MOLECULAR CYTOGENETIC ANALYSIS OF NON- SMALL CELL LUNG CARCINOMA: BY COMPARATIVE GENOMIC HYBRIDIZATION AND SPECTRAL KARYOTYPING

Catherine Yuen Yee Luk

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto

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Comparative Genomic Hybridization (CGH) was used to screen 22 non-small ce11 lung carcinoma ce11 lines and primary tumors. Al1 chromosomes were involved in imbalance. Gains of DNA were much more muent than losses. The most fiequently gained chromosome arms were Sp (73%), 8q (68%), 15q (50%), Iq (45%), 20q (45%),

3q, 1 1 q and 19q (36%). DNA losses were found in chromosome 9 (36%), 18q (36%), 6q

(27%), 5q 12-32, 13q and 19p (1 8%). High copy number amplifications were found on

2p23-24, 3q24-27, 5p, 6cen-p2 1.1, 6q26, 7p2 1, 7q3 1, 8q, 1 l q 13-qter, 20q 12- 13.2.

Seventy one percent of the ce11 lines showed an overrepresentation of 8q in their CGH profile, with four cases showing amplification in the 8q24 region. MYC aneuploidy was observed in NSCLC ce11 lines by FISH analysis. In most of the ce11 lines, only low ievel

MYC amplification was detected. Detected MYC signals not only localized to normal chromosome 8, but also increased in copy number by translocation to various marker chromosomes. Spectral Karyotyping (SKY)was performed in two ce11 lines where G- banded analysis was also available. SKY was able to provide additional cytogenetic information on chromosomal translocations, identification of rnarker chromosomes and characterization of complex structural rearrangernents that could not be identified by conventional banding. Dedication

To my family and friends for their love and support. Acknowledgements

I gratefully thank Jane Bayani and Ajay Pandita for their technical assistance and for their kindness and helpfulness. 1 thank Sandra Johnson for the excellent help with the G- banding analysis of the ce11 lines. 1 thank Christine To for her support, encouragement and fiiendship. 1 thank al1 my committee members (Dr. Frances. A. Shepherd, Dr.

Suzanne Kamel-Reid, Dr. Dittakavi SR Sma) for their advice and guidance throughout this project. 1 thank Winston Siu for his help with al1 the cornputer assistance and love. I would like to thank my parents for their support and understanding. Finally, 1 would like to thank Dr. Ming-Sound Tsao and Dr. Jeremy Squire for their patient instruction and providing me a chance to participate as a graduate student in their laboratories. List of Tables Page

Table 1. WHO histologicai classification of lung tumors 11

Table 2. Revised TNM definitions and stage grouping for NSCLC 13

Table 3. Human cancers associated with DNA virus infection 16

Table 4. Histological subtype and copy number imbaiances detected 58

by Comparative Genomic Hybndization in 22 Non-small ce11

lung carcinomas.

Table 5. Summary of MYC FISH results in 12 NSCLC ce11 lines 63

Table 6. Candidate genes mapped to chromosomal imbalance regions 73

identified by CGH List of Figures Page

Figure 1. Multi-stage mode1 of carcinogenesis 3 Figure 2. Turnor ce11 kinetics 6 Figure 3. Method of detecting loss of heterozygosity 2 1 Figure 4. Schernatic outline of CGH technique 29 Figure 5A,B. Fluorescence image of the MGH7 ce11 line 55 Figure 5C. Comparison of red-to-green fluorescence intensities 55 dong the metaphase chromosomes in the MGH7 ceIl line Figure 6. Schematic representation of the genetic imbalances detected 56 by CGH analysis in 22 NSCLCs Figure 7. Schernatic representation of the genetic imbalances detected 57 by CGH analysis in 14 NSCLC ce11 lines Figure 8. Schematic representation of the genetic imbalances detected 60 by CGH analysis in 8 pnmary NSCLCs Figure 9. Comparison of CGH the profiles for SQCC and ADC 62 Figure 10. Exarnples of the MYC FISH results 65 Figure 1 1. Representative karyotype for MGH7 ce11 line 66 Figure 12. Representative karyotype for A549 ce11 line 67 Figure 13. Example of SKY for MGH7 ce11 line 68 Figure 14. Swnmary of aberrant chromosomes after G-banding 69 and SKY in MGH7 ce11 line Figure 15. Surnmary of aberrant chromosomes afier G-banding 70 and SKY in A549 ce11 line Figure 16. Comparison between SKY and MYC FISH results for 71 MGH7 ce11 line Figure 17. Comparison between G-banding, CGH and SKY in 73 MGH7 ce11 line Page

Abstract 1

Dedica tion i i .. Acknowledgements 111

List of tables iv

List of figures v

1. INTRODUCTION

1.1 Effect of smoking on the incidence and mortality of lung cancer

1.2 Lung carcinogenesis

1.3 Diagnosis of lung cancer

1.4 Screening of lung cancer

1.5 Histological classification of lung cancer

Non-small ce11 lung carcinoma

Squamous ce11 carcinoma Adenocarcinorna Large cell undifferentiated carcinoma Carcinoids

r Small ce11 lung carcinoma

1.6 Staging for lung cancer

Staging for NSCLC m Staging for SCLC

1.7 Genetic events in cancer development O Proto-oncogenes a Tumor suppressor genes r Mismatch repair genes

1.8 Advances in molecular cytogenetic analysis in study of tumor

Conventional cytogenetic analysis a Fluorescence in situ hybndization Comparative Genomic Hybridization Spectral Kwotyping

1.9 Molecular biology of non-small ce11 lung carcinoma

2. RATIONALEMYPOTHESIS and OBJECTIVE 3. METHODS and MATERIALS

A. Cell lines and tumor tissues preparation

B. Isolation of genomic DNA from tissue and ce11 lines

C. Normal metaphase preparations

Normal lymphocytes isolation and culture r Preparation of metaphase chromosome spreads from lymphocytes

D. Comparative Genomic Hybridization

DNA labeling by nick translation Metaphase slides pretreatment Hybridization Post hybridization wash and detection r Digital image analysis for CGH

