oral cancers and premalignant lesions for alteration to markers on the 1 l q arm i n order to better define the timing and frequency o f alterations at 1 l q . A second goal was to determine whether there was an association o f alteration at 1 l q with clinical information and prognosis. A final objective was to use microsatellite analysis to begin to define novel regions o f alteration i n oral premalignant lesions that could later be studied for candidate tumor genes located at 1 l q .  The following introduction begins with a summary o f the clinical and histological alterations that occur during oral carcinogenesis and the limitations o f histopathological criteria i n identifying outcome for lesions with no dysplasia or with low-grade dysplasia. A brief summary o f genetic alterations associated with oral tumorigenesis is then given followed by a more specific presentation o f 1 l q alterations and cancer, with an emphasis on oral squamous cell carcinomas and premalignant lesions.  3  1.2.  Clinical, histological and molecular alterations during oral carcinogenesis  1.2.1. Clinical alterations during oral carcinogenesis  A premalignant lesion, as defined by the W o r l d Health Organization ( W H O ) , is 'morphologically altered tissue i n which cancer is more likely to occur than i n its apparently normal counterpart' ( W H O , 1978). This definition was reaffirmed i n 1994 by the International Collaborative Group on Oral White Lesions ( A x e l l et al, 1996).  In the oral cavity, premalignant lesions most frequently present as leukoplakia and erythroplakia ( A x e l l et al, 1996). Leukoplakia (Figure 1, A ) means "white patch". It occurs on membranes such as the mucosa o f the oropharynx, larynx, esophagus, and genital tract. The term leukoplakia should not be applied to all the white patches o f these mucosal surfaces. The W o r l d Health Organization (1978) defines leukoplakia i n 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 b y rubbing. Usually, a definitive diagnosis o f oral leukoplakia is made as a result o f the identification, and i f possible, elimination o f suspected etiological factors ( A x e l l et al, 1996).  Likewise, the W H O defines erythroplakia  (Figure 1, B ) as a fiery red patch that cannot  4  be characterized clinically as any other definable lesion (Pindborg et al, W H O , 1997). In contrast to leukoplakia which are often benign in nature, lesions with erythroplakia are generally either already malignant or are at high-risk for transformation to malignancy (Bouguot and Ephros, 1995; Mashberg, 1977; Waldron and Shafer, 1975).  It is generally believed that a premalignant lesion that most frequently presents clinically as leukoplakia or erythroplakia often precedes oral cancer. However, the majority o f leukoplakias w i l l not progress to cancer.  Figure 1.  Clinical presentation of oral premalignant lesions  A B A : Leukoplakia on right latero-ventral tongue; B : Erythroplakia on right latero-ventral tongue.  A number o f clinical factors have been found to affect the malignant risk o f oral premalignant lesions. These include the appearance, size, site and duration o f the lesion, the consumption o f tobacco and alcohol, and history o f head and neck cancer, or o f Candida infection (van der Waal et al, 1997). The following is a brief discussion o f these attributes.  Leukoplakia can be classified as either homogeneous or non-homogeneous, according to  5  the clinical appearance o f oral premalignant lesions. Homogeneous leukoplakias are those lesions showing a consistent color and texture; these lesions have a lower risk for malignant transformation compared to non-homogeneous leukoplakia ( A x e l l et al, 1996; Pindborg et al, W H O , 1997).  Leukoplakia occurs throughout the oral cavity, with those i n the buccal and mandibular sites being the most common. Leukoplakia located on the floor o f the mouth, ventrolateral surface o f the tongue and soft palate have an increased cancer risk (Schell and Schonberger, 1987; Mashberg and Meyers, 1976). Hence, these regions are called high-risk areas whereas the other oral sites are called low-risk areas (Schell and Schonberger, 1987; Mashberg and Meyers, 1976).  Clinical risk factors help clinicians decide whether an oral lesion should be biopsied for histopathological evaluation and also help i n the overall judgment o f malignant risk o f the lesions. However, these clinical factors have a limited ability to precisely predict cancer risk. Currently, histological criteria represent the gold standard forjudging the risk o f malignant transformation for oral premalignant lesions.  6  1.2.2. Histological alterations during oral carcinogenesis 1.2.2.a.  Normal oral mucosa  The oral cavity is lined by oral mucosa, which consists o f stratified squamous epithelium and a layer o f connective tissue called the lamina propria. The stratified squamous epithelium can be divided largely into basal and prickle cells. The lamina propria lies underneath the epithelium and contains blood vessels and lymphatic vessels, small nerves, fibroblasts, collagen and elastic fibers. It functions to nourish and support the epithelium. A t the boundary o f the epithelium and connective tissue is a layer o f basal cells. Some o f these basal cells are "stem cells" o f the tissue which possess the ability to replicate themselves to replace cells lost, during tissue turnover. W h e n a basal "stem" cell divides, it may give rise to new basal cells or differentiate to form the larger polyhedral-shaped prickle cells. A s the prickle cells mature, they push towards the surface where they shrink i n size, become long and flat, and are eventually desquamated. Oral epithelium is usually non-keratinized except for mucosa lining the attached gingiva, hard palate, dorsal surface o f the tongue, and lips. The majority 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 ) .  7  I.2.2.b. Oral premalisnant lesions and squamous cell carcinoma (SCC)  Oral S C C is believed to evolve from normal tissue that develops premalignant lesions with these lesions increasing i n severity and eventually becoming malignant. T o assess the risk o f malignant transformation o f leukoplakia or erythroplakia, a biopsy is taken o f the clinical lesion and examined for the presence and degree o f dysplasia. The W o r l d Health Organization has established the following criteria for histological diagnosis o f oral dysplasia (1978):  1.  Loss o f polarity o f the basal cells  2.  The presence o f more than one layer having a basaloid appearance  3.  Increased nuclear/cytoplasm ratio  4.  Drop-shaped rete-ridges  5.  Irregular epithelial stratification  6.  Increased numbers and abnormality o f mitotic figures  7.  The presence o f mitotic figures i n the superficial half o f the epithelium  8.  Cellular pleomorphism (variation i n shape and size)  9.  Nuclear hyperchromatism (dark staining nuclei)  10.  Enlarged nucleoli  11.  Loss o f intercellular adherence  12.  Keratinization o f single cells or cell groups i n the prickle cell layer  8  Architecturally, 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 a lesion i n which the dysplastic cells are confined to the lower one third o f the epithelium. Moderate dysplasia is a lesion in which the dysplastic cells are evident i n about half the thickness o f the epithelium. Severe dysplasia is a lesion i n which the dysplastic cells have filled the lower two-thirds o f the epithelial thickness. In carcinoma in situ (CIS), the dysplastic cells occupy the entire thickness o f the epithelium (bottom to top changes) although the basement membrane is still intact (Lumerman et al, 1995). 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 .  A histological progression model has been established for oral cancer (Figure 2). In this model, oral cancers progress through hyperplasia and increasing degree o f dysplasia (mild, moderate and severe) to CIS, and finally break through the basement membrane and become S C C s . Severe dysplasia and CIS usually are grouped together as high-grade dysplasia, because both are late stage, preinvasive lesions and the distinction between them is often difficult and does not appear to be o f practical value in the management o f oral mucosa (Pindborg et al, W H O , 1997).  The presence and the degree o f dysplasia are 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 have higher malignant risk than those without dysplasia  9  (Waldron and Shafer, 1975; Lumerman et al, 1995). A large clinical study by Silverman et al. (1984) found that during a mean follow-up period o f 7.2 years, more than 36% o f leukoplakia lesions with epithelial dysplastic features eventually underwent malignant transformation, whereas those leukoplakia without dysplasia only demonstrated a malignant rate o f 15%. The relationship o f the malignant risk and degree o f dysplasia is further demonstrated by parallel studies from uterine cervix and other systems and organs including skin and respiratory system (Boone et al, 1992; Braithwaite and Rabbitts, 1999; Geboes, 2000; Pinto and Crum, 2000; Shekhar et al, 1998). A s a result, the gold standard forjudging the malignant potential o f premalignant lesions i n these organs and systems, including the oral cavity, is the presence and degree o f dysplasia.  It is important to note, however, that there are limitations i n the use o f histological criteria to predict malignant risk o f oral premalignant lesions. These criteria have a good predictive value for high-grade dysplasia, severe dysplasia and CIS, which have a high chance o f progressing into invasive lesions (Banoczy and Csiba, 1976; Schepman et al, 1998). However, histology is a poorer predictor o f malignant risk for low-grade dysplasia (mild and moderate dysplasia). Low-grade lesions that ultimately progress to tumors appear histologically mimic to those that regress or remain unchanged over extended period o f time. Since such lesions represent the majority o f premalignant lesions, a way to differentiate progressing from non-progressing would have a large impact on prevention o f oral cancer. Advances i n molecular techniques may provide a new direction to solving this problem.  10  X  H  1.2.3. Molecular alterations during oral carcinogenesis  1.2.3.a. The importance of studying genetic changes in cancer progression  It is now widely accept that cancer develops through a series o f genetic events that parallel the histopathological progression o f a lesion through premalignant stages to carcinoma in situ, and, finally, into an invasive lesion. Molecular progression models outlining these genetic changes were established i n colon cancer first (Vogelstein et al., 1988), and then developed for many other solid tumors. According to these models, different genetic changes occur i n different stages o f the disease process and play an integral role i n the progression o f the lesion to cancer. M a n y o f these genetic events take place well before a given tumor produces clinical symptoms and often before a benign lesion or focus o f dysplasia develops into an invasive cancer (Sidransky, 1997). This suggests that i f such changes could be used to distinguish lesions with an elevated risk o f developing into cancer.  A s an example o f such an approach, there is substantial evidence that microsatellite analysis can be used to predict outcome for oral premalignant lesions (Califano et al, 1996; Partridge et al., 1998; 1999). In a recent report from this laboratory, it was shown that loss o f heterozygosity ( L O H ) at 3p and/or 9p was one o f the earliest changes associated with an increased risk o f malignant transformation (Rosin et al., 2000). L o w grade dysplasia with L O H at 3p and/or 9p had approximately 4 times the risk o f  12  developing into cancer compared to those without such loss. Cases that had L O H at 3p and/or 9p with additional loss on other chromosome arms (4q, 8p, 1 l q , or 17p) had a 34fold increase i n relative cancer risk. These results are supported b y a number o f studies from other laboratories (Partridge et al, 1998; 1999; Lippman and Hong, 2001) and strongly support the use o f molecular markers to predict cancer risk o f premalignant lesions. This article w i l l be discussed i n more detail i n section 1.2.3.f.  Genetic alterations have also been found to correlate with clinical parameters o f cancer, such as recurrence, metastasis and poor prognosis. This has been observed for many types o f cancer. For example, L O H o f loci at 3p25.1, 13ql2 or 17pl3.3 have been associated with lymph node metastasis and poor prognosis o f breast cancers (Hirano et al, 2001). Deletion at 3p25.3-p23 has been shown to be associated with metastasis o f endocrine pancreatic carcinoma (Barghorn et al., 2001). A putative tumor suppressor gene (TSG) located at 2 0 p l 1.23-pl2 has been reported to be involved i n the development of prostate cancer metastases (Goodarzi et al., 2001). L O H at 5q21-22 has been reported to be linked to known oral S C C etiologic factor (human papilloma virus) and the prognosis o f patients (Mao et al, 1998). Another study has concluded that microsatellite analysis for 18q21.1 and 9p21-22 is capable o f predicting the clinical outcome o f bladder cancer patients (Uchida et al, 2000). A l l these papers have shown that genetic alterations can be used to predict clinical outcomes and help to decide treatment.  Another reason for studying genetic alterations is to facilitate the development o f new regimen such as treatment o f gene therapy for premalignant lesion and cancers. The  13  efforts i n cancer gene therapy focus on four types o f approaches: chemogene therapy, such as the introduction o f genes that confer susceptibility to chemotherapeutics; immunogene therapy, which involves modulation o f the patient's immune response capacity; tumor suppressor gene-mediated inhibition o f tumor growth promoters like oncogenes and cytokines; and inhibitors o f tumor angiogenesis and invasiveness (Vogelstein and Kinzler, 1998). Each o f these approaches requires a basic understanding o f the genetic background o f a patient's lesion. For example, i n gene therapy, the transfer o f T S G s to cancer cells to suppress tumorigenesis requires the identification o f such genes in a patient's lesion.  A final example o f how studies o f genes altered in tumorigenesis can be used to tailor intervention and treatment strategies is exemplified by carriers o f APC (adenomatous polyposis coli) mutations. Such individuals have a higher risk o f developing colon cancer (Nagase et al, 1992; M i y o s h i et al, 1992; Powell et al, 1992; Smith et al, 1993). In such situations, a prophylactic colorectomy is used to reduce the incidence o f this disease dramatically (Tomlinson et al, 1997).  I.2.3.b.  