E. MYC fluorescence in situ hybndization

F. Spectral Karyotyping

r Metaphase slides pretreatment for SKY O Chromosome and probe denaturation m Detection - Digital image analysis SKY

vii 4, RESULTS

4.1 CGH analysis for ce11 lines and pnmary tumors 4.2 MYC analysis for NSCLC cell lines 4.3 Conventional G-banding analysis 4.4 SKY analysis for MGH7 and A549 4.5 Comparison between SKY and MYC FISH analysis in MGH7 4.6 Comparison between G banding, CGH and SKY analysis in MGH7

5. DISCUSSION

6, CONCLUSION 7, FUTURE STUDIES

9. APPENDIX

viii Introduction

1.1 Effect of Smoking on the Incidence and Mortality of Luag Cancer

In the late 181h century, lung cancer was one of the lest reported hurnan malignancies (10). The incidence began to nse exponentially, and today it is the cornmonest malignancy and the leading cause of cancer death in North Amenca and

Worldwide (4,14). Several risk factors related to lung cancer have been identified (e.g. exposure to asbestos, radon gas, radiation, some organic chernicals and environmental tobacco smoke). Although there are numerous causes for lung cancer, the predominant risk factor is cigarette smoking (1,2,3). This accounts for 80085% of al1 lung cancer cases

(2,3). Therefore, most lung cancers are preventable.

The mortality rate for lung cancer has started to decline arnong men, but is nsing rapidly among women. In the United States between 1990 and 1994, lung cancer deaths decreased 1.4% per year in men (4), but lung cancer deaths in women increased about

1.7% per year (4). The discrepancy between the mortality rate for lung cancer in different genders may largely be attributed to a change in the smoking behavior among men and women. The increase in consumption of tobacco products in women has resulted in a marked increase of lung cancer death among women (12). In contrast, the mortality rate of hgcancer in men has leveled off in the mid- 1980s and is still consistently declining, reflecting their drop in tobacco consumption beginning in the mid-1960s. In 1998, the estimateci number of deaths for lung cancer in women is 6,500 compared to 5,300 deaths for breast cancer (14). Among men, lung cancers deaths (10,600) far exceed the number of deaths due to prostate cancer (4,300),the second leading cause of male cancer death in

Canada ( 14).

The current five-year sunival rate for lung cancer is 15% as compare to 6% in

1950-1954 (5,19). Despite advances in lung cancer research by scientists and clinicians, the survival rate has not improved dramatically over the past 40 years. This is putatively due to the fiequent occurrence of blood borne metastasis dunng the early stages of lung cancer development. Understanding of the biology and mechanism of multi-stage lung carcinogenesis especially at the molecular level, may soon have a potential impact on early diagnosis and prevention of lung cancer, hence overall survival.

1.2 Lung Carcinogenesis

Lung cancer develops more fiequently in the upper than in the lower lobes, and in the right more than in the left lungs. The anterior segment of the right upper lobe is the most common location (6). Most cancers of the lung are derived fiom the epithelial tissues in the bronchi and are called bronchogenic carcinomas. Sarcomas and lymphomas of the lung are very rare (6).

Since there is a latent period between the onset of cigarette smoking and the first signs of lung cancer, this leads to the assumption that not one but several molecular events must take place during the cancer development (13), hence the concept of multi- stage carcinogenesis. Figure 1 illustrates a proposed schema of multi-step carcinogenesis in a mouse skin carcinogenesis mode1 (1 3). Four distinct phases have been identified: 1)

Initiation, 2) Promotion, 3) Conversion, 4) Progression.

(clonai &pansion)

rt Turnor initiaam uschomisrk wMch cmcame cancer Merirdmindfemd rlone. '* Tumr promaors themrekes are nat Waqenic. Howcuer, tumor prmcdors cm cru88 cancer 8elsctiueîy in an area thrt hrbsai preuiourly erpmed to a tumor initiator

Nde: hm the abence of treatment Wh 8 promotirbg ageM when 1he dome ofthe iriiliding cwchagen im nat large, tumor8 vvlW nat appew. Ka luge ch88 Mthe kiithting -nt ir giuen, howeuer, tumors dl u8udip devskp in the abence of 0 promuter. Figure 1. Multistage model of carcinogenesis (Adapted from Reddel RR, Harris CC. Carcinogentsis. in: Roth JA, Ruckdeschel JC, Weisenburger TH, eds. Thoracic Oncology. Philadelphia, W .B. Saunders, 1989; pp. 16-37).

Studies of respiratory tract carcinogens have failed to assign them an unequivocal role as turnor initiator and tumor promoter, as strictly defined in the mouse skin model.

This is because the initiating doses used to date have not been low enough and have resulted in the appearance of tumors in the absence of a promoter (13). Nevertheless, interesting information has emerged from these studia. Polycyclic hydrocarbons, especially benzo[a]pyrene (BP) and compounds of the N-nitroso class (e.g., N-nitroso-N- methylurea, NMU) cause carcinomas in hamster trachea and bronchi (13). The skin tumor promoter 12-O-tetradecanoylphorbol-1 3-acetate (TPA) enhance tumor formation in rat trachea initiated with dimethylbenzo[a]anthracene (DMBA); the does of DMBA administered were not subcarcinogenic in these expenments, but the TPA caused a marked enhancement in tumor numbers and a decrease in the latency period ( 13). DMBA and TPA appear to represent the closest approximation so far in respiratory epithelium in vivo to the classic sequential administration of initiator and promoter.

1.3 Diagnosis of Lung Cancer

Diagnostic investigations in lung cancer can be separated into two parts. First, it is necessary to diagnose whether a cancer is present. Then there is a need to determine the stage and histological subtype of the tumor in order to decide appropriate management and treatment for the patient.

Early stage hgcancer patients are usually asymptomatic. As the tmor grows it starts to cause symptoms, which include persistent cough, hemoptysis (coughing up blood), chest pain, hoarseness, weight loss and shortness of breath. Chest X-ray perfonned on these patients may suggest the presence of a lung tumor but diagnosis requires tissue confirmation by biopsy. Samples for microscopic examination may be obtained by different methods

depending on the location of the tumor. Approximately two-thirds of the lung cancer

patients with central tumor are usually diagnosed by cytology examinations of multiple

sputum sarnples (9). A greater accuracy and confidence may be gained by examining a

piece of the tumor tissue using either bronchoscopy or fine-needle aspiration

histologically.