Oncogenes and tumor suppressor genes (TSGs)  N o w e l l proposed i n 1976 that neoplastic transformation occurred in a single cell that had a critical genetic alteration that gives it a growth advantage over its neighboring cells (Nowell, 1976). This was followed by successive rounds o f mutation and expression  14  with the accumulation o f multiple genetic alterations during the progression o f the disease. In the head and neck region, 7-10 independent genetic events are believed to be involved i n the production o f invasive S C C (Renan, 1993). This theory has been supported by a genetic progression model for head and neck cancer developing by Califano et al. (1996). Based on this model, specific genetic alterations occur during the progression from normal mucosa through increasing degrees o f dysplasia and finally to invasive S C C . These genetic events include the mutation o f a number o f critical genes that control the processes o f cellular proliferation and differentiation i n a tissue. The genes associated with carcinogenesis are classified into two main groups: oncogenes and tumor suppressor genes (TSGs).  Oncogenes, which originally were identified as the transforming genes i n viruses, are altered forms o f normal cellular genes called proto-oncogenes. In human cancers, protooncogenes are frequently located adjacent to chromosomal breakpoints and are targets for mutation. The products o f proto-oncogenes play a key role i n regulating the cascade o f events that maintains the ordered processes through the cell cycle, cell division, and differentiation. M o r e than 50 different proto-oncogenes have been identified, coding for proteins that function as growth factors, growth factor receptors, cytoplasmic second messengers, protein kinases, nuclear phosphoproteins, transcription factors and others. They can be roughly subdivided into two groups. One class o f genes rescues cells from senescence and programmed cell death, acting as immortalizing genes. A second class o f genes reduces growth factor requirements and induces changes in cell shape that results in a continuous proliferative response (Vogelstein and Kinzler, 1998).  15  A number o f mechanisms have been described for the mutation o f these proto-oncogenes to oncogene (called activation) including point mutations, gene amplification and chromosome translocations (Vogelstein and Kinzler, 1998). These alterations can involve the coding region o f the gene, resulting i n alteration o f structure and activity o f coded proteins. Alternatively they can alter the regulating region o f a gene, resulting i n the inappropriate expression o f the gene. This resulting genetic alteration is autosomal dominant, meaning that only one o f the two gene copies needs to be changed for an effect to be observed.  The mechanism o f gene amplification requires further comment at this time, since the region to be studied i n this thesis (1 l q l 3 ) has often been reported as being amplified during carcinogenesis. The term gene amplification refers to an increase i n copy number o f a gene or a specific, subchromosomal region. Gene amplification almost always results in the overexpression o f one or more genes contained on the amplicon. The amplification and consequent overexpression o f a number o f genes can act to confer a selective advantage on a cancer cell.  In contrast to oncogenes, tumor suppressor genes (TSGs) are a group o f genes encoding proteins, which, through a variety o f mechanisms, function to negatively regulate cell growth and differentiation pathways. The functions o f T S G s must be lost i n order for tumorigenesis to occur. Loss o f a T S G ' s function requires the inactivation o f both gene copies (maternal and paternal) through mutation, deletion, or other mechanisms such as methylation (Knudson et al., 1977, 1985, 1993). The most commonly reported process o f  16  T S G loss in sporadic tumors involves the inactivation o f a gene b y a point mutation followed by inactivation o f the second copy by any one o f many mechanisms including deletion. A commonly used procedure to identify such deletion is the microsatellite assay, to be discussed in a later section. This assay is the primary techniques used i n this thesis.  M a n y tumor suppressor genes have been localized and identified, including p53, KB (retinoblastoma), VHL (the gene responsible for von Hippel-Lindau syndrome), FHIT (fragile histidine trial), pi6, DPC4, APC (adenomatous polyposis coli), doc-1 (deleted i n oral cancer), TSC2, BRCA1, NF-1, NF-2 and WT-1 (Mao et al, 1996; Reed et al, 1996; Gleich et al, 1996; Todd et al, 1995; Largey et al, 1994; Pavelic et al, 1997; U z a w a et al, 1994; M a o et al, 1996; and K i m et al, 1996; Latif et al, 1993; Kanno et al, 1994; Sparks et al, 1998). Although the cellular functions o f tumor suppressor proteins, such as p l 0 5 - R B , p53 and p l 6 , are becoming increasingly well understood, others remain largely undefined. It is clear, however, that the tumor suppressor proteins w i l l exhibit a variety o f functions within the cell.  Functional loss o f T S G s is one o f the most common genetic alterations during carcinogenesis including that o f the head and neck region. Thus, the defining o f chromosomal regions that harbor biologically important suppressor genes may have broad practical implications not only on tumor progression, but also on the clinical management o f cancers and premalignant lesions.  17  I.2.3.C. Oncogenes and TSGs in oral premalisnant and malignant lesions  Few oncogenes have been identified as showing mutation i n head and neck or oral squamous cell carcinoma, although changes i n the expression o f many potential oncogenes has been reported. These genes include ras, cyclin-Dl, myc, erbB, bcl-l, int2, CK8 and CK19 (Kiaris et al, 1995; Lese et al, 1995; Saranath et al, 1993; Warnakulasuriya et al, 1992; W o n g et al, 1993; Bartkova et al, 1995; X u et al, 1995; Masuda et al, 1996; Riviere et al, 1990). Our knowledge about the frequency o f mutation o f these genes i n different populations is still somewhat limited. For example, ras and myc mutations appear to be more prevalent i n head and neck tumors occurring in the Far East, possibly due to the use o f chewing tobacco and betel quid b y these populations (Anderson et al, 1994; Clark et al, 1993; Paterson et al, 1996; Saranath et al, 1993). Mutations o f K-ras have been identified i n approximately 35% o f tumors i n the latter group; however, the prevalence o f these mutations i n Western patients is only five per cent (Kiaris et al, 1995; Matsuda et al, 1996; Sakata, 1996). In addition, very few studies have included an analysis o f mutation frequencies i n premalignant lesions. The few studies available tend to use immunohistochemical analysis and look at increased expression o f the gene, not mutation. F o r example, H o u et al (1992) reported a progressive increase in c-erb-2/neu expression as premalignant lesions advanced to malignant lesions. However, it is not known whether this effect was due to a mutation o f the gene itself or to a dysregulation o f the expression o f this gene resulting from a downstream effect o f another mutation.  18  O n the other hand, many studies have focused on the role o f T S G s i n oral carcinogenesis. Some o f the T S G s involved i n head and neck cancers include p53, Rb  (retinoblastoma),  andpl6INK4A (Gallo et al, 1999; Gleich et al, 1996; Jares et al, 1999; Liggett et al, 1996; Papadimitrakopoulou et al, 1997; Partridge et al, 1998 and 1999a; Pavelic and Gluckman, 1997; Reed et al, 1996; Sartor et al, 1999). Other potential candidates are FHIT (Fragile histidine triad), APC (adenomatous polyposis coli), doc-1 (deleted i n oral cancer), VHL (the gene responsible for von Hippel-Lindau syndrome) and TfiR-II (the gene coding for transforming growth factor type II receptor) (Croce et al, 1999; Largey et al, 1994; M a o et al, 1996c; M a o , 1998; Todd et al, 1995; U z a w a et al, 1994; Waber etal,  1996).  Recent advancement i n molecular analysis techniques has rapidly revolutionized the ability to look at these genetic alterations. Most studies on T S G s , particularly those i n oral premalignant lesions, use microsatellite analysis to identify loss o f heterozygosity ( L O H ) i n D N A extracted from epithelial cells belonging to these lesions. This is also the major technique used to conduct research for this thesis.  I.2.3.d.  Microsatellite analysis amd loss of heterozygosity (LOH) studies  Microsatellite analysis is a powerful molecular technique for identifying and studying T S G s . It can detect changes from as small as a few thousand nucleotides to as large as a whole chromosome i n one o f a pair o f chromosomes. The assay is designed to assess  19  alterations to polymorphic chromosomal regions that map close to or within putative or known T S G s .  Two methods are available for the study o f L O H : restriction fragment length polymorphism ( R F L P ) analysis and microsatellite analysis. The advantages o f using microsatellite analysis are twofold. First, microsatellite repeat markers are highly polymorphic and w e l l distributed throughout the human genome. They show levels o f heterozygosity between 30-80%, significantly above the level observed with the R F L P analysis based on base substitutions at endonuclease recognition sites. Second, this [yp ] end-labeled PCR-based approach is much more sensitive than R F L P analysis and 32  requiring only a small amount o f D N A (5 nanograms or less per reaction). This aspect is critical for the study o f premalignant lesions given the small size o f such lesions. Another advantage is that microsatellite makers can be used on D N A extracted from paraffin-embedded archival samples i n addition to fresh or frozen samples. This allows one to use the large number o f specimens i n hospital archives to perform retrospective analysis o f lesional progression. The following is a brief description o f this procedure.  Microsatellites contain runs o f short and tandemly repeated sequences o f di-, tri-, or tetranucleotides, such as - G T G T G T - , - G T A G T A G T A - , o r - G T A C G T A C G T A - (Figure 3). The number o f such tandem repeats is highly polymorphic in the population. Thus, individuals often contain a different number o f copies o f the repeat i n maternal and paternal alleles. These regions are well interspersed throughout the human genome (e.g., estimated every 30-60 kilobase pairs (kb) for C A repeats) and are highly conserved  20  through successive generations (Ah-See et al, 1994; Beckman and Weber, 1992).  The assay involves amplification o f region containing microsatellites with polymerase chain reactions ( P C R ) using radioactively labeled nucleotides. The reaction products are separated on polyacrylamide gel, with separate bands being produced whenever on individual is heterozygous for a region (i.e., when paternal and maternal copies o f the region contain different numbers o f repeats). The intensity o f the two alleles is compared for D N A isolated from normal and tumor or dysplasia cells in a tissue. A n alteration i n intensity o f the bands i n the lesion D N A compared to normal D N A is scored as L O H (see section V.13. for details).  The observation o f a high frequency o f L O H i n a microsatellite region i n a set o f tumors or dysplasias is suggestive o f the presence o f a putative suppressor gene nearby. However, it is important to note that microsatellite analysis cannot distinguish between duplication or low-level amplification o f an allele and deletion o f an allele, particularly i f there are contaminating normal cells within the tumor (Ah-see et al, 1994). Both alterations would result in a change in the relative signal intensities for the two alleles in the lesion D N A . To determine whether a deletion or amplification has occurred, other assays are used. One approach involves the use o f fluorescence in situ hybridization (FISH) on cell or tissue preparations to determine the number copies o f the region in cells. Alternatively, Southern blots could be used with specific gene probes. Both procedures are time-consuming. The latter procedure requires significantly more D N A than the microsatellite analysis. Other procedures involve sequencing the gene to  21  determine whether a mutation exists. This process also requires large quantities o f D N A orRNA.  Because o f the difficulty in differentiating amplification from deletion as a source o f alteration to microsatellite signals, many investigators use the term allelic imbalance ( A l ) to describe the change o f signal intensity observed with microsatellite analysis. I w i l l use both terms indiscriminately throughout this thesis.  22  C^-H  O  d o  o  '3b  CO  u in  8 .a  U Pi  EL •§  .3  CO  3 o £  1  d  8  43  1 *  id  d d  T3 C  «  •—  3  -  O  M  <D  O  Oh  e o so o  M  ea  "3 s-  1 . 1  u  1  "3 ?  CM  O  a  _o +3 CO  3  m  a  U  E  r j m  a  <u  ^  o  o =  O  1  d  I  1 *  iJ § d g  efl d 0  u  §  1 §  la  as  3  i r  3 CD  *  nj  t+H  _ • i  j g a o  T<3u  §  co  C  H  n  t3  <  CD  "  P  CO CU  c3 3  * '3 u c  o  CO CD  J3 •^J O  CU  c3 13  _c  CO  Ifl  13  U  o  d "5 d o  'S  .3  CO  CO  cU  3 3 M  o o co CO o CO £ ea  ci3  60  CD  o u  r-  J  co  o d  I D  H o o '  3 3 x .3 3  tN  i s  1-1 O m co  M  ^  CO  1  0  '55  f t ;  J=  OX)  Oh  0 <u  S3  d .3 Pi O tO 03 ^ O  3  o  i-  bo e  .3 U  « S  •M  d  oj O 5 d  "c3  o JO  CO  "c3  P o  * J  <  1.2.3.e.  Microsatellite analysis of oral cancer and premalisnant 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 in oral S C C s (Ah-See et al, 1994; Nawroz et al, 1994; Ogawara et al, 1998; M a o et al, 1998; M i y a k a w a et al, 1998; Nakanishi et al, 1999; Partridge et al, 1999; Yamamoto et al, 1999). Such chromosomal regions include: 3p, 4q, 5q, 8p, 9p, lOq, 1 l q , 13q, 14q, 17p, 18q and 22q (Califano et al, 1996; el-Naggar et al, 1995; Pershouse et al, 1997; Shah et al, 2000; U z a w a et al, 1994; Resto et al, 2000; Field et al, 1995; Partridge et al, 1994, 1996, 1999; Okami et al, 1998; Lee et al, 1997; Papadimitrakopoulou et al, 1998; M a o 1998; Ishwad et al, 1999; M i y a k a w a et al, 1998).  In contrast, there are fewer studies o f genetic changes i n oral premalignant lesions. This is partly due to the greater difficulty in accessing premalignant lesions as compared to oral S C C i n the larger hospitals. In addition, oral premalignant lesions are generally much smaller than S C C , and yield smaller quantities o f D N A . M o s t o f the studies on oral premalignant lesions are limited in either the number o f samples used and/or the number o f regions assayed. In addition, often the degree o f dysplasia is not mentioned. The following is a discussion o f a few critical articles that have been keynote to the study o f progression o f oral premalignant lesions.  24  I.2.3.f. Genetic progression model for head and neck cancer  In a hallmark study by Califano et al. (1996), L O H patterns were examined at 10 loci i n a wide spectrum o f lesions o f the head and neck region, including hyperplasia, dysplasia, CIS and S C C i n order to determine whether there was an association o f such alteration with the histological progression o f the disease. The study showed that L O H occurred early i n disease development. Loss o f at least one locus occurred i n nearly all samples o f dysplasia and CIS but was present in one third o f hyperplasias. Although the authors suggested that it was the accumulation and not necessarily the order o f genetic events that determined progression, they also suggested that some losses were more likely to occur early in the development o f the disease oral cancer. Others are at later stages. A molecular progression model was proposed with L O H at 9p an early event associated with the transition from normal to benign hyperplasia. L O H at 3p and 17p were associated with development o f dysplasia, while CIS and S C C were characterized by additional loss at 4q, 6p, 8, 1 l q , 13q and 14q.  A major drawback o f the study is that all the dysplasias were grouped together. It is well accepted that with increasing degrees o f dysplasia there is an increasing risk o f malignant transformation. While the majority o f mild dysplasia w i l l not progress into cancer, severe dysplasia, similar to CIS, has a much higher probability o f cancer progression. Identification o f genetic profiles i n early dysplasia and late dysplasia is therefore critical  25  to understanding progression i n the disease. Our research lab has further refined this molecular progression model for oral SCC by investigating all degrees o f oral dysplasias using multiple primers on 8 chromosome arms (Rosin et al, 2000; P o h et al, 2001; manuscript i n preparation) (see Table 1, Figure 4).  Table 1.  A l in oral lesions  Degree of dysplasia  Chromosome  SCC  Hyperplasia region  3pl4  4/31 (13)  a  Mild  Moderate  Severe  10/29 (34)  11/24 (46)  10/23 (43)  28/34 (82)  4q26-31  0/31 (0)  2/28 (7)  2/20 (10)  8/21 (38)  13/33 (39)  8p21-23  0/32 (0)  2/28 (7)  6/23 (26)  5/23 (22)  17/34 (50)  9p21  2/32 (6)  11/31 (35)  14/24 (58)  18/23 (78)  26/34 (76)  llql3-22  1/32 (3)  4/30(13)  2/23 (9)  2/23 (9)  18/34 (53)  13ql2-14  1/33 (3)  1/30 (3)  1/24 (4)  4/21 (19)  15/33 (45)  14q31-32  0/33 (0)  7/28 (25)  10/23 (43)  7/23 (30)  12/33 (36)  17pll-13  0/34 (0)  4/30(13)  8/24 (33)  16/23 (70)  21/33 (64)  Values given as number o f samples showing loss/total number o f informative cases. Values i n parentheses are percentages. a  26  The data support the findings o f the Califano study that an accumulation o f genetic alterations is critical for tumor progression, and that there are preferred patterns o f allelic loss associated with different degrees o f dysplasia. These patterns include the following:  Loss associated with lesions without dysplasia or low-grade dysplasia: A s shown in Table 1, the most common change in hyperplasia occurred at loci on 3p (13% o f hyperplasia). The most frequent loss in mild dysplasia occurred at 3p, 9p, and 14q. Frequencies o f loss at 9p and 14q are significantly higher i n mild dysplasia compared to hyperplastic lesions (P < 0.01). Loss at 3p approached significance (p = 0.07, probably w i l l be significant with larger sample number).  Loss associated with high-grade lesions: L O H at 17p and 4q associated with transitions between moderate and severe dysplasias/CZS. L O H in severe dysplasia/CIS are significantly higher than in moderate dysplasia at 17p (p = 0.02) and at 4q (p = 0.07, approaching significance).  Loss associated with invasion: Frequencies o f Loss at 4 loci increase significantly between severe dysplasia/C/S and S C C : 3p (p = 0.0038), 8p (p = 0.05), 1 l q (p < 0.001), and 13q (p = 0.04). These data suggest the possibility that genes i n these regions play a significant role with late stage events such as invasion and metastasis.  27  1.2.3. g. Prediction of risk of progression for oral premalignant lesions (OPL)  The aforementioned studies suggest that L O H is a common event i n O P L s and can occur early i n carcinogenesis. A few reports support the use o f this assay to improve our ability to predict risk o f malignant transformation for oral premalignant lesions.  In 1996, M a o et al. (Mao et al., 1996) examined 84 oral leukoplakia samples from 37 patients enrolled i n a chemoprevention study. The samples were analyzed for L O H at 9p21 and 3 p l 4 . L O H at either or both loci was observed i n 19 o f these patients and this loss was strongly associated with cancer progression. Seven (37%) o f the 19 positive cases later developed S C C . In contrast, only 1 o f 18 cases (6%) without L O H progressed to cancer.  Partridge and co-workers also looked for an association between L O H pattern and progression. In an early study i n that laboratory, they reported an association between multiple L O H i n oral premalignant lesions and increased progression risk (Partridge et al., 1998). This result was again observed in a more recent study from that laboratory (Partridge et al., 2001) involving 78 oral premalignant lesions, histologically diagnosed with hyperplasia or dysplasia, all from patients with no prior history o f oral cancer. In half o f the patients, the lesions progressed, usually at or adjacent to the original site. Progressing lesions were characterized b y the presence o f multiple regions o f L O H , with loss o f 9p or 3p present in 94% o f lesions.  29  A s mentioned previously in section 1.2.3./., we recently have completed a retrospective study that restricted the focus to lesions without - or with minimal - dysplasia (Rosin et al., 2000). These are the lesions that are the most difficult for clinicians to manage. One hundred and sixteen biopsies o f oral premalignant lesions were examined to see i f a correlation existed between L O H at 7 chromosome arms (3p, 4q, 8p, 9p, 1 l q , 13q, and 17p) and progression risk. None o f the patients had a history o f cancer prior to the studied hyperplasia or mild/moderate dysplasia. Twenty-nine o f the 11.6 O P L s progressed to cancer. The progressing lesions showed not only a significantly higher number o f L O H s , but also characteristic L O H patterns (Figure 4). L O H at 3p &/or 9p was present i n 97% o f progressing lesions, suggesting that loss at these arms may be a progression prerequisite. However, since many non-progressing lesions also showed losses at 3p and/or 9p, such loss alone is probably insufficient for malignant transformation. Indeed, cases with L O H at these arms but no others showed only a 3.8fold increase in relative risk for cancer development. In contrast, individuals with additional losses at 4q, 8p, 1 l q , 13q, or 17p showed a 33-fold increase i n relative cancer risk. In non-progressing cases, additional losses were uncommon. These results suggest that L O H patterns may differentiate 3 progression risk groups: L o w , with retention o f 3p and 9p; intermediate, with losses at 3p and/or 9p; and high, with losses at 3p and/or 9p plus losses at 4q, 8p, 1 l q , 13q, or 17p (Rosin et al., 2000).  30  1.3.1. Genetic changes on l l q in a variety of cancers  Genetic alterations on chromosome 1 l q are very common in a variety o f cancers. The majority o f these genetic changes have been found to be located i n two regions: one is at 1 lq22-25 and the other at l l q l - 3 - 1 4 (Table 2).  Uq22-25 region. H i g h frequencies o f A l have been observed at this region using microsatellite analysis (Table 2). The involvement o f several putative tumor suppressor genes has been suggested for this region based on these data. F o r example, U z a w a indicated i n his paper that two putative T S G s might be located at 1 lq23 and 1 lq25 and play a role i n oral cancer (Uzawa et al, 1996). Herbst reported a high frequency o f L O H at 1 lq23.1-23.2 and 1 lq23.3 in cutaneous malignant melanoma and suggested putative T S G s i n these two loci (Herbst et al., 1999). Koreth proposed two discrete tumor suppressor loci, 1 lq23.1 and 1 lq25 (Koreth et al., 1999) based on their data. Finally, Skomedal indicated that a putative T S G at 1 lq23.1 might be involved i n carcinogenesis o f cervical cancer (Skomedal et al., 1999). It should be noted that L O H i n this region has been shown to correlate with clinical outcome. For example, L O H o f 1 lq22-qter in esophageal squamous cell carcinoma is associated with lymph node metastasis (Tada et al, 2000); L O H at 1 lq24.1-25 i n young woman is associated with poor survival in breast cancer (Gentile et al., 1999); and L O H for 1 lq23-qter is associated with poor survival in ovarian cancer (Gabra et ah, 1995, 1996), etc.  31  I l q l 3 - 1 4 region. There are several critical tumor genes that have been localized in this region and which may play a role in carcinogenesis in a variety o f human tumors. B y applying microsatellite analysis, a high frequency o f L O H was observed for this region i n numerous tissues (Table 2). The most important, numerically, are carcinomas o f breast and head and neck regions.  32  Table 2.  Tumor types  Breast cancer  Cervical carcinoma  Ovarian cancer (Early)  Lung cancer  Cutaneous malignant melanoma  B cell prolymphocytic leukemia  AI on l l q in various cancers as detected by microsatellite analysis  llq23  Reference  Cases  Location  326/776 (42%)  a  Launonen et al, 1999  llq22-23.1  31/49 (63%)  K o r e t h e / a / . , 1997  llq25  23/45 (51%)  K o r e t h e / a / . , 1997  llql3  24/36 (67%)  Zhuang etal,  llq  19/44 (43%)  Hampton et al, 1994  1122-23  22/57 (39%)  Carter ef al, 1994  llq23  34/81(42%)  M u g i c a - V a n et al, 1999  llq23.1-23.2  20/33 (60.6%)  Pulido et al, 2000  Ilq22-q23  4/13 (31%)  K o i k e ef al, 1999  llql3  4/16 (25%)  Weitzel et al, 1994  Hql3  72/81(89%)  Dhar et al, 1999  Ilq23-q24  20/28 (71%)  Wang et al, 1999  l l q 13 (MEN1)  4/11 (36%)  Debelenko et al, 1997  Ilq23.1-q23.2  11/44(25%)  Herbst et al, 1999  llq23.3  7/27(26%)  Pruneri et al, 2000  llql3  12/48(25%)  Pruneri et al, 2000  llq23.1  7/18(39%)  Lens etal, 2000  1995  Values given as number o f samples showing loss/total number o f informative cases. Values i n parentheses are percentages. a  33  1.3.2. Genetic alterations at l l q in head and neck squamous cell carcinoma (HNSCC)  Genetic changes at 1 l q are frequently observed in head and neck S C C , including its subset, oral S C C and other aerodigestive tract cancers. Most o f the genetic changes are found i n two regions: One is at 1 lq22-23 and the other is at 1 l q l 3 (Table 3).  l l q 2 2 - 2 3 region. To date only a few papers have reported data for this region i n H N S C C . Most o f these studies have employed the microsatellite assay. Deletion o f one or several T S G s is suggested by results obtained with this assay (Nunn et al., 1999; U z a w a et al., 1996; Steenbergen et al., 1995). From the standpoint o f association with outcome, A l at 1 lq22-23 has been found to be associated with lymph node metastasis (Tada et al., 2000) and recurrence (Lazar et al, 1998) i n H N S C C .  l l q 13 region. A high frequency o f A l has been reported at multiple loci on 1 l q l 3 i n H N S C C (Table 3). This suggests that several candidate T S G s are located i n this region. Venugopalan suggested the presence o f a T S G at int2-DHS533  in H N S C C  (Venugopalan et al, 1998) and Jin, suggested a T S G at 1 l q l 3 - 2 3 i n H N S C C (Jin et al, 1998).  34  Table 3.  Location  A l at loci on l l q in H N S C C s  Reference  Cases  Dwighte? al, 2000  llql3  13/33 ( 3 9 % )  llql3  6/20 (30%)  Ah-See etal, 1994  \\qU(int2-DllS533)  9/23 (39%)  Venugopalan et al, 1998  llql3-14  12/15 (80%)  D ' A d d a e r a / . , 1999  llq23-25  14/25 (56%)  U z a w a et al, 1996  llq23  13/52 (25%)  Lazaret al, 1998  l l q l 4 (D11S901)  7/26 (26.9)  a  llq22-23 (D11S2000)  13/36 (36.1%)  llq23.2-24 (D11S934)  10/29 (34.5%)  llq23 (D11S490) & l l q l 3 (int2)  14/23 (61%)  H u i etal, 1996  Nawroz etal, 1994  Values given as number o f samples showing loss/total number o f informative cases. Values in parentheses are percentages. a  35  1.3.3. Mechanism of alteration at llq!3 and llq22-23  Mechanism o f alteration at 11 q 13. The alterations at 1 l q l 3 are very complicated. Both deletions and amplifications have been observed. Most studies at 1 l q l 3 region suggest that amplification rather than deletion is responsible for alterations in this region (Table 4). Some o f these studies used fluorescence in situ hybridization (FISH), one o f the most efficient and powerful techniques for studying amplification. The advantage o f F I S H is that it can readily distinguish amplification and deletion by the observation o f copy numbers o f fluorescence labeled probe in cells i n tissue after hybridization with samples. The reported amplification rate is 20% or higher i n H N S C C i n most studies (Williams et al., 1993; Lese et al., 1995; Meredith et al., 1995; Jin et al., 1998; M u l l e r et al., 1997;Izzo et al., 1998; Wang et al., 1999). These studies also suggested that there is an amplicon, int2-cyclinDl CCND1/PRAD1,  amplicon that involved several oncogenes (including  int2/FGF3,  HST1/FGF4,  EMS1), located at l l q l 3 , which is involved in  the amplification.  The amplification and consequent overexpression o f critical genes could confer a selective advantage on a cancer cell with such genetic changes. Although amplification is observed frequently in cancer, the precise mechanisms underlying this change are still not well understood (Vogelstein and Kineler, 1998). According to previous studies, the gene amplification can be observed as either extra-chromosomal amplification, i n the form o f double-minute chromosomes (dmin), or intra-chromosomal amplification, i n the  36  form o f homogeneously staining regions (hsrs). In the case o f 1 l q l 3 amplification in H N S C C , intra-chromosomal amplification at the entopic 1 l q l 3 site seems to be the preferred route, as evidenced by the presence o f hsrs containing 1 l q l 3 sequences localized to chromosome 11 (Shuster et al, 2000; Roelofs et al, 1993; Lese et al, Jin et al, 1998) or frequent duplication o f 1 l q l 3 (Jin et al, 1993; V a n D y k e et al,  1995; 1994;  Shuster et al, 2000). A study by Coquelle and his colleagues i n 1997 suggested a model, breakage-fusion-bridge ( B F B ) , for intra-chromosomal genes amplification involving an initial distal breakage event, perhaps at a fragile site, followed by sister-chromatid fusion at the break and proximal breakage o f the dicentric bridge at mitosis (Coquelle et al, 1997). In 2000, Shuster and his colleagues explained the mechanism o f 1 l q l 3 amplification in oral S C C by applying the B F B cycle model (Figure 5). According to his study, there is an initial break distal to CCND1,  possibly at a fragile site that initiates the  process, followed by breakage at a fragile site between RIN1 and CCND1 which promotes the B F B cycles (Shuster et al, 2000). B F B could cause the amplification o f several critical genes at 1 l q l 3 , including int2, CCND1, key role i n oral cancer carcinogenesis.  