In those patients where enlarge, suspicious lymph glands are noted on imaging

studies, exploratory rninor mediastinoscopy/mediastinotomy is undertaken to biopsy

lymph glands for microscopic examination.

1.4 Screening of Lung Cancer

Because the prognosis for lung cancer is best if it is detected in an early

asymptomatic stage, screening of high-risk subjects has been attempted to detect asymptomatic cancers. Screening tests include sputum cytology and chest X-ray.

Although screening trials could detect tumor at an earlier stage, no evidence shows that screening (with sputurn cytology and chest X-ray) can reduce the mortality rate for lung cancer patients (7). The Arnerican Cancer Society ha recornrnended against the use of chest radiographs and sputum cytologies as screening tools (8).

Tumor ce11 kinetics model proposed by DeVita and colleagues able to illustrates the challenge of lung cancer in early detection (1 1). Even with the best available diagnostic tools (generally the chest X-ray); it can only identiQ a lesion at least 1 cm in diameter

(containing approximately 1XI O' tumor cell). Clinically, a 1-cm tumor is considered to be very favorable. However, this early stage primary hunor actually represents a very

late phase in its natural history of lung cancer development. Since only a few more

twnor ce11 dmblings will result in a metastatic tumor (Figure 2). Currently, even with the

best diagnostic approaches, less than 20% of patients have disease limited to the lung by

the time of diagnosis. Therefore, improvement in lung cancer survival will be dependent

upon signifiant advances in the detection of tumors at a much earlier stage.

0 Zone of cllnical # rountine detection

Figure 2. Tumor ce11 kinetics A clinically early stage prirnury mmor actualiy represen ts a vev late phase in the natural history of the lung cancer deveiopmeni.

(Adapted from Mulshine JL, Szabo E, Scoot F, Quinn K, Zhou J, Avis Il Miller MJ, Vos M, Treston AM, Cunina F and Shaw GL. The sming for and early deteçtion of lung cancer. In: Carney DN, ed. Lung Cancer. Arnold, 1995; pp. 5 1. More and more studies are now concentrated on genetic changes occurring in pre-

malignant lung tissues. It has been indicated that such DNA alterations may serve as

molecular genetic markers, providing additional information for early diagnosis as well

as prognostic factors.

1.5 Histological Classification of Lung Cancer

The prognosis and choice of treatment is determined by multiple factors including the

histological subtype of lung cancer, the stage of the cancer, the patient's symptoms, and

the patient's general health (3). The most widel y recognized pathologie classification

system for lung cancer was published by the World Health Organization (WHO) in 1967

and revised in 1982. Histological classification is based on light rnicroscopic

examination of stained tissue sarnples. It is a qualitative assessrnent whereby a tumor is

categonzed according to the normal tissue type it most closely resembles. In addition to

histological typing, tumor can be graded according to cytological difkrentiation. This is

a measure of the differentiation of the tumor expressed as the extent to which a tumor resembles the normal tissue at that site. Turnor grading is expressed in numerical grades of differentiation fiom most differentiated (Grade 1 to least differentiated (Grade 4).

WHO classification comprises over 50 different lung neoplasms. In practice and according to the diffaences in prognosis and treatment option, lung cancers are grouped into two major categories: small ce11 (SCLC) and non-small ce11 lung carcinomas

(NSCLC). SCLC accounts for 20% to 25% of al1 new cases of lung cancer worldwide.

The remaining 75% to 80% belongs to the major subtypes of NSCLCs: squamous ce11 carcinoma (SQCC), adenocarcinorna (ADC), large ce11 undifferentiated carcinoma

(LCC),and infrequently occumng carcinoids (1 5).

Sauamous Ce11 Carcinoma (SOCC]

Squamous ce11 carcinoma is more commonly found in smokers and used to be the

predominant fonn of lung cancer (20). A slight decrease in the relative fiequency of

squamous carcinomas has been noted over the past 20 years; perhaps due to the rise of

lung cancer in women, who tend to develop adenocarcinornas more commonly than

squamous ce11 carcinoma (26). SQCC now accounts for approximately 30% of al1 lung

cancers (45). SQCCs mise from the basal cells of the bronchial epithelium and progress

through varying degrees of dysplasia, carcinoma in situ, and finally invasive carcinoma.

Most of them (75%-95%) occur in the large bronchi. The tumor cells produce keratin, a

substance nomally found in skin and hair. Since they tend to develop into large centrally

obstructing symptomatic masses before metastasis, 60 % of them are usually surgically

resectable and therefore have a better prognosis than 0thsubtypes (2 1).

Adenocarcinorna IADCl

ADC accounts for approximately 35% of ail lung cancers (45). Adenocarcinornas can be recognized by its attempt to form glandular structures. These tumors usually aise in the periphery of the lung and alveolar lining epithelium. Adenocarcinorna grows slower than other bronchogenic carcinoma subtypes but tends to metastasize early in its

development. Around 40 % of ADCs are resectable. Smoking does not seern to

predispose this type of lung cancer (21). According to the WHO classification,

adenocarcinomas can be subclassified into four major subtypes: acinar, papillary, and

bronchoalveolar subtypes, as well as a solid carcinoma with mucus production.

Well-differentiated acinar adenocarcinoma is composed of glands lined by

columnar cells, which oAen secrete a mucinous substance into the lumen. Tumor cells

are usually large, have prominent nucleoli, and abundant cytoplasm (23). Papillary

adenocarcinoma foms papillary projections and may produce significant arnount of

mucin. Bronchoalveolar carcinoma is defined as an adenocarcinoma that grows along the

alveolar septa (the alveolar architecture is preserved) (23).

Large Ce11 Undifferentiated Carcinoma [LCCl

Large-cell undifferentiated carcinoma is a poorly differentiated non-small ce11 lung carcinoma that does not dernonstrate readily obvious features of squamous, adeno or small ce11 differentiation. This twnor tends to be penpherally located (23). Some of these tumors may actually represent poorly differentiated fom of SQCC and ADC. Carcinoids

Carcinoid is carcinoma-like tumors that more commonly occurred in the proximal

bronchi of young patients (45). They constitute 1 to 2% of lung tumors. It has no

association with smoking (22,25). It was initially classified as an adenornas, but a small

proportion of these tumors has the ability to metastasize. It is now included under

malignant epithelial tumors in the WHO nomenclature. The 5-year sunival rate after

resection is over 90% (22).