37  EMS1, GARP, etc, which play  CD  2 ^  o O  J 3  -2  o o o  Q a 0) °O cS CD o 'SH +3  CD  CD  °a 2  "•3 o o •H  O  u  3  -I c3  cii a  E o a  .5  +-» CD  C4H  O  O  a  o  CD  V)  CO  I  U  t-i  v  S O  a o«  ;=s o a 3  5 &  -si 2 -s ^  -4-*  PQ o  CD  w  T3  co J 3  H  . .  CO  CD  3  S3 CD  m $  o  o o  CO  CD .a  ^  CD  Q  gp a  J 3  CM  o cd o o  S3 O "§  (3 o o (D l-l O u °  I  PQ U  !3 CD CD  J3 Oo Xb>0 o CD S3 P o  to t/>  2I  PQ  2 3O  CD  7L  (3  O  3  o CJ  »  -id  M  T 3  CD  •a  CD CJ h-1 cd  < 8  cs  3,  c  cj u IS  a E  .2 CO  3  3  11 a o  O  S T3 S3 C3  u  ^!  PQ to PQ  pa  en CD ha S  T3 cu  o u U  cu  m  OH  CU  JS H  CU  s DC  o  m  00  According to Shuster's study, these two fragile sites play critical roles i n initiating the B F B cycle to cause the amplification of 1 l q l 3 , as well as in determining the size and genetic content o f amplified units. One possible fragile site involved i n gene amplification o f 1 l q l 3 in oral cancer, FRA11A,  is found to be located between RIN1 and  CCND1 (Shuster et al, 2000). Further evidence to support the occurrence o f a second break distal to CCND1 is the observation that 1 l q l 3 amplification is usually accompanied by distal deletions between Y A C s 55G7 and 749G2 (Jin et al., 1998).  Since the genes i n an amplicon always amplify together, some believe that samples with int2 amplification should reflect the amplification of other closely localized genes. Consequently the amplification of int2 gene has been used as a marker by some to study the amplification o f other oncogenes at 1 l q l 3 , including cyclin Dl (Izzo et al., 1998; WmgetaL,  1999).  However, other individuals have suggested that some o f the alterations at 1 l q l 3 could involve deletions. These studies used microsatellite analysis and found A l i n the 1 l q l 3 region, including int2, MEN1, 1 l q l 3 . 1 P F G M , DUS4946  and D11S913 loci (Bockmuhl  et al, 1996; H u i et al, 1996; D ' A d d a et al, 1999; Bikhazi et al, 2000; Iwasaki, 1996;Chakrabarti et al, 1998; Dwight et al, 2000; Guo et al, 2001; N o r d et al, 1999). However, as discussed before, the limitation of microsatellite analysis is that it cannot distinguish deletion from amplification. Ah-See and his co-workers (1994) have shown that PCR-based assays cannot readily distinguish duplication or low-level amplification  39  of an allele from loss o f heterozygosity, particularly i f there are contaminations o f normal cells within the tumor. Nawroz et al. (1994) have stated that the L O H on 1 l q might i n fact represent amplification at this region (1 l q l 3 ) . In contrast, i n 1994, Ah-See et al used microsatellite analysis to score for A l and Southern blots to evaluate the amplification o f CCND1 at the same time. The results demonstrated amplification i n only one o f 20 tumors but A l i n 9 o f the 20 tumors. Such results would suggest that loss o f T S G s at this region by deletion or mitotic recombination is a more likely scenario than amplification. In 1996, by using microsatellite assay and immunohistochemical analysis, M u r a l i and coworkers found A l at 1 l q l 3 i n 9 o f 23 cancers, but amplification i n only one o f the 9 cancers, again pointing to a deletion theory.  A l l these literature suggest that both amplification and deletion occur at 1 l q l 3 . One possible interpretation is that because T S G s (MEN1 etc) and oncogenes (CCND1,  INT2,  EMS1 etc) are both locate i n this region, there might be different pathways o f carcinogenesis, either involving amplification o f oncogenes at l l q l 3 , or involving deletion o f T S G s at l l q l 3 .  N o matter whether the changes at 1 l q l 3 are deletion or amplification, the alterations have been correlated with clinical outcome, such as poor prognosis (Gebhart et al., 1998; Akervall et al, 1997; Akervall et al, 1995) and metastasis (Alavi et al, 1999). However, although many studies have investigated 1 l q i n cancer, few have examined A l profiles i n premalignant lesions, which is an open area for further research, and w i l l be a focus for this thesis.  40  Table 4.  Location  l l q l 3 (CCND1)  Amplification at l l q l 3 as identified with FISH in H N S C C  Reference  Cases  13/23 (56.5%)  a  Fujii et al, 2001  llql3  8/20 (40%)  Ott et al, 2002  l l q l 3 (Zn*2)  12/21 (57%)  Bayerllein et al, 2000  5/20 25%)  A l a v i et al, 1999  llql3  31/85 (36%)  Williams etal, 1993  llql3  146/282 (52%)  Muller etal, 1997  llql3  22/56 (39%)  Meredith etal, 1995  llq!3  11/50(20%)  Fortin etal, 1997  l l q l 3 (CCND1)  Values given as number o f samples showing loss/total number o f informative cases. Values i n parentheses are percentages. a  Mechanism o f alteration at 1 lq22-23. In contrast, most o f studies for the region o f 1 lq22-23 indicate that alterations at 1 lq22-23 involve deletion. Besides studies using microsatellite analysis (Table 2 and Table 3), there are a few i n w h i c h F I S H technique was used to study this region. Most o f these F I S H studies were investigating leukemias. Only deletion was observed at the region spanning 1 l q 2 2 . 3 - l l q 2 3 i n some o f these studies (Leblanc et al, 1996; Dohner et al, 1997; Zhu et al, 1999; Lens et al, 2000; Zhu  41  et al, 2000; Cuneo et al, 2002). Interestingly, there are five articles that also reported amplification o f M y e l o i d Lymphoid Leukemia gene (MLL gene) or 1 l q 2 3 , where the MLL gene is located, i n leukemias by applying F I S H (Michaux et al, 2000; Cuthbert et al., 2000; Streubel et al, 2000; Reddy et al, 2001; Avet-Loiseau et al, 1999).  In H N S C C , there is only one study applied F I S H to detect the alteration o f 1 lq22-25 region. In this study, 5/16 (31%) o f adenomas and 2/25 (8%) o f primary hyperplasia were observed deleted at 1 l q 2 3 , but no amplification was observed. To date, no one has reported amplifications or oncogenes located at l l q 2 2 - 2 5 involved i n H N S C C .  42  1.3.4. l l q alterations in head and neck premalignant lesions  Most o f previous studies o f 1 l q reported that alteration at 1 l q occurred late i n carcinogenesis, between severe dysplasia and S C C (Table 2). So far, few papers have investigated l l q alterations in head and neck premalignant lesions. B y applying microsatellite analysis, el-Naggar et al. (1995), Califano et al. (1996) and P o h et al. (2001) have demonstrated A l at 1 l q in head & neck premalignant lesions (Table 5). In 2000, Rosin et al. (2000) indicated that L O H at 1 l q was strongly associated with an increase risk for progression. Their P-value was 0.062 and 0.011 for hyperplasia and low-grade dysplasia separately. A s we know, there are two hot spots at l l q , l l q l 3 and 1 lq22-23 regions, which play different roles i n oral carcinogenesis. The limitation o f this study is that it scored A l at these two sites together. This study needs to be confirmed using a larger number cases and scoring the two sites at 1 l q separately.  Amplification at 1 l q l 3 has also been observed i n head & neck premalignant lesions. In 1998, Izzo used F I S H to show that 7/9 (77.7%) o f dysplasia had amplification o f CCND1 (Izzo et al, 1998). In one study, amplification o f int2 gene (also done with F I S H ) was observed in 1/4 (25%) during hyperplasia to dysplasia transition and 2/4 (50%) o f dysplasia (Roh et al, 2000). These two papers suggested that the int2-cyclinDl  amplicon  was amplified i n the early stage i n carcinogenesis o f H N S C C and that cyclinDl  might be  involved i n early regulation. However, the case numbers i n both o f these two papers are  43  very small and further study is required to confirm the results.  These few papers demonstrate one or more o f the following limitations: (1) they had limited number o f cases; (2) they grouped all premalignant lesions together without separating them into different stages (e.g. different degree o f dysplasia) o f premalignant lesions, and (3) they scored genetic alterations at 1 l q l 3 and 1 l q 2 3 together (Table 5). It is still not well understood whether gene alterations at 1 l q precede carcinoma development or results from the unstable nature o f tumors. Based on these limitations, more studies o f genetic alterations at 1 l q i n O P L need to be done to better understand the timing and mechanism o f alterations o f 1 l q i n O P L and their clinical value.  Table 5.  Genetic alterations at l l q of head and neck premalignant lesions  Location  Stages  Frequency  Reference  Microsatellite analysis  llq  Premalignant lesions  9/31 ( 2 9 )  Califano et al, 1996  Microsatellite analysis  Hq  Premalignant lesions  15/75 (20)  Rosin et al, 2000  Microsatellite analysis  llq  Non-invasive lesion  1/15 (7)  el-Naggar et al, 1995  Hyperplasia  2/44 (5)  Low-grade dysplasia  6/52 (12)  High-grade dysplasia  6/37 (16)  Analysis applied  Microsatellite analysis  llql3-22  a  P o h et al, 2001  Values given as number o f samples showing loss/total number o f informative cases. Values i n parentheses are percentages. a  44  1.3.5. Main genes on chromosome l l q  Table 6 contains a list o f genes that are referred to i n articles on 1 l q l 3 as possible oncogenes/TSGs i n that region. A t 1 lq23.1, the genes myeloid lymphoid leukemia gene (MLL gene), ALL-1,  and Homology o f Trithoraz (HEX), are thought to play an important  role in hematogenous malignancies with ATM being a key candidate for mutation in solid tumors, including H N S C C . A brief discussion o f seven o f these genes is given below.  45  Table 6.  CCND1  FGF4/HSTF1  List of genes located at l l q l 3  GENE  Location  Function  Cyclin Dl  54917530-  Regulating progress through the cell  54930900  cycle  Fibroblast growth  55049454-  Heparin-binding growth factor  factor 4 precursor  55051829 55086382-  Stimulate the proliferation of  55087115  fibroblast and endothelial cells  Fas associated via  55347924-  Universal adapter protein in  death domain  55351947  apoptosis that mediates signaling of  FGF3/int2  FADD  all known death domain-containing members of the T N F receptor superfamily EMS1  RIN1  Cortactin  ras inhibitor  55543127-  Involved in the restructuring of the  55581159  cortical actin cytoskeleton  57291330-  R A S inhibitor  57295778 MEN1  VEGFB/VRF  Multiple endocrine  59347865-  Mutated form is responsible for  neoplasia 1  59355064  Multiple endocrine neoplasia  Vascular endothelial  59706252-  Vascular endothelial growth factor  growth factor  59709238  46  GENE  PPP1CA/PPP1 A  Location  Function  61059778-  Serine/threonine-specific protein  61063386  phosphatases, A candidate T S G  GST-pi  CPT1A  Xenobiotic detoxification  Anionic glutathione  61182590-  S-transferase  61185105  Liver carnitine  62546480-  K e y enzyme in the carnitine-  palmitoyltransferase  6260940  dependent transport across the mitochondrial inner membrane  I 75601775-  NUMA1  Cell-cycle related protein  75679417  PAK1  p21-activated kinase  81163598-  Regulate cytoskeletal dynamics by  1  81233157  decreasing M L C K activity and myosin light-chain phosphorylation  GARP  Glycoprotein A  81886288-  A candidate oncogene at 1 l q l 3 . 5 -  repetitions  81898610  llql4  predominate Location: From Human Genome project draft ( U C S C ) Dec. 22, 2001 browser (http://genome.cse.ucsc.edu/index.html).  47  L3.5.a.  CCND1 (cyclin Dl, PRAD1) sene at  llgl3  The fidelity o f cell division requires that an accurate copy o f a complete genome be passed on to each daughter cell. This means that earlier events i n the cell cycle, such as completion o f D N A replication, must be accomplished for later events such as mitosis and cytokinesis to occur. Eukaryocytic cells have developed feedback controls called checkpoints to monitor and regulate various steps i n cell-cycle progression.  The CCND1 gene (cyclinDl  gene) is a proto-oncogene that codes for a protein that is  strongly implicated in cell cycle control. CCND1 binds to and activates a cell cycle kinase that controls phosphorylation o f the retinoblastoma protein (pRb) (Donnellan et al., 1998). Phosphorylation to Rb gene causes it to release proteins such as transcription factor E 2 F . This factor can i n turn bind to regulatory region i n the D N A for specific genes and promote their transcription. This i n turn drives cells through the checkpoint i n late G l phase.  Substantial evidence suggests that the level o f cyclin D l protein is critical to proper cell cycle progression and that deregulated expression o f this gene may disrupt cell cycle control and contribute to genomic instability (Almasan et al., 1995). Indeed, cells overexpressing cyclin D l have a shortened G l phase, reduced dependency on exogenous mitogens, and abnormal proliferative characteristics (Quelle et al, 1993; Jiang et al.,  48  1993). These cells also demonstrate a higher frequency o f gene amplification, especially under conditions o f genotoxic stress (Zhou et al, 1996). Moreover, overexpression o f cyclin D l has been shown to be associated with tumor transformation i n in vitro studies in normal fibroblasts and primary embryo cells transfected with this gene (Jiang et al., 1993; Hinds et al., 1994; Uchimaru et al, 1996), and increased tumorigenesis in in vivo studies using transgenic mice that over-express this protein (Wang et al., 1994).  CCND1 is commonly found amplified and overexpressed i n almost all types o f cancers, such as lung cancer (Reissmann et al., 1999), ovarian cancer (Dhar et al., 1999), myeloma (Hoechtlen-Vollmar et al., 2000), colorectal cancer (Mckay et al., 2000), prostate cancer (Kaltz-Wittmer et al., 2000), mantel-cell lymphoma (Remstein et al., 2000), breast cancer (Rennstam et al, 2001), and H N S C C (Julie et al, 1998).  1.3.5.b.  int2 (FGF3) gene at Hal3  The gene int2 is also located i n the chromosome 1 l q l 3 region. It is the first oncogene recognized as a member o f fibroblast growth factors (FGFs). Activation o f the int2 gene is a result o f transcriptional deregulation, resulting i n constitutive overexpression o f the normal polypeptide products. The gene int2 encodes a growth factor known as F G F 3 , which is structurally related to other F G F family proteins and participates i n several biological processes such as cell differentiation, motility, proliferation and angiogenesis (Dickson and Peters, 1987). A l l F G F s , including F G F 3 , have oncogenic potential. They  49  are synthesized by many tumor cells and can induce blood vessel formation (Burgess and Maciag, 1989; Goldfarb, 1990). Transferring int2 gene into mice results in hyperplasia o f the mammary gland and prostate; however, tumor formation is rare (Muller et al, 1990; Ormtz etal,  1991).  