Small Cell Lunn Carcinoma

Small ce11 carcinoma usually anses centrally but occasionally develops in the

periphery (25). It has a rapid growth rate and tends to spread to lymph glands and bloodstream early. Current effective chernotherapy has improved the median sunival of patients to 1 year or more and the 5-year survival has been increased to 5-1 0% compared to less than 1% before systemic therapy is used (25). This tumor is cornposed of unifomly mal1 cells with dense round or oval nuclei and sparse cytoplasm. The tumor cells express many neuroendocxine (NE) markers such as L-dopa decarboxylase and neural ce11 adhesion molecule (NCAM). They are found more commonly in men than women and have a strong relationship with cigarette smoking. A. Bcnign

B. Dysplasia / carcinoma in situ

C. Malignant

2. Small-ccll carcinoma a. Oat-ccll carrinoma b. Intermediatccell type c. Combined oat-ccll carcinoma

3. Adenocaminorna a. Acinar b. Papillary c. Bmnchoatvcolar d. Solid carcinoma with mucin formation

4. Largeîcll carcinoma a. Girint-crll carcinoma b. Clcar-ce1l carcinoma

6. Carcinoid Nmor

7. Bronchiril-gland carcinoma

II. Soft Tissue Tumors

III. Merothelial Tumon

IV. Miscellrneous Tumars

A. Benign B. Malignant

V. Semndrry Tumon

W. Unchulfieâ Tumars

VII.Tumor-like Lesions 1.6 Staging for lung cancer

Staging is a process that detemines how extensive the cancer cells has spread. The

treatrnent and the prognosis of cancer depend to a large extent on its stage. The staging

system use for SCLC is different fiom the one use for NSCLC because of the high frequency of distant metastases in SCLC. An overall stage is assigned based on the size of the untreated primary cancer (T), the regional lyrnph node involvement (N) and whether distant metastasis (M) has occurred.

Staging for NSCLC

NSCLC is more resistant to chemotherapy and radiotherapy when compare to SCLC, hence surgery remains an important of treatment option. The single most important prognostic factor for NSCLC patients is the stage. The Revised International System for

Staging Lung Cancer was adopted in 1997 by the American Joint Comrnittee on Cancer and the Union Internationale Contre le Cancer (See table 2). After assigning the T, N, M categories, the various TNM subsets are then grouped into stages. Patient in the same stage group will have a similar life expectancy. The overall 5-year survival rates are 70% for stage 1, 45% for stage II, 10020% for stage III and 0% for stage IV. Tumor proven by the presence of malignant cells in sputum or bronchial washings but not visualized by imaging or bronchoscopy; or any tumor that cannot be assessed as in a pretreatment staging.

1 T" No evideace of primary tumor TI8 Carcinoma in situ

Tl Tumor e 3 cm diameter without invasion more proximal than the lobar bronchus (i.e., not in the main bronchusl* T2 Tumor . 3 cm in diameter or tumor of any size with any of the following: invades visceral pleura; atelectasis of less than entire lung, proximal extent at least 2 cm f rom carina

T3 Tumor of any size with any of following: invasion of chest wall; involvement of diaphragm, mediastinal pleural, or pericardium; atelectasis involving entire Lung, proximal extent within 2 cm of carina

TI Tumor of any size with any of following: invasion of mediastinum, heart, great vessels, trachea, esophagus, verteral body, carina; or separate tumor nodules in the same lobe; or tumor with a maligaant pleural effusion +* *Note: The uncamon euperficial tumor of any size with ite irnmeive conipo~3entlimited to the branchial dl, which may extend proximal to the abah brouchue, io kLuo clasaifiad ae Tl.

++Hote: Most pleural effusions aaeociated with lung cancer rue due to tumor. H-r, the= are a feu patitatr in wham multiple cytopathologic axaaiinationrr of pleurai fluid are ~cgative for tumor, In these C~LICB, fïuid ie nom-bloody anci ia nkt an unidate. Whea theae elements aad clhical judgement dictate that the effusion ie not relateâ to the tumar, the effusia Bhould be excluâod a6 a rrtaging elhaiarit and the patient ehould be etagad a6 Tl, Ta, or T3.

HI Regioaal lymph nodes cannot be assessed 10 No regional lymph node metastasis 11 Metastasis to ipsilateral hilar lymph nodes 12 Metastasis to ipeilateral mediastinal and/or subcarinal lymph nodeIs) 13 Metastasis to contralateral mediastinal or hilar nodes, or ipsilateral or or contralateral supraclavicular nodes Diatant metaataaio (II)

Distant metastasis cannot be assessed No distant metastasis Distant metastasio present

Occult Carcinomm TX Btrga O TIS 8t.g. 8t.g. IA Tl Staga In T2 Btagm XIA Tl Btaga IXB T2 T3 Tl T2 T3 T3

Stagm IIXB T4 MY N MO -Y T N3 MO Stage IV hYT &Y N Ml Adapted hmAJCC Cancer Staging ManuaUAmerican Joint Comminee on Cancer 5& ed. Lippincott-Raven, 1 997. Staging for SCLC

SCLC is an aggressive tumor. At the time of diagnosis, most of the tumors have

spread widely in the chest or to other parts of the body, therefore surgery is rarely an

option. Fortunately, chemotherapy and radiotherapy have significantly prolonged the

survival of SCLC patient. As it is not so important to decide who may benefit from

surgery, staging for SCLC is very simple.

A two-stage (limitai and extensive) classification adopted by the Veterans

Administration Lung Cancer Study Group has been wide1y used today. Limited-stage disease means the tumor is confined to one hemithorax and its regional lymph nodes.

Extensive disease is defined as tumor extending beyond these boundaries. About two- thirds of the people with small ce11 lung cancer will have "extensive" disease at the time their cancer is first found ( 149).

1.7 Genetic Events in Cancer Development

Most carcinomas are rare under the age of 30; however, the incidence rate increases dramatically (10~-104times) with age (24). From this observation, it is thought that approximately three to seven mutations are needed for malignant transformation of a normal ce11 (24). The estimated spontaneous mutation rate of a given gene is about 10'~ per ce11 division. The rate of spontaneous mutation alone is too slow to be accounted for the changes necessary to produce a metastatic tumor ceIl during the lifetime of a host. To account for the incidence of cancer it has been suggested that mutations in genes give a

ce11 an increased growth advantage compared to others, therefore creating an expanded

target clone for the next mutation (148). Other mutations in the DNA damage repair

genes and the p53 gene in some tumors result in genomic instability, and therefore

increase the mutation rate.

Turnors result from loss of control/inappropnate expression of genes that are

responsible for regulating ce11 growth. The loss of the normal control mechanisms arises

fiom the acquisition of mutations in three broad categones of genes: (1) protooncogenes,

(2) tumor suppressor genes, (3) mismatch repair genes.

Pro to-oncogenes

Proto-oncogenes are discovered through the study of critical genes in the acutely

transfoming retroviruses (54). Some RNA tumor viruses (retroviruses) cany a distinct

nucleotide sequence that is responsible for their oncogenic properties. This distinct viral

nucleotide sequence is a mutated fonn of a normal cellular gene, which has been picked

up by the vinises fiom its natural animal host during the course of viral evolution (54).

The first acutely transforming retrovirus identified was the Rous sarcoma (src) virus, which only infects chickens. The cellular src gene (c-src) in the chicken encodes a plasma membrane protein playing a regulatory role in ce11 adhesion and migration. The

Wus carries a mutated form (viral-src or v-src) of the chicken gene which is constitutively active and responsible for its oncogenic effects (54). Studies of the acute transforming retroviruses have revealed more than 50 different oncogenes. Although retroviruses are histoncally important, they are rare causes of cancer in hurnans (54).

DNA oncogenic viruses on the other hand are important causative agents in human cancer (Table 3). These viruses introduce cellular genes that have few homologies

(similar arnino acid sequences) with known marnmalian proteins.

Cancer Virus

Burkitî's lymphoma Epstein-Barr virus

Nasopharyngeal cancer Epstein-Barr virus

Cervical carcinoma Human papilIomavimses

Hepatocellular carcinoma Hepatitis B virus

Protooncogenes are normal cellular genes encoding cytoplasmic and nuclear proteins. These genes encode a variety of proteins involved in ce11 proliferation, mitogenesis and differentiation, which are organized into a cascade of reaction (54). The mutated form of a proto-oncogene (or oncogene) is considered dominant in its action because a single altered allele is sufficient to transfomi cells in the presence of a normal allele. Besides virus infection, there are other mechanisms, that may lead to proto- oncogene activation include (1) point mutation, (2) gene amplification, (3) translocation

(DNA remangement) or (4) intragenic deletion. Changes could be quantitative (increase gene expression without changing the protein product) or qualitative (change the gene to produce an oncogenic protein product). (1) Activation by point mutation (missense mutation): Point mutation may lead to substitution of one amino acid for another, resulting in an altered oncoprotein. The

prototype oncoprotein produced by point mutation is the activated RAS protein (54). The

oncogenic RAS gene found in tumors contains point mutation that resulted in a constitutively active and transfonning fom of the protein, when a substitution of arnino acid at position 12, 13, or 6 1 occurs (54).

(2) Oene amplification: Amplification increases the copy numbers of a gene, which cm lead to excess production of the normal gene product. Many types of oncogene are activated by gene amplification, such as MYCN in neuroblastoma (55) and c-erbB-2 in breast cancer (56). Amplified genes may be present in two types of microscopically abnomal chromosomal structure known as double minutes (DMs) or homogeneously staining regions (HSRs). DMs are small extrachromsomal chromatin bodies that segregate randomly dunng mitosis and are not linked to a centromere. HSR is presented as an expanded chromosomal region, which inserted within a normal chromosome.

HSRs do not exhibit the usual banding pattern of nomal chromosome. Since it is linked to a centromere, it segregates normally during mitosis. In some cases where the amplification is not presented as DMs or HSRs, different oncogene specific probes could be used to check for gene amplification.

(3) Translocation: Activation by chromosomd translocations can be qualitative or quantitative. Activation by qualitative change involves the creation of a novel chimeric gene, which may generate a stnicturally altered protein that has an abnomal transforming property. The best known example is the Philadelphia translocation t(9,22) in chronic myelogenous Ieukemia (CML) (57). The breakpoint on chromosome 9 is within an intron of the ABL oncogene. The translocation joins most of the ABL genomic sequence on to a gene called BCR (breakpoint cluster region) on chromosome 22 creating a novel fusion gene. This chimeric BCR-ABL fusion gene on the Philadelphia chromosome encodes a tyrosine kinase, which has an elevated tyrosine kinase activity and this may contribute to its oncogenic action (64). Activation by quantitative change involves the transposition of the oncogene to an active chromatin domain. Instead of creating a novel chimeric gene, the oncogene is inserted to an actively transcribed chromatin region, which lead to overexpression of the oncogene. An example of this is the translocation between chromosomes 8 and 14 in Burkitt's lymphoma results in deregulated expression of a structurally normal MYC gene (54). The MYC gene from chromosome 8 is translocated into the immunoglobulin heavy chain gene. In this case, the regulation of expression of the translocated MYC allele is abnormal and is influenced by factors controlling the expression of the immunoglobulin gene to which it has been joined. In B cells this region is actively transcribed, leading to overexpression of MYC (54).

(4) Intragenic deletion: Small intragenic deletion may result in the activation of a proto-oncogene via the rernoval of a protein domain that normally inhibits the protein's activity. Alternatively, removing the protein domain may induce conformational changes that mimic those normally brought about by stimulatory signals. An in-fiame deletion is observai in the epidermal growth factor receptor gene (EGFR) in a portion of the brain tumors (58). The deletion results in the removal of exons 2-7, which encode part of the

extracellular domain of the EGFR. Functional studies of this truncated EGFR protein are

in progress, but it is thought that it contributes fiirther to the constitutive activation of the

protein.