Genetic alteration o f int2 has been reported mainly i n H N S C C and breast cancer (Watatani et al, 2000; Selim et al, 2001, 2002; Lese et al, 1995; R u b i n et al, 1995). A few papers also reported alteration olint2 i n prostate carcinomas (Latil et. al, 1994) and ovarian cancer (Foulkes et al, 1993). In H N S C C , a few papers reported alteration at int2 as deletion (Friedman et al, 1989; Thakkar et al, 1989; Ah-See et al, 1994; H u i et al, 1996). In contrast, alterations at int2 were recognized as amplification b y more papers (Williams et al, 1993; Lese et al, 1995; Rubin et al, 1995; M u l l e r et al, 1997; Jin et al, 1998; Wang et al, 1999; Shuster et al, 2000; R o h et al, 2000).  I.3.5.C.  HST1 sene at  llql3  HST1 (Heparin secretory transforming factor 1, FGF4) is a human transforming gene originally detected by N I H 3T3 fibroblast transfection with D N A from human stomach tumors (Terada et al, 1986). It also belongs to the fibroblast growth factor family. Activation o f HST1 is a result o f transcriptional deregulation, resulting i n constitutive overexpression o f the normal polypeptide products.  50  In H N S C C , HST1 has been reported to be co-amplified with some genes at 1 l q l 3 (Sugimura et al, 1990; Lese et al, 1995; Meredith et al, 1995; Shuster et al, 2000). It is believed to be involved in H N S C C carcinogenesis (Lese et al, 1995) and angiogenesis (Schweigerer, 1989).  I.3.5.d.  EMS1 sene at llgl3  EMS1 [mammary tumor and squamous cell carcinoma-associated (p80/85 src substrate)] gene has been mapped to 1 l q l 3 (Schuuring et al, 1992). It encodes human cortactin, an acting-binding protein possibly involved in the organization o f the cytoskeleton and cell adhesion structures (Shuster et al, 2000). Since amplification o f the 1 l q l 3 region has been associated with an enhanced invasive potential o f these tumors (Adnane et al, 1989; Borg et al, 1991; Kitagawa et al, 1991; Schuuring et al, 1993), overexpression and concomitant accumulation o f the EMS1 protein in the cell-substratum contact sites might contribute to the invasive potential o f these tumor cells.  EMS1 is generally believed to be an oncogene (Schuuring, 1995; J i n et al, 1998). Amplification oiEMSl  gene has been observed i n breast cancer (Schuuring et al, 1992;  Brookes et al, 1993) and H N S C C (Williams et al, 1993; Jin et al, 1998; Shuster et al, 2000). Moreover, i n H N S C C , 1 l q l 3 amplification, including EMS1, is associated with poor prognosis (Meredith et al, 1995; Rodrigo et al, 2000).  51  L3.5.e.  MEN1 eene at  llal3  Multiple endocrine neoplasia typel (MEN!) is an autosomal dominant disorder that is associated with endocrine tumors o f the parathyroid, the endocrine tissues, and the anterior pituitary (Wermer, 1954; Weber et al, \99A). Additional associations include foregut carcinoid, facial angiofibroma, and lipomas.  Larsson et al. (1998) initially made the critical observation that two malignant insulinomas from brothers with. MEN1 showed loss o f the entire copy o f chromosome l l q that was inherited from their parents without MEN1.  This study suggested that the  w i l d type MEN1 gene functions as a T S G . MEN1 was mapped to chromosome 1 l q l 3 i n later studies (Nakamura et al, 1989; Bystrom et al, 1990; Janson et al, 1991). Other evidence supporting a role for MEN1 as a tumor suppressor gene comes from microsatellite studies that show L O H o f the normal allele at the MEN1 locus (Vogelstein and Kinzler, 1998). Depending on the probes used, L O H has been shown to be frequent in MEN1 tumors o f the parathyroid, approaching 100% (Lubensky et al, 1996; Zhuang et al, 1995). 1 l q l 3 L O H has also been found i n 85% o f nongastrinoma pancreatic islet tumors and i n 40% o f gastrinomas (Debelenko et al, 1997).  1.3.5. f.  RIN1 eene at 11a 13  Studies using F I S H on 10 oral S C C cell lines, which have been identified with  52  amplification o f int2, HST1 and CCND1 shown that RIN1 (Ras interaction/interference) gene is co-amplified (7 out o f 10 cell lines) with other critical oncogenes, such as int2, CCND1 and HST1 i n oral cancer (Shuster et al, 2000). Human RIN1 was first characterized as a R A S (an oncoprotein) binding protein based on the properties o f its carboxyl-terminal domain (Han et al, 1997). Through a separate domain, RIN1 has also been shown to interact with and serve as a substrate for the tyrosine kinase A B L (an oncoprotein) (Afar et al, 1997)). The intimate relationship o f RIN1 with both R A S and A B L oncoproteins suggests the possibility o f a direct or indirect role for RIN1 i n naturally occurring tumors (Shuster et al, 2000).  I. 3.5.g.  A TM eene at Hq23.1  ATM (Ataxia Telangiectasia Mutated) gene was found to be located on chromosome band 1 lq23.1 and its mutated form is responsible for ataxia telangiectasia ( A T ) .  The A T M protein plays a key role i n signaling cell cycle arrest i n response to D N A double-strand breaks (Kastan et al, 1992; M e y n et al, 1994). Without this surveillance mechanism, cells are prone to replicate damaged D N A templates i n S-phase and segregate damaged chromosomes through mitosis (Meyn et al, 1995). A T M protein also has been shown to interact with other proteins (Shafman et al, 1997) and to play a role i n controlling cell cycle and apoptotic pathway (Barlow et al, 1997) as w e l l as signal transduction (Keegan et al, 1996). Wild-type A T M protein is required for up-regulation  53  o f p53 tumor suppressor protein in response to ionizing radiation and other D N A damaging agents (Westphal et al, 1997; H a w l e y et al, 1996). Cells lacking the A T M protein show a reduced and delayed activation o f the tumor suppressor gene p53 i n response to D N A damage. The proposed function o f the A T M protein points to a potential role o f ATM as a tumor suppressor gene.  A T is a hereditary autosomal recessive disorder with a variety o f different clinical manifestations, including progressive cerebellar ataxia, oculocutaneous telangiectasis, immunodeficiency, chromosome instability, radiation sensitivity and an increased susceptibility for the development o f various malignancies (Gatti et al, 1991). The incidence o f the disease is estimated to be 1/40,000-100,000 (Telatar et al, 1998; Gatti et al, 1991; Sedgwick et al, 1991). A T patients also exhibit severe hypoplasia o f the thymus and lymphoid tissues, moderate to severe hypogonadism and atrophy o f the cerebellum. A n estimated 1 i n every 100 A T children from the age o f 10 onward w i l l develop a new cancer each year (Morrell et al, 1986; Taylor, 1992). The risk o f developing cancer is 61 to 184 times higher i n A T homozygotes than i n the general population.  More than 250 mutations in the v47Mgene have been identified i n A T families (Vogelstein and Kinzler, 1998). L O H o f ^ T M h a s been reported i n breast cancer (Waha et al, 1998), cervical cancer (Skomedal et al, 1999), adult acute lymphoblastic leukemia (Haidar et al, 2000), ovarian cancer (Launonen et al, 1998), lung cancer (Murakami et al, 1999), and also H N S C C (Lazar et al, 1998).  54  Although these data suggest the possibility that the ATM gene could be acting as a T S G for these cancers, definitive proof is not available. For H N S C C , there have been no reports o f mutation in this gene.  55  II.  STATEMENT OF PROBLEMS  II.l. Where are the additional tumor genes at l l q l 3 located?  Although a number o f tumor genes including CCND1 and int2 have been established i n the 1 l q l 3 region, additional tumor genes, particularly tumor suppressor genes, are suspected to be located at the region. It has been proposed that there are many additional genes in the 1 l q l 3 region, and that this region may play a key role i n controlling chromosomal instability and progression o f tumors (Bekri et al, 1997, Izzo et al, 1998, 1999; Zhou et al, 1996; Gebhart et al, 1998). Identification and location o f these potential genes w i l l contribute to the understanding o f tumorigenesis. A major problem in using tumors to locate tumor genes is that cancers are most often accompanied by an intrinsic genetic instability that results i n a cascade o f gene alterations i n the tumor, many of which are just random changes that are not critical to tumor development.  This thesis used a spectrum o f tissue samples including S C C , dysplasia and hyperplasia to search for novel regions o f alterations i n the 1 l q l 3 region. The rationale for using preinvasive lesions is that alterations occurring i n these early lesions are more likely to be a driving force for carcinogenesis rather than due to the random genetic instability seen i n later lesions including tumors. Consequently, studies o f preinvasive lesions are more likely to reveal the small region o f loss that contains critical tumor genes.  56  II.2.  At what stages of oral cancer development does AI occur for int2 (llql3), D11S1778 (llq22.3) and any novel loci identified in this study?  Information obtained from allelic imbalance studies has dual merit. The finding o f frequently lost regions during cancer development can lead to discovery o f new T S G s . In addition, allelic imbalance findings can provide critical information on the role o f the presumptive tumor genes even before the cloning o f the tumor gene. For example, allelic imbalance studies have shown that there are three discrete regions o f loss at 3p, suggesting that each o f these three regions contains at least one tumor suppressor gene (Partridge et al., 1996). While we have yet to identify the genes involved at 3p, studies on the timing o f such loss during histological progression have already lead to the conclusion that loss on at least one o f the regions, 3 p l 4 , is an early critical event for cancer development. A s discussed i n section 1.2.3.f, L O H at 3 p l 4 can serve as an important molecular marker for predicting risk o f malignant transformation for oral premalignant lesions (Rosin et al., 2000).  While the mapping studies in this thesis may yield information on location o f putative tumor suppressor genes at 1 l q l 3 - 1 4 , it may be some time before such genes can be identified. Acquisition o f information on at what stage o f oral cancer development the new candidate gene is altered w i l l not only provide information on the possible roles o f  57  the gene, but may also facilitate the process o f identification o f the gene.  In addition to doing temporal studies for any novel loci showing frequent A l i n the 1 l q l 3 - 1 4 region, this thesis w i l l further explore the timing o f loss for the 2 regions previously studied i n this lab (1.2.3.f). These regions are defined by markers int2 which amplifies sequence with the int2 gene at 1 l q l 3 and by marker D11S1778, which amplifies sequence that is 0.6 M b from the ATM gene at 1 lq22.3. Our early studies fused the data from these two loci (1.2.3.f). These studies suggested that alteration to 1 l q l 3 22.3 occurred with formation o f S C C . In this thesis, the 2 regions w i l l be examined independently to confirm the association with histological progression.  58  III.  OBJECTIVES  To use microsatellite analysis to examine D N A extracted from severe dysplasia, carcinoma in situ (CIS) and S C C for novel alterations i n the 1 l q l 3 region. This region should be distinct from the int2 (1 l q l 3 ) and D11S1778 (1 lq22-23) regions o f alteration previously studied in this laboratory.  If a novel region o f loss is identified, to determine at what stage o f oral cancer development the alteration occurs by performing microsatellite analysis on a spectrum o f stages o f oral premalignant lesions (hyperplasia, m i l d dysplasia, moderate dysplasia, severe dysplasia, CIS) as w e l l as invasive S C C .  To determine at what stage o f oral cancer development the int2 (1 l q l 3 ) and D11S1778 (1 lq22.3) alteration occur by performing microsatellite analysis o f the same oral premalignant and malignant lesions and compare the data obtained with that seen for any novel regions o f loss identified i n this study.  To determine the significance o f L O H at int2 (1 l q l 3 ) and D11S1778 (1 lq22.3) loci to the progression o f oral premalignant lesions by comparing frequencies o f loss for different locus i n low-grade dysplasia with known outcome, i.e., low-grade lesions that did not progress into cancer with morphologically similar lesions that did develop into SCC.  59  IV.  HYPOTHESES  Microsatellite analysis o f microdissected oral premalignant and malignant samples w i l l reveal novel loci in the 1 l q l 3 region, which harbors one or several putative tumor genes.  A l l e l i c loss at int2 (1 l q l 3 ) and D11S1778 (1 lq22-23) occur mainly at the tumor stage.  Progressing low-grade lesions (those without dysplasia or with low-grade dysplasia) have increased frequencies o f allelic imbalance at int2 (1 l q l 3 ) and D11S1778  (1 lq22-23) in  this thesis compared to morphologically similar non-progressing lesions, suggesting a role for this alteration i n cancer progression.  60  V.  MATERIALS AND METHODS  V.l.  Sample collection  This thesis used paraffin-embedded archival samples from the provincial Oral Biopsy Service o f British Columbia, located at the Oral Pathology D i v i s i o n o f Vancouver General Hospital and Health Sciences Center. This service receives 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. The use o f these samples was approved by the University Ethics Committee.  V.2. Sample sets  Two different sample sets were used.  The first set had 4 groups in it, as follows: primary S C C , severe dysplasia/CZS, mild/moderate dysplasia and hyperplasia (Table 7).  61  Table 7.  Histological groups in sample set 1  Number of cases  Lesion type  Hyperplasia (without dysplasia)  33  Mild & moderate dysplasia (low-grade dysplasia)  54  Severe dysplasia & CIS (high-grade dysplasia)  56  Primary S C C  91  234  Total  The second sample set included 2 groups o f cases. Both groups included cases with hyperplasia (without dysplasia), mild dysplasia and moderate dysplasia (Table 8). In 1 group (called "progressing lesions") the lesions later progressed to CIS or S C C . N o progression occurred i n the second group (called "non-progressing lesions").  62  Table 8.  