Turnor Sumressor Genes

Tumor suppressor genes involve in the control of abnormal ce11 proliferation. Their

genes code for products that enhance neoplastic transformation when their activity is lost

(59). These genes are recessively inherited and generally both alleles of the tumor

suppressor genes must be inactivated or loss for transformation to occur (60). However,

a dominant negative effect has also been observed, where damage of one allele results in an altered tumor suppressor protein that inhibits the normal protein of the other allele

(61 )*

There are differences in the patterns of mutation between oncogenes and turnor suppressor genes. Oncogenes are usually mutated in a consistent manner either by point mutation (RAS) or translocation (KR-ABL) or gene amplification (MYCN). However, there are less constrained in the type and the position of the alterations that inactivate a tumor suppressor gene. A number of mechanisms may be involved in the inactivation tumor suppressor genes, including point mutations (both missense and nonsense mutations), deletions, rearrangernents and hypennethylation. Point mutations that inactivate RB are located on a large number of positions within the gene in contrast to the three codons that are point mutated in the activated USgenes. The APC (adenornatous polyposis coli) gene, which is mutated in a large proportion of the colonic tumors, is

subject to a preponderance of nonsense mutations such as stop codons, or small deletions

and insertions, which introduce fiameshifis. Translation of the APC protein is usually

prematurely tenninated and hence the protein is inactive (62). In contrast, p53 gene tends to have missense mutations, which result in a full length protein with just a single amino acid substitution. Some p53 proteins with missense mutations can interact with the normal p53 protein to inactivate of the normal p53 protein of the remaining normal allele

(63).

Both alleles of the hunor suppressor genes must be inactivated or lost for transformation to occur. The mutation that inactivates the first (usually inherited) allele of a tumor suppressor gene is usually confined to the gene itself (i.e. by point mutations, deletions or rearrangements). In contrast, the second mutation more commonly involved the loss of a large part or even the entire chromosome upon where the second allele is located.

Based on the fact that the second mutation in a tumor suppressor gene involves loss of part or the entire chromosome, a molecular assay - loss of heterozygosity (LOB) is ofien use to detect the presence of a turnor suppressor gene. DNA polyrnorphisms are naturally occumng variations in DNA sequence. There is usually at least one polymorphic site close to any gene locus. If a selected polymorphic marker is near the tunor suppressor gene of interest, individuals who are heterozygous for a polymorphism in a genetic marker appear homozygous if one of the aileles is lost. So by screening paired blood and tunor sarnples with polymorphic markers spaceù across the genome,

the candidate locations for himor suppressor genes could be detected (figure 3).

Tumor

Blood Tumor

rnarker A marker B

Figure 3. Loss of heterozygosity analysis

M is the nrutated allele and m the normal allele of the cancer-predispoition gene. A marker near to the cancer-predisposition locus has two alleles, A and B. This individual is heterozygous for these markers (i.e. is AB). Analysis of the marker in blood DNA would therefore confinn that the individual is AB. In the tumor, Ioss of the normal allele, m, is accompanied by loss of B as it is nearby on the chmmosome. Analysis of the tumor would therefore indicate that the individual is A, i.e. the hetero~ygusstate has been lost in the tumor cells (so-called Ioss of heterozygosity).

(Modified hmStrachan T, Read AP: Human molecular genetics BIOS Scientific Publishers Limited, Wiley Lis, 1996). Mismatch-Repair Genes

Mismatch-repair genedmutator genes are responsible for rnaintaining the integrity of

the genome and the fidelity of information transfer (120). Errors arise during DNA

replication, which are not corrected by the 395' exonuclease proofieading activtiy of

the DNA polymerase, may be corrected by a process called mismatch repair. This

involves the recognition of mismatched bases in the newly replicated strand, followed by excision and replacement with a correct base (1 20).

Mismatch-repair genes were first discovered in E.cofi including the mutH, mutL and mutS genes (12 1). Mutation in the MutHLS error correction system generates a mutator phenotype in which spontaneous mutagenesis is enhanced at many loci (120). Human

DNA mismatch repair genes (MSHî, MSH3, MSHo. MLHI. PMSI and PMSZ) were first detected in hereditary non-polyposis colon cancer (HNPCC)by studying microsatellite instability.

Microsatellites are short sequences of DNA (1 to 6 bp long), repeated between 10 to

50 times. They are inherited in a stable manner but the length of repeat may vaty individual to individual (122). In the LOH studies (using microsatellite markers), some

HNPCC samples showed gain of extra alleles rather than loss of alleles when comparing the tumor to its corresponding nonnal tissue at multiple unrelated loci throughout the genome. These tumors are said to display a RER+ (replication error) phenotype, which may irnply they have a defective mismatch repair system (1 20). Recent evidence shows that inherited mutations in MSHZ and MLHl account for the majority of cases of HNPCC (124). In families with HNPCC, a mutation in one gene

copy is inherited, and a somatic mutation in the second copy is associated with microsatellite instability in the turnor. This parallels the mechanism seen with turnor suppressor genes, where both copies are inactivated in the tumor (124). Other cancers such as stomach, endometrium and lung also display microsatellite instability ( 124).

1.8 Advances in molecular cytogenetic analysis in study of tumor

Non random structural and numerical alterations have been identified in a large number of leukemias and solid tumors. Cellular oncogenes, tumor suppressor genes and

DNA mismatch repair genes can al1 be affected by translocations, inversions, deletion, or gene amplification. The identification of recunent chromosomal abnormalities not only improves the understanding of oncogenesis by identi@ng genes involve in cancer development but also help the diagnostic and prognostic assessment.

Conventional cvtogenetic analvsis

The traditional way to detect structural and numerical chromosomal abnormalities is by karyotype analysis. This technique can provide information including 1) modal chromosome number (quivalent to DNA index obtained by flow cytometry), 2) numerical abnomalities (e.g. monosomies, trisomes, etc.) 3) structural abnormalities (detecting addition, deletion, translocations, presence of double minute chromosomes and homogeneously staining regions). Metaphase spread fiom tumor is pretreated with trypsin and then stained with Giemsa to produce a banding pattern characteristic for each chromosome. Since metaphase spread is needed, this technique is difficult to apply to solid tumors because the number of mitotic events tends to be low in solid tumor. In addition, karyotypes can be extremely complex complicating efforts to identiS recurrent changes.