Histological groups in sample set 2: the progression test series  Lesions not progressed  Lesions later progressed  to CIS or S C C  to CIS or S C C  33  6  Mild dysplasia  31  9  Moderate dysplasia  23  14  Lesion Type  Epithelial hyperplasia (without dysplasia)  The criteria for choosing samples for the non-progressing group included confirmation o f histological diagnosis by two pathologists using criteria established by the W o r l d Health Organization ( W H O collaborating Reference centre 1978) and the provision that the sample was large enough to yield sufficient D N A from both the epithelium and from the connective tissue for multiple microsatellite analyses. The third criterion was confirmation that these patients had no prior history o f head and neck cancer and, for hyperplasia and dysplasia, that they did not subsequently develop such cancer. This confirmation was obtained from hospital records and by using a computer linkage with the British Columbia Cancer Registry. A l l but three o f these cases had at least 3 years o f follow up.  63  The inclusion criteria for progressing group included confirmation o f histological diagnosis by 2 pathologists, sufficient sample size, and no prior history o f H N S C C . A final provision was that both the primary hyperplastic or dysplastic lesions and their matching CIS or S C C had to be from the same anatomical site as recorded on pathology reports and patients charts and the interval between the primary lesions and later CIS or S C C had to be longer than 6 months. The later criterion was used to exclude cases where the appearance o f the CIS or S C C might be due to inadequate biopsy or sampling o f the index (first) biopsy error. The time interval chosen for exclusion was arbitrary.  V.3. Diagnostic criteria for the samples  The W H O diagnostic were used, which have been reviewed i n section I.2.2.b. The diagnosis was confirmed independently by D r . R. Priddy and D r . L . Zhang, oral pathologists at the B C provincial biopsy service. O n l y those cases i n which the two pathologists agreed on the diagnosis were used for the study.  V.4. Clinical information  The following clinical data were obtained for the cases studied by examining pathology reports and hospital charts: smoking habit, age and gender o f the patients and anatomical  64  location o f the lesions. Some o f this information was not recorded for some cases (see Result section).  V.5. Slide preparation  Tissue blocks for cases were chosen for study removed from the archive and one 5micron-thick section was cut from each block, stained with H & E (hematoxylin and eosin) and coverslipped for reference. Further sections for microdissection were then cut at a 10 to 12 microns thickness with approximately 15 sections per sample. These sections were also stained with H & E but left uncoverslipped. The H & E procedure is described below:  1.  Slides were baked at 37°C overnight in an oven, then at 60 to 65°C for 1 hour, and left at room temperature to cool.  2  Samples were deparaffmized by two changes o f xylene for 15 minutes.  3  Dehydration i n gradient alcohols (100%, 95, 70% ethanol).  4  Hydration by rinsing in tap water.  5  Slides were placed i n G i l l ' s Hematoxylin for 5 minutes then rinsed i n tap water.  6.  B l u e d " with 1.5% (w/v) sodium bicarbonate, then rinsed i n water.  65  7.  Slides were lightly counterstained with eosin, dehydrated, and cleared for coverslipping.  8.  Thick sections to be dissected were stained by the above procedure without the dehydration step then air-dried prior to microdissection.  V.6. Microdissection  Microdissection o f the specimens was either performed or supervised by D r . L . Zhang. Areas o f hyperplasia, dysplasia and S C C were identified using the mounted H & E stained sections. Epithelial cells i n chosen areas were meticulously microdissected from adjacent non-squamous epithelium tissue or cells under an inverted microscope using a 2 3 G needle. Genomic D N A from normal tissue was obtained b y dissecting out the underlying stroma i n these sections. This D N A was used as control D N A for each case (Zhang  al. 1997).  V.7. Sample digestion and DNA extraction  The microdissected tissue was collected i n a 1.5 m l eppendorf tube and digested i n 300 ul of 50 m M T r i s - H C L (pH 8.0) containing 1% sodium dodecyl sulfate (SDS) and proteinase K (0.5 mg/ml) at 48°C for 72 or more hours. During incubation, samples were spiked with 10 or 20 p i o f fresh proteinase K (20 mg/ml) twice daily. The D N A was then  66  extracted 2 times with P C - 9 , a phenol-chloroform mixture, precipitated with 100% 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 quantification (Rosin et al 1997; Zhang et al 1997).  V.8.  DNA quantification  Fluorescence analysis with a Picogreen kit (Molecular Probes, Eugene, Oregon) was used to quantify D N A . This method used 2 standard curves. The low concentration standard curve was used for samples with 1 to 20 ng/u.1, while the high concentration standard curve was used for concentrations between 10 and 400 ng/ul. Absorbance was read with a S L M 4800C spectrofluorometer ( S L M Instruments Inc. Urbana, IL). The sample D N A concentration was then determined from one o f the standard curves depending on its concentration, hence absorbance. A series o f dilutions were done subsequently to adjust the concentration o f D N A to 5 ng/ul with L O T E buffer (Rosin et al 1997; Zhang et al 1997).  V.9.  Primer extension preamplification (PEP)  I f the concentration o f D N A was low (less than 100 ng total), a procedure called P E P was performed. P E P involves amplification o f multiple sites o f the genome using random  67  primers and low stringency conditions, thus increasing the amount o f total D N A for microsatellite analysis. The P E P reaction was carried out i n a 60 p.1 reaction volume containing 20 ng o f the D N A sample, 900 m M o f T r i s - H C L , ( p H 8.3), 2 m M o f d N T P where N is A , C , G and T, 400 pJVI o f random 15-mers (Operon Technologies, California), and 1 u l o f Taq D N A polymerase (Introgen, Gibco). 2 drops o f mineral o i l were added prior to the reaction. The amplification was done on an automated thermal cycler (Omigene H B T R 3 C M , Hybaid Ltd.) and involved 1 cycle o f pre-heat at 95°C for 2 minutes, followed by 50 cycles of: 1) denaturation at 92°C for 60 s, 2) annealing at 37°C for 2 min, and 3) polymerization at 55°C for 4 m i n (Rosin et al 1997; Zhang et al 1997).  V.10. Coding samples  A l l samples were coded i n such a way that the analysis o f allelic imbalance would be performed without the knowledge o f the sample diagnosis.  V . l l . End-Labeling  One more step prior to microsatellite analysis was end-labeling o f one member o f the chosen microsatellite primer pair. Those reactions were carried out i n a total volume o f 50 u l which contained 38 ul o f P C R grade water, 5 ul o f 10 x buffer for T4 polynucleotide kinase (New England BioLabs, Beverly, M A ) , 1.2 u l o f 100 x B S A , 100  68  ng o f one o f the primer pair, 3 u l o f T4 polynucleotide kinase (New England BioLabs, Beverly, M A ) , and 2 u l o f [y- P] A T P (20 u C i , Amersham). The P C R reaction was 1 32  cycle at 37°C for 60 m i n run on the thermal cycler (Rosin et al 1997; Zhang et al 1997)  69  V.12. Microsatellite analysis: PCR amplification  The microsatellite markers came from Research Genetics (Huntsvile, A L ) and mapped to the following regions: 1 l q l 3 (D11S4207, D11S916 and int2) and 1 lq23  (D11S1778).  Markers int2 was used to amplify D N A sequence within the int2 gene (need to confirm). Marker D11S1778 is 0.6 M b from the ATM gene and has been used as the marker for ^47Mgene i n many publications (Laake K et al., 1997; 1999).  P C R amplification was carried out in a 5 ul 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 (Invtogen, Gibco), 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 (P-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 m i n ; 40 cycles o f 1) denaturation at 95 C° for 30s, 2) annealing at 50-60 C° (depending on the primer used) for 60s, and 3) polymerization at 70 C° for 60s; 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 7% urea-formamide-polyacrylamide gels, and visualized b y autoradiography. The films were coded and scored for allelic imbalance (Zhang et al., 1997).  70  V.13. Scoring of allelic imbalance  For informative cases (meaning both alleles are o f different length and thus could be distinguished from one another by electrophoresis), allelic loss is scored i f the signal intensity o f the band is at least 50% less than its normal control counterpart (connective tissue D N A ) (Rosin et al 1997; Zhang et al 1997). A l l samples showing allelic imbalance were subjected to repeat analysis after a second independent amplification and re-scored whenever the quantity o f D N A is sufficient.  71  VI.  RESULTS  VI.l. Choice of microsatellite markers for this study  There is strong evidence supporting the involvement o f alterations i n the 11 q l 3 region i n ' cancer development and the potential involvement o f numerous genes has been suggested (see Table 6 for list). However, with the exception o f CCND1,  the evidence for a role o f  these genes i n oral carcinogenesis is largely speculative. W h e n the research in this thesis was first started, a decision was made to focus on the D N A sequence between cyclin Dl (CCND1) and int2 i n the 1 l q l 3 region, since many o f these putative oncogene/TSGs mapped to that region, and to use the microsatellite assay to screen for novel regions o f alterations (distinct from CCND1).  The rationale was that such novel alterations could be  used to localize genes playing a role in oral carcinogenesis. The Human Genome Database (http://www.gdb.org/) was used to identify potential microsatellite markers between CCND1 and int2. O f the markers chosen for the study, D11S4207 (AFMal03zf9)  proved to be highly informative with frequent allelic loss in tumor D N A  from microdissected oral S C C samples. It was thus chosen as a starting point for the study and the majority o f the work described below used this marker. It should be noted that during the course o f this research, there has been a vast improvement i n sequence analysis for the genome. A s shown i n Figure 6, this has resulted i n the current localization o f D i 1S4207 to a region that is 20 M b telomeric to the CCND1-Int2 region  72  [sequence localization by University o f California Santa Cruz database (http://genome.cse.ucsc.edu/index.html) using the Human Genome project draft ( U C S C ) dated Dec. 22, 2001]. A s shown below, this marker identifies a novel region o f frequent alteration in oral S C C and premalignant lesions that occurs independent o f alterations at CCND1-Int2.  The evidence in support o f this statement is given below.  73  2  CO  rt  CO  CO  cd  "EH "EH CO  a o  Retenti  u $ CO CO  ©  3  Q  o  # ©  o  O  ^  cd  "I  «  <; cp  o o c  CU  CO  >> CD  *0  Cd r^5  o  ^  uo  =0  cu  t\  •o a  i  ^  o m oo cu Co os 0\ „-<  C3  SO SO  •x  s  ©  r - os co co -  •X  t  §  ©  ©  @  @  ©  ®  © © © © ®  S S S I I  ©  ©  — coVO O o co  CO  (X  <—i  co  •4-1  co co  o  SO OS  co  @©©0000§0i©®00©0  co co • IX  SO  S  00000  00  668086 8 6 © 000060 0 00000  00  oo oo  cu  co  Cd cu u  .O  Os i-i >X to  CU  !.  OS  M  wo r oo  Os (N  <x so SO —< co u-i no  3  CU  cu et  OO  —  CO  OO  00  O i-  u  o  L/~t U O  M  I t  I  §  1 9 1 1 8  t  i@©®o©©  @©@©©  © ®  Q Q O t-- — oo oo o oo — (N  x i  i l S I I I  •  22  oo so ° °  0§©i©0O©§  co — so r o  M  ^  O S«J C \  w  O O co O OS OS •* •*  »o uo  s ©  cu u  a  . p H H H H H H H Ht—'Tfso — u - i c N f M s o  OJD  ^ l O w w w t s i n ^ ' t  <  Q Q os co oo  Q Q P Q Q -3- so <N o so <N t— oo — — co co so  Q Q oo r io <N —  X  oo <N —  VI.2. A l at D11S4207 in oral SCCs and premalignant lesions  In order to determine the frequency o f A l at D11S4207, tumor cells were isolated by microdissection from archived paraffin blocks o f 91 cases o f oral S C C . The D N A was extracted and the locus was amplified using the D11S4207 primers. The underlying stroma served as a source o f normal control D N A . The amplified products were separated on polyacrylamide gels. 74 (81%) o f these cases were informative (showed 2 bands), 35 (47%) o f which showed A L A picture o f the alteration i n band intensities ( A l ) at this locus is shown for several cases in Figure 7.  75  Microsatellite analysis of S C C cases at D11S4207 and int2  Figure 7.  342T  a  • T  c  436T  a  b  b  • • T  c  T  c  C  T  215T  61T3 a  b  a  b w m  f  •*  c  T  c  1 T  c  T  114T  wr  it  i m  c  T  C  T  c  b  a  b  1  fll  43T  a  |  mm  |  |  •  T  c  T  3  2  C  T  D11S4207 and wrt2 were amplified from areas o f epithelium o f S C C (T) and normal connective tissue (C). a: Images showing A I at D11S4207; b. Images showing retention at int2.  76  To determine whether this alteration was also frequent i n premalignant lesions, the same primers were used to amplify D N A isolated from 33 epithelial hyperplasias, 54 low-grade dysplasias and 56 high-grade dysplasias. It should be noted that these samples all came from cases i n which there was no prior history o f oral cancer (i.e. primary lesions). The data is shown i n Table 9.  Table 9.  Allelic imbalance at D11S4207 in a spectrum of primary lesions with different histological diagnoses  Diagnoses  Hyperplasia  Al"  Number of cases  Informativity  33  19/33 (58)  7/19 (37)  54  29/54 (54)  10/29 (34.5)  56  38/56 (68)  18/38 (47)  91  74/91 (81)  35/74 (47)  2  Low-grade dysplasia  High-grade dysplasia  SCC  Informativity: Number o f cases informative for this locus (showing 2 bands)/total case number. Numbers i n parentheses are percentages. a  b  Number o f cases showing Al/total number o f informative cases. Numbers i n parentheses are percentages.  77  H i g h frequency o f A l at D11S4207 was present i n hyperplasia cases (37%) and lowgrade dysplasia cases (34.5%) and slightly elevated i n high-grade dysplasia (47%) and S C C cases (47%). Figure 8 provides examples o f band appearance o f gels showing D N A from hyperplasias with A l . Figure 9 compares the frequencies observed at this locus with those seen with microsatellite markers at 3 p l 4 (D3S1228, D3S1234, D3S1285 and D3S1300) and 9p21 (INFA, D9S1751, D9S171 mdD9S1748).  The latter regions are  those that have been previously reported to be among the earliest alteration i n oral carcinogenesis (see section 1.2.3.f. for evidence). A s a comparison, A l at 8p (D8S261, D8S262, D8S264) is infrequent i n hyperplasias, and both low-grade and high-grade dysplasia but increases significantly in S C C s (see section 1.2.3.f. for evidence).  78  Figure 8.  