Fluorescence in situ hvbridization (FISH)

Pardue and Gall in 1969 developed the in situ hybridization technique (ISH).

This technique allows specific nucleic acid sequences to be detected in morphologically preserved chromosomes, cells or tissue sections. At that time, only radioisotopes were available for labeling nucleic acid sequence. Although radioactive labeled probes are sensitive; there are some disadvantages such as probe instability due to radiolysis, relatively long delay for the autoradiographic exposure, and the position in different focal planes of the signal and the underlying chromosome. To overcome these problerns, nonradioacitve in situ hybndization techniques have been developed. Non-radioactive in situ hybndization (?WH)is considerably faster, usually has greater signal resolution, and provides many options to simuItaneously visualize different targets by combining various detection methods. Today the most popular protocol for NISH is the fluorescence in situ

hybndization (FISH). As the name implies, FISH employs fluorescent DNA probes for

detecting structural and numerical chromosomal abnormalities for clinical settings as well

as for mapping specific genes. A variety of DNA probes has been developed for

anaiyzing whole ce11 genome, whole chromosomes or chromosome bands, centromeric

regions, and specific genes. Probe for specific gene is cloned into plasmid, phage,

cosmid, or yeast arti ficial chromosome (YAC) vectors depending on the insert size.

Probes derived from different vectors represent different sequence lengths and thus

produce different signal intensities. FISH cm be performed on metaphase spreads,

interphase nuclei, fresh or paraffin-embedded archival tissue sections, and cytological preparations from exfoliated or fine-needle-aspirated cells, offering a range in flexibility which karyotyping analysis cannot deliver.

To perforrn FISH analysis, DNA probe has to be first labeled. Haptens (such as biotin or digoxigenin) or fluorochromes can be used for labeling. Before hybridization, both the double stranded target DNA and the probe(s) have to be denatured.

Hybndization allows the labeled DNA probe to reanneal with the denatured target DNA at its precise location. Large probes often contain intersperseci repetitive sequences (IRS) such as SINE and LINE sequences that can cause background staining because of the wide distribution of these sequences throughout the targeted genome. This background staining cm be suppressed by hybridization with an excess of unlabeled repetitive DNA.

This procedure has also been known as chromosomal in situ suppression (CISS) (1 15). AAer hybridization the nonspecifically bound probe is washed from the target specimen, followed by the application of antifade solution and counterstain.

FISH analysis of genetic aberrations in tumors is based on scoring of signals within the interphase nuclei of the tumor. By using specific centromeric probes, a normal ce11 gives two signals. Therefore, one signal indicates only one copy of the chromosome

(monosomy) is present whereas three signals mean there is an extra copy (trisomy) of the specific chromosome. Based on the sarne principle, gene amplification and chromosome deletions cm also be detected with specific geneAocus probe. FISH can also help to detect chromosome translocation by using two different color probes that are close to specific translocation break points. Chromosome translocation will generate "color fusion" signal on the fusion chromosome. FISH cm also use to map and order DNA clone(s) on normal or abnomal chromosome. At present the maximum resolution of conventional FISH procedures on metaphase chromosomes is 2 to 3 Mb. To obtain higher resolution, DNA probes are hybndized to the naturally extended interphase chromosomes or to artificially stretched chromatin or DNA fibers prepared by a variety of different methods (1 16). These preparations permit extremely hi& mapping resolutions: fiom 700 kb to under 5kb.

However, FISH analysis of genetic aberrations does have some limitations. For example, it can only provide information on one chromosomal locus/gene at a time.

Therefore, this method is less suitable for obtaining an oveniew of genetic changes in the whole genome. Also, the investigator has to have some prior knowledge regarding the nature of the aberrations present in a given tumor. If the cytogenetic abnormalities are unknown, it will not be possible to select a suitable probe for FISH detection in metaphase or interphase nuclei.

Comparative Genomic Hvbridization

Comparative genomic hybridization (CGH) is a relatively new FISH-based technique, which can detect the relative copy number of gains and losses of whole chromosomes and subchromosomal regions on the entire tumor genome in a single experiment (1 8). Since CGH does not rely on cellular preparations of tumor specimen, it is particularly usefûl for analyzing solid tumors. CGH can be performed with genomic

DNA from many different sources such as fresh, frozen, fornalin-fixed or even paraffin- embedded tumor tissue. CGH can also perform on DNA that is amplified fiom universal

PCR from very small tissue samples.

In typical CGH expenments tumor DNA and normal control DNA from cells such as fibroblasts are differentially labeled and then hybridized simultaneously to nonnal chromosome metaphase spreads. In order to prevent nonspecific cross-hybndization due to the interspersecl repetitive sequences that are present in the genomic DNA, excess udabeled human Cot-1 DNA is added. The hybridization is detected with two different fluorochromes (18). Tumor DNA is labeled with biotin and detected with fluorescein

(green fluorescence), the control DNA is labeled with digoxigenin and detected with rhodamine (rd fluorescence). Regions of gain or loss of DNA sequences in the -or, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. An arnplified sequence will generate increased green tluorescence, whereas a deletion will shi fi the redlgreen ratio towards red. For low copy number amplifications and deletions, this change in fluorescence ratio is difficult to distinguish by eye and requires specialized image analysis software. For exarnple, when there are two copies of the chromosomes in the two genomes, it should give a redlgreen ratio of 1 (1 17). In case of monosomy and

îrisomy, the rdgreen ratio will be 0.5 and 1.5, respectively. Figure 4 outlines the CGH technique. Comparative Genomic Hjhidhation Differential lrbcling of DNAs (Sûûbp-UWr)bp)

Reference DNA Test DNA Hut~~at~Cet-1 DNA =

In-situ irybndizaüon to normal+ chromosomes

f Fiuorescence microscopy and image aaalysis

Figure 4. Schematic outline of CGB technique

Turnor and reference DNA are lubeled with a green and reci jluorochrome. respective&, and hybridized ?O normal metophaîe sprearis. Images of the fluorescent signals are captured, and the green to red ratios are digitalij quant~ijedfor each chromosome homologue. An omplified sequence in the tumor sample will generate increased green fluorescence, whereas a deletion will sh@ the redgreen ratio towards red. Bulanced chromosomal regions will look orange under fluorescence microscope.