Microsatellite analysis of hyperplasia cases at D11S4207  102  28  C  H  144  C H 141  #p  up  C  H  C  H  150  C  H  103  C H  171  C  H  D11S4207 were amplified from areas o f epithelium o f hyperplasia (H) and normal connective tissue (C).  79  Figure 9.  Comparison of A l frequencies observed at D11S4207 with those at 3pl4,9p21 and 8p  80  •3p Total  60  -9p Total  <  "5  /  40  y  /  • 8p Total -D11S4207I  20  H y p e r p i as i a  Low-grade dysplasia  HI gh-gr ade dy s pi as i a  Microsatellite markers at 3 p l 4 (D3S1228, D3S1234, D3S1285 andD3S1300), 9p21 (INFA, D9S1751, D9S171 smdD9S1748) and 8p (D8S261, D8S262 mdD8S264).  80  VI.3. The timing of induction of A l at D11S4207, int2 and D11S1778 during histological progression.  In this study, the 143 cases o f primary oral premalignant lesions studied in the previous section for D11S4207 were further assayed using the markers int2 and D11S1778.  The  purposes were two folded, the first was to determine whether or not the alteration at D11S4207 was distinct from that occurring at these 2 loci that we have previously studied in this laboratory (see section I.2.3.f.) with a small number o f cases, and the second was to determine the temporal changes o f these two loci i n different stages o f oral premalignant and malignant lesions. The data suggest that the stage i n which alterations occur i n these 3 regions is different for D11S4207 compared with the other 2 regions (Table 10)  In contrast to the results obtained with D11S4207, A l was rarely observed i n hyperplasia and low-grade dysplasia for the markers int2 and D11S1778.  A l was present at  D11S1778 i n only 1 o f 27 (4%) hyperplastic lesions and 3 o f 51 (6%) low-grade dysplasias; for int2 theses frequencies were 2 o f 23 lesions (9%) and 2 o f 36 (6%) respectively. F o r both int2 and D11S1778, there was a significant increase i n A l i n highgrade dysplasias, with the alteration occurring i n 9 o f 42 (19%) lesions for D11S1778 (P = 0.0325) and in 12 o f 41 (29%) lesions for int2 (P = 0.0081). Further increases in A l were noted with invasion for both D11S1778 and int2, with A l being present i n 24 o f 73  81  (33%) S C C s and 29 o f 62 (47%) S C C s , respectively. However, these increases i n A I between high-grade dysplasias and S C C were not significant (Table 10).  A l l e l i c imbalance data for these three loci were also plotted i n Figure 10. This figure clearly shows the low A I frequencies i n early lesions and the sharp rise o f these frequencies from low-grade dysplasia to high-grade dysplasia, and then to invasive S C C s for both D11S1778 and int2. In contrast, D11S4207 displays high A I i n the earliest lesions and only a slight elevation o f these frequencies between low-grade and high-grade dysplasia.  When A I frequencies are compared i n S C C s for the 3 primers, there is no significant difference suggesting that at this stage a similar frequency o f alteration is present in all 3 loci. In contrast, A I frequencies at D11S4207 were significantly higher than those at either D11 SI 778 or int2 for all earlier stages: hyperplasia, low-grade dysplasia, and highgrade dysplasia (Table 10).  82  Figure 10.  Comparison of AI frequencies observed at D11S4207 with those seen with microsatellite markers at D11S1778 and \nt2  83  H  M  r-  .s 10/29 (34.5)  c  7/19 (37)  CU  •x  DUS4207  S * - H  2/36 (6)  0.0081  0.3265  18/38 (47)  b  12/41 (29)  5 0.0325  &  9/42 (19)  "« s©  2/23 (9)  "«  35/74 (47)  0.1003  29/62 (47)  grade dysplasia  0.2077  co  24/73 (33)  (n = 91)  CU  3/51 (6)  cu i-  (n = 56)  "a B  (low- vs. highgrade dysplasia)  -o a  D11S1778  .5 see  «  (n = 54)  cs  (n = 33)  CO  Locus  c  High-grade dysplasia  'vi  P value  VI  a O  Low-grade dysplasia  M  Hyperplasia  u u  a  "-H  co cu co  fi  _OJD  co CD co ca o  CM  O  CX)  CM  O s-  CD  J  CU  u  o  ,fi  o  CO  CO  0>  I  CM  CO  O ii  1  CD  fi  CO  a fi  >  fi c3  -a  CD M CD  _*o  'co fi O o co  o o  V  OH CO  u  CD  CD  '5b  >  PQ  -fi O  VI.4. Further evidence in support of the AI at D11S4207 being an independent event  Another way o f demonstrating that the A I at DUS4207  is occurring independent o f  alteration at the other 2 regions is to determine how frequently these alterations occur together or independent o f each other i n the same samples. Table 11 compares patterns o f A I for D11S4207 and intl. Table 12 provides similar comparisons for D11S4207 and Dl 1 SI 778.  A s shown in Table 11, i n the majority o f samples i n which A I occurred at either D11S4207 or intl, the alteration i n these 2 regions was not 'synchronous', that is, the alteration occurred i n only 1 o f the 2 primers and not both at the same time. This was true for 75% o f hyperplasias, 100% o f low-grade dysplasias73% o f high-grade dysplasias, and 56% o f S C C s .  85  Table 11.  Patterns of alteration at D11S4207 and int2: frequencies at which these alterations occur together or independent of each other  Hyperplasia  # of cases  A l at  A l at int2  A l at both  Total # cases  informative  D11S4207  only  D11S4207  with different  at both loci  only  & int2  pattern (%)  13  2  1  1  3/4 (75%)  22  5  1  0  6/6 (100%)  28  7  4  4  51  9  9  14  a  Low-grade dysplasia  High-grade dysplasia  SCC  11/15 (73%)  18/32 (56%)  Value given as number o f samples showing A l for one primer but retention for the other (% o f cases i n parentheses).  a  Table 12 gives similar data for D11S4207 and D11S1778.  Again, the alteration to these 2  regions was most often not synchronous. The alteration occurred i n only 1 o f the 2 regions i n 100% o f hyperplasias, 80% o f low-grade dysplasias, 60% o f high-grade dysplasias and 70% o f S C C s .  86  Table 12.  Patterns of alteration at D11S4207 and D11S1778: frequencies at  which these alterations occur together or independent of each other  Hyperplasia  # of cases  Alat  Alat  A l at both  Total # cases  informative  D11S4207  D11S1778  D11S4207&  with different  at both loci  only  only  D11S1778  pattern (%)  15  5  0  0  5/5 (100%)  29  8  0  2  8/10 (80%)  8  1  6  9/15 (60%)  18  8  11  26/37 (70%)  a  Low-grade dysplasia  High-grade 32 dysplasia  SCC  63  Value given as number o f samples showing A l for one primer but retention for the other (%> o f cases in parentheses).  a  VI.5. Fine-mapping at D11S4207  The next logical step i n this research was to fine-map the region around D11S4207 (using further primers) i n order to determine the smallest region o f A L That region would then be examined for putative oncogenes/TSGs. A n attempt was made to do this early i n the development o f the thesis. Two primers were selected for study that originally mapped to  87  either side o f D11S4207: D11S916 and D11S4119.  The initial data suggested that we had  successfully established telomeric and centromeric boundaries for this region o f alteration. Unfortunately, a month ago, the location o f these markers changed. The December 22,2001 Human Genome project draft shown in the University o f California Santa Cruz database (http://genome.cse.ucsc.edu/index.html) now places the markers as shown i n Figure 6.  Future studies w i l l require the careful mapping o f primers onto tiled B A C arrays, a more stringent way o f localizing markers. However, at present our data suggests that there may be a telomeric boundary at D11S4119 which is located approximately 0.5 megabase pairs from D11S4207.  The data supporting this boundary is still weak with only 4 cases  of S C C (161T, 414T, 448T and 529T) showing A l at D11S4207 and retention at D11S4119.  VI.6. Allelic imbalance at chromosome l l q and malignant progression risk  A n important question that must be answered with each marker that is proposed to be associated with progression is whether or not the presence o f this alteration increases the risk o f a premalignant lesion to transform into an invasive S C C .  In order to answer this question, I compared A l frequencies in early premalignant lesions  88  with known outcome. Prior data on these samples has been presented i n a recent published paper (Rosin et al. 2000) and is described below. A decision was made to look for associations o f intl and D11S1778 to progression risk. D11S4207 was not studied because it requires further mapping to better localize the region o f alteration prior to using the primers on these very precious samples.  The lesions consist o f two groups o f hyperplasia and oral low-grade dysplasia. One group (n - 87) is from patients with no subsequent history o f head and neck cancer. A l l but three o f these cases had at least 3 years o f follow up time. W e refer to these cases as 'non-progressing'. The other group (n = 29) is from patients that later progressed to CIS or S C C at the same anatomical site. The interval between the primary lesions and later CIS or S C C had to be longer than 6 months. W e refer to them as 'progressing'.  A s shown i n Table 13, there was no significant difference between the progressing lowgrade dysplasias and those without a history o f progressing i n terms o f gender (56% male in progressing cases vs. 57% o f those without a history), age (mean age 58 years i n progressing cases versus 55 i n those without a history), site, and smoking history (of those with known habits, 78% o f progressing cases vs. 85% o f 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. This lengthy follow-up time is to ensure that progression did not occur.  89  Table 13.  Demographic information of patients with low-grade dysplasia  Non-progressing  Progressing  (n=54)  (n=23)  Age (mean, years)  55  58  0.416  Sex (% male)  57  56  1  % with smoking history  85  78  0.170  Follow-up (mean, months)  96  37  0.0001  P-value  Features  Table 14 shows the association o f A l at D11S1778 and int2 with progression. For nonprogressing low-grade lesions, A l was rioted i n only 4/78 (5%) at D11S1778 and 4/59 (7%) at int2. In contrast, A l was present 9/23 (39%) at D11S1778 and 6/21 (29%) at int2 in those morphologically similar but progressing lesions. The difference between progressing and non-progressing low-grade lesions is o f statistical significance both at D11S1778 (p = 0.0002), and at int2 (p = 0.0175).  90  Table 14.  Allelic imbalance of D11S1778 (ATM) and int2 (int2-cyclin Dl) in  progressing and non-progressing hyperplasia and low-grade dysplasia  Low-grade lesions  Non-progressing  (n=87)  D11S1778  4/78 ( 5 )  Int2  4/59 (7)  Progressing  p-value  (n=29)  a  9/23 (39)  6/21 (29)  0.0002  b  0.0175  Value given as number o f samples showing loss/total number o f informative cases (% of cases i n parentheses). B o l d means p< 0.05, was considered significant. a  b  The data were also examined for association with disease progression b y using the Kaplan-Meier method. Time-to-progression curves were plotted as a function o f A I at D11S1778 or at int2 (figure 11). A significant difference was observed for each o f the loci. These data suggest that D11S1778 and int2 both mark regions i n the D N A with genes that are associated with risk o f progression to cancer.  91  Figure 11.  Probability of having no progression to cancer, according to A l at D11S1778 or at int2  VISITYR  P = 0.0032  i.i  1.0'  1  I I I I I I -I—I—(-  .9  INT2 D  1.00  +  1.00-censored  co  |  M  .00  CO  E 3  .7  4  -10  0  10  VISITYR  P = 0.0054  92  20  .00-censored  VII.  DISCUSSION  To the best o f m y knowledge, this thesis has for the first time investigated a large number o f premalignant lesions for genetic alterations at l l q . Three loci (Dl 1S4207, D11S1778 and intl) at l l q have been studied. D11S4207 is a hot locus newly identified by this thesis; whereas D11S1778 and int2 are two loci that are widely studied i n cancer but only limited information is available regarding their occurrences i n oral premalignant lesions. For a better flow o f the discussion, I w i l l first discuss the temporal changes o f the 3 loci during the multistage oral carcinogenesis, and then discuss the relationship between cancer progression o f oral premalignant lesions and l l q changes, and finally the new locus, D11S4207, as a potential new hot spot containing tumor gene(s).  VII.l.  Allelic imbalance of genes at D11S4207, D11S1778 and int2cycline Dl during multistage oral carcinogenesis  The histological progression model for head and neck S C C is w e l l established. There are strong evidences indicating that accumulations o f changes to critical control genes (oncogenes and T S G s ) underline the progression o f lesions from hyperplasia to increasing degree o f dysplasia (mild, moderate, severe), and to CIS and finally to invasive S C C . Currently a number o f tumor genes and even more potential chromosome loci containing tumor genes have been established i n oral S C C but information on these genes  93  and loci in the early stage o f oral lesions are limited in number and scope due to the difficulty o f obtaining suitable specimens for analysis and to technical problems associated with working with very small lesions and minute amounts o f D N A (Zhang et al, 1997; Califano et al, 1996; R o z et al, 1996; M a o et al, 1996b; E m i l i o n et al, 1996). Although a molecular progression model has been first proposed by Califano et al. (1996), and later refined by Rosin et al. (2000), genes and chromosome loci investigated in the model are limited. Understanding o f the additional genetic changes and o f their timing during the molecular progression o f oral cancer is critical for our further understanding o f the mechanisms o f the tumor progression and prediction o f cancer risk o f oral premalignant lesions as well as intervention and management o f high-risk oral lesions.  This thesis has studied a large number o f oral premalignant lesions at different stages o f progression for alterations for chromosome 1 l q at D11S4207, D11S1778 (1 lq22-23) and intl (int2-cyclin Dl region) to determine the timing o f the alterations at these loci.  VII.1.1.  Temporal changes of the 3 loci at l l q  This thesis provides a great deal o f genetic information, for the first time, o f timing and frequency o f A l at these three loci in oral carcinogenesis, which is essential to help better understanding o f the mechanism o f oral carcinogenesis and the role o f 1 l q .  94  Temporally, allelic imbalance at D11S4207 (the new gene site) is markedly different from those at D11S1778 (1 lq22-23) and intl (int2-cyclin Dl region). A I for D11S4207 (the new gene site) occurred very early during the multistage carcinogenesis with only slight increase i n the frequency o f A I with progression o f the oral lesions from low-grade to high-grade lesions and finally to invasive S C C . In oral hyperplasia, 7/19 (37%) demonstrate allelic imbalance for the new gene site. This rate is much higher than that seen for 3p and 9p losses in oral hyperplasia, both o f which have been shown to occur early i n the development o f oral and many other solid cancers (Rosin et al, 2000). However, unlike the changes seen for 3p and 9p losses, which steadily increase with progression o f oral lesions, the rate o f allelic imbalance seen i n oral hyperplasia at D11S4207 (the new gene site) has not markedly increased with progression o f the oral lesions. Even when the rate o f the allelic imbalance o f the invasive oral S C C (35/74, 47%) is compared with that o f the oral hyperplasia (7/19. 37%), the result is far from significantly different (P = 0.4506).  Unlike the new gene locus, allelic imbalances at Dl 1 SI 778 ( l l q 2 2 - 2 3 ) and int2 (int2cyclin Dl region) are rare at low-risk oral premalignant lesions. Both o f these markers show markedly increased allelic imbalance with advent o f high-grade dysplasias, which contain significantly increased alterations at these markers compared to low-grade dysplasia and hyperplasia (P = 0.0352 for D11S1778 and P = 0.0223 for intl). in the frequency o f allelic imbalance continued i n the invasive S C C s .  95  The rise  VII. 1.2.  Significance of the changes of the 3 loci at 11 q  Studies o f temporal changes o f allelic imbalance during multistage oral carcinogenesis can provide critical information on the role o f a presumptive tumor gene i n the development o f oral cancer even prior to the identification o f the actual tumor gene. For example, i f a gene were mainly altered during the transformation o f preinvasive lesions to invasive lesions, it would suggest that the gene plays a role i n the tumor invasion.  Vn.1.2.1. Sienificance of Al at D11S4207  The finding o f a high frequency o f A l at D11S4207 (the new gene site) i n oral lesions with very low cancer risk (37% o f hyperplasia) and the finding o f a similar frequency or only slightly higher frequency o f A l i n oral lesions with higher cancer risk (34.5% lowgrade dysplasia and 47% o f high-grade dysplasia) or even i n S C C (47%) are unusual. Our previous studies on other chromosome regions in oral lesions generally demonstrate a significant rise i n the losses at a specific stage(s) o f oral cancer development. For example, losses at 3p and 9p were found to be significantly (or approaching significantly) increased from hyperplasia to low-grade dysplasia, and then continued to be markedly increased at later stages (high-grade dysplasia or S C C ) . O n the other hand, a significant increase in L O H at 17p occurred with the formation o f high-grade dysplasias, whereas significant increases in L O H at 8p and 13q occurred with the advent o f invasive S C C (see Table 1 at Section 1.2.3.f).  96  Is such early occurrence o f a high frequency o f A l at D11S4207 without obvious increase in the frequency o f A l with progression o f oral lesions exceptional? In a recent study, we have noticed similar temporal changes o f A l i n another chromosome region with presumptive tumor genes during oral carcinogenesis. A l at 14q31-32 was shown to be high in low-grade dysplasia (17/51, 33%), but the frequency o f A l did not increase with progression o f oral lesions: A l was noted in 10/23 (30%) o f high-grade dysplasias and 12/33 (36%>) invasive S C C s (unpublished data from this lab).  The significance o f such early occurrence o f A l without accompanying increase with progression o f oral lesions remains speculative. Since demonstration o f A l b y microsatellite analysis is an evidence o f clonal expansion o f cells with growth advantages (required by tumorigenesis), the presence o f A l , even i n hyperplasia, could not be ignored. The explanation for lack o f further increase i n the frequency o f A l could be complicated, including alternate modes o f inactivation/alteration such as epigenetic silencing o f gene expression by promoter methylation (Thiagalingam et al., 2002), and different cancer development pathways, or simply because only one primer,  D11S4207,  has been used to probe the region, and an increased number o f primers for the region may identify further cases with the alteration.  Whatever function or significance the new locus has, it is clear that A l at D11S4207 occurred independently o f A l at int2 (D11S4207 is localized at a region 20 M b telomeric to CCNDl-int2  region), and o f A l at D11S1778 (1 lq22-23). The results i n Section V I . 4  97  c\  showed that the majority o f samples did not show A I at D11S4207 simultaneously or synchronously with either A I at int2 or at D11S1778 (see Tables 11 and 12). A t int2, the discordance with D11S4207 occurred in 50% o f hyperplasias, 100% o f low-grade dysplasias, 73% o f high-grade dysplasias, and 56% o f S C C s . A t D11S1778,  the  discordance D11S4207 occurred in 100%) o f hyperplasias, 80% o f low-grade dysplasias, 60%> o f high-grade dysplasias and 70% o f S C C s . The rates should be even higher given the fact that the assay does not yield any information on whether or not the maternal or paternal allele is being altered i n samples showing A I . Thus, estimates on the actual percentage o f cases in which 2 events are giving rise to alterations at these 2 loci are even lower. A n alteration could occur on the maternal allele o f one locus and on the paternal allele o f another loci and still be recorded as a single event.  Several studies have suggested the existence o f fragile sites at 1 l q l 3 region (Conquelle et al, 1997; Jin et al, 1998; Shuster et al, 2000), including the region harboring D11S4207 (Jin et al, 1998). One speculation for the early occurrence o f A I at D11S4207 is that cells with such fragile site alterations have an increased genomic instability, which subjects the cells with the A I to further genetic changes. In our recent publication, it has been shown that A I at 3p &/or 9p occurred early and were found i n almost all oral premalignant lesions that later progressed into cancer, suggesting A I at these loci are essential for cancer formation. Table 15 examines the relationship between A I at D11S4207 and A I at 3p &/or 9p, the proposed essential changes for oral cancer development, to determine the potential importance o f A I at  98  D11S4207.  O f the 29 cases o f low-grade dysplasia, 10 cases have A l at D11S4207, whereas 19 have no alteration at the site. A l at 3p &/or 9p was noted i n a significantly higher proportion of lesions with A l at D11S4207 as compared to lesions without A l at D11S4207: 9 (90%) of 10 lesions vs. 7 (37%) o f 19 cases (P = 0.0084). Such results suggest an association between genetic alterations at D11S4207 and those at 3p and 9p.  Table 15.  Association of allelic imbalance at D11S4207 and 3p &/or 9p in lowgrade dysplasias  3p and/or 9p P-value  Al  Al  Retention  9  1 0.0084  D11S4207 Retention  12  7  VII.1.2.2. Significance ofAIatDHS1778  (11Q22-23) and int2 (int2-cyclin Dl region)  Although A l at 1 l q l 3 - 2 2 has been extensively investigated in head and neck cancer, this is the first study to investigate int2-cyclin Dl genes at 11 q 13 and 1 lq22-23 region separately i n oral premalignant lesions with a large number o f oral lesions at different stages o f cancer progression. A l at the two loci was rare i n hyperplasia and low-grade dysplasia. Both D11S1778 (1 lq22-23) and int2 (int2-cyclin Dl region) showed significant increase i n the frequency o f A l  with formation o f high-grade oral  99  dysplastic lesions, and the increase continued with formation o f S C C . Such results indicate that lesions with these changes have high-risk for cancer progression, and also support the literature that genes at 1 l q l 3 - 2 2 play important roles i n tumor instability and invasion.  VII.2.  Allelic imbalance at D11S1778 and int2 is associated with cancer risk.  A salient advantage for microsatellite analysis is that data from microsatellite analysis not only could provide clue for the identification o f target genes, but also could be used as markers for diagnosis and prognosis, even prior to identification o f the actual target genes. The latter is particularly important as from a clinical point o f view; the ultimate significance o f molecular studies is that molecular markers can be used to guide clinical management, including prediction o f risk o f cancer progression o f oral premalignant lesions.  Despite o f the obvious significance and importance o f linking molecular results with clinical outcome, few such studies are available for premalignant lesions. Genetic studies o f oral premalignant lesions are difficult even without the requirement o f the clinical outcome. 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 A I at D11S1778 and int2 in order to determine the potential roles o f these gene loci i n the  100  progression o f low-grade oral premalignant lesions.  A s discussed above, the results o f one o f my studies show that allelic imbalances at D11S1778 and int2 occur mainly in high-grade oral dysplastic lesions and invasive oral S C C s . These results would suggest that these molecular markers could serve as risk markers for cancer progression since high-grade dysplasias are known to have a high-risk for cancer progression.  Low-grade lesions (those without dysplasia or with low-grade dysplasia) were chosen for this study because the majority o f these lesions w i l l not progress into cancer. Currently it is not possible to identify the small percentage o f progressing low-grade lesions from the majority o f morphologically similar but non-progressing lesions. Since A l at D11S1778 and int2 were rare in the low-grade lesions in m y study, i n this study we asked whether A l at D11S1778 and int2 indicate a risk o f cancer progression, and whether those lowgrade dysplasias with A l at the two loci had increased cancer risk.  The study showed that non-progressing low-grade lesions (without dysplasia or with lowgrade dysplasia) had significantly lower frequencies o f allelic imbalance at both D11S1778 and int2, as compared to the non-progressing hyperplasia (Table 14). For non-progressing low-grade lesions, A l was noted in only 4/78 (5%) at D11S1778 and 4/59 (7%) at int2, significantly lower than those morphologically similar but progressing lesions: 9/23 (39%) at D11S1778 (P = 0.0002) and 6/21 (29%) at int2 (P = 0.0175). Such study results lend strong support to the hypothesis that A l at both D11S1778  101  (1 lq22-23) and int2 (int2-cyclin Dl region) is associated with high cancer risk for oral premalignant lesions and that morphologically low-grade lesion with A I at either o f the two loci may indicate high cancer risk despite o f a low-risk morphology. This thesis provide solid evidence that A I at D11S1778 and int2 could be used as potential markers to identify high-risk lesions at early stage, which may have important impact on the clinical diagnosis and management.  VI1.3.D11S4207, a new hot spot at llq!3  A s mentioned in literature review, despite o f discovery o f a number o f genes at the 1 l q l 3 region, it is generally believed that many more have yet to be identified from the site, which is a major region that plays a key role in controlling chromosomal instability and progression o f tumors (Bekri et al, 1997, Izzo et al, 1998, 1999; Zhou et al, 1996; Gebhart et al, 1998). Identification and localization o f these potential genes w i l l contribute to the understanding o f the roles o f 1 l q l 3 region i n tumorigenesis, including oral carcinogenesis.  This thesis has identified a new hot spot, D11S4207, for tumor gene at 1 l q l 3 region. O f the 91 oral S C C investigated, 74 were informative and 35/74 (47%) demonstrated allelic imbalance at Dl 1S4207.  Subsequent to the discovery o f the new locus, I investigated the boundaries o f the region  102  o f alteration. M y data had indicated successful establishment o f telomeric and centromeric boundaries for the region o f alteration with microsatellite markers D11S916 and D11S4119, until one month ago when the location o f these markers changed (http://gonome.cse.ucsc.edu/index.html), although two cases o f S C C did suggest that there may be a telomeric boundary at D11S4119, which is located approximately 0.5 megabase pairs from D11S4207.  Despite o f the need for further fine mapping o f the region, existing data do support that this is a new hot spot containing tumor gene(s). Not only there is a high frequency o f A l at D11S4207 in oral S C C s , a high frequency o f A l at D11S4207 was also noted in preinvasive lesions including low risk oral hyperplasias. Cancers are characterized by an intrinsic genetic instability that frequently results i n a cascade o f nonspecific genetic alterations, which make the identification o f alterations to critical control genes difficult. Consequently it could be argued that A l at D11S4207 in oral S C C s could be non-specific, even though the frequency o f A l (47%) is too high for that argument. The presence o f A l at the same region i n preinvasive lesions, however, strongly support that the alteration is not random 'gun-shot' effects, but rather true hot spot containing tumor gene(s).  The identification o f this new hot spot and further fine mapping o f the region w i l l lead to pinpoint the location o f putative tumor genes for further analysis and is critical for further sequencing to identify new genes and related functions, and add further understanding to the role o f 1 l q l 3 i n the cancer development.  103  VIIAEnding Mark  Although m y study identified a new locus and also provides A I frequencies o f three loci at 1 l q : D11S4207, int2 and D11S1778 in oral cancer and premalignant lesions, there are some limitations as well. One major limitation is that there was only one primer used for each locus o f the three loci at 1 l q . This is insufficient since each o f the loci probably contains multiple genes; hence the data provide only information o f 'global' changes on the region. The use o f only one primer for each locus also increases the percentages o f cases studied being non-informative, which lowers the sample size with data and increases the possibility o f Type II error in statistical analysis. F o r example, 14 (42%) out o f 33 o f hyperplasia cases were non-informative at D11S4207.  There are many exciting things I would like to continue on the project i n the future. This w i l l include fine map the locus of D11S4207 to localize and ultimately sequence and identify the candidate tumor genes in this region. Currently it seems that careful mapping o f primers onto tiled B A C arrays would be the choice o f techniques although other newer techniques could be out very soon with the rapid advances i n technologies. The identification o f the gene(s) could be followed by immunohistochemical studies o f the gene products to investigate the protein changes o f the genes. 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