(Adapted from Hauldsworth J and Chaganti RSK: Comparative Genomic Hybridization: An overview. AJP 1994: 156, l253-I2!%). Since normal metaphase chromosomes are used in CGH experiment, this technique is limited by the resolution of the hybridization target. Genetic changes are detected and mapped on chromosomes when the size of the chromosomal region affected

exceeds 5- 1O Mb. Changes affecting regions smaller than this are only detectable in case of high-level amplifications (e.g. 5- 10 fold amplification of 1 Mb). Also, CGH cm only detect sequence copy number changes if greater than 50% of the cells analyzed contain a chromosomal gain or loss. This means that copy number changes in tumors that have many heterogeneous populations of normal and stroma1 cells may escape from detection

(1 18). In addition, CGH cm recognize gains and losses of DNA regions only with respect to the average copy number of the complete tunor specirnen. The nimor sarnple showing copy number changes can have any ploidy level (e.g. diploid, triploid or tetraploid) (118). While CGH serves as a powerfùl technique in identimng chromosomal gains and losses, it cannot identiQ balanced chromosomal rearrangernents

(e.g. inversions or translocations) which do not affect sequence copy number changes.

Also, CGH cannot provide any information about the structural changes involved in the gains and losses of DNA regions and how these segments have been translocated or invertecl ( 1 18).

The limitations of CGH can be overcome by a new molecular cytogenetic technique called spectral karyotyping (SKY) if tumor metaphase spread is available

(1 19). SKY can assign individual chromosome a unique color, allowing identification of al1 chromosomes in a single experiment. It has the ability to identiS, bdanced

rearrangements; cryptic translocations and marker chromosomes in tumors where

metaphase spreads are available (1 19). Three major steps involve when doing SKY. 1)

Preparation of metaphase fiom the tumor specimen 2) Hybndization of the chromosomal

preparation to probe cocktail 3) Analyze result by combining fluorescence microscopy,

CCD-imaging and Fourier spectroscopy. The commercially available probe cocktail is

made fiom flow-sorted human chromosomes. By PCR, the flow-sorted chromosomes

were directly labeled with nucleotides conjugated to five different fluorochromes or

combinations thereof. The probe cocktail is then blocked by the addition of an excess of

unlabeled human DNA e~chedfor repetitive sequences (Cot- 1 DNA). This composite

probe set containing al1 24 fluorescently labeled human chromosomes is then hybridized

to the -or metaphase spreads. Finally, an image displaying a spectral karyotype of al1

chromosomes can be visualized through spectral imaging, in which the overlapping

fluorochromes are separated and classified according to the mission spectra produced

fiom each pixel. The limit of sensitivity for metaphase chromosome analysis with

currently available painting probes is between 0.5 Mb- 1.5 Mb ( 1 19).

1.9 Molecular Biology of non-smaii ceii lung carcinoma

To improve the survival of lung cancer patients, advances are needed in early diagnosis and in the development of new therapeutic strategies. These goals require a better understanding of the molecular mechanisms that underlie ce11 transformation and tumor progression. Although incidence of lung cancer is high, detailed cytogenetic analyses are rather limited for this neoplasm compared to the hematological malignancies. In general, performing cytogenetic analysis of solid tumors, such as NSCLC, has proven to be difficult and time consuming. Good quality metaphase preparations are hard to prepare, and the karyotypes obtained can be very complex with many stmctural and numencal changes, including numerous unidentifiable marker chromosomes.

Cytogenetic analysis of short-terrn culture of NSCLC primary tumors and ce11 lines have revealed a number of recurrent chromosornai aberrations. According to these studies, gain of 5p,7 and loss of 3p, 9p, 17p and Y chromosome were the most frequently reported changes (33-37). Balanced translocations are rarely reported in NSCLCs, whereas losses due to missing chromosomes or unbalanceci rearrangements (deletions and derivative chromosomes) are fiequently observed. Allelic losses detected by LOH in more than 600 NSCLCs have identified several regions of consistent chromosomal deletion at 2q, 3p, Sq, 8p, 9p, 1lp, 13q, 17p, 17q, 18q and 22q which potentially contain tumor suppressor genes (38-44). The most intensively studied turnor suppressor genes are located on chromosome 3p, 9p and 17p.

Loss of chromosome 3p

LOH on chromosome 3p was identified in 45985% of the NSCLCs in different studies (97, 98, 99, 43). NSCLC patients with 3p deletion tend to have a poorer prognosis, but not to a statistically signifiant extent. The high incidence of genetic loss indicates the presence of hunor suppressor gene involved in lung cancer on this chromosome. Also, it occurs more frequently on SQCC than ADC, their prevalence being 69% and 35% respectively (43). Deletion of 3p is also observed in a number of other malignancies, including renal ce11 carcinoma, ovarian cancer, utenne ceMcal carcinoma and head and neck cancer (100, 101, 102, 103). Thus, loss of 3p may represent a generalized hlmorigenic event common to various solid tumors, including

NSCLCs. There are three distinct regions on 3p appear to be frequently deleted in lung cancer. They are 3p25, 3821.3 and 3pcen-14 (104, 43,99, 98, 97). One of the tumor suppressor genes that located on 3p25 is von Hippel-Lindau (VHL). This gene is responsible for a dominantly inherited familial cancer syndrome--von Hippel-Lindau disease. Affected individuals are predisposed to a variety of tumors particularly hemangioblastomas of the central nervous system, retina, and renal ce11 carcinomas

(105). However, this gene appears not to be involved in development of lung cancer, since it is rarely mutated in lung cancer ce11 lines (1 OS).

Recent attention has focused on the human FHIT gene at 3p14.2 cloned by Ohta et al (106). FHIT is a member of the histidine triad gene farnily. It spans at least 500 kb, with 10 exons, five of which (exon 5-9) constitute the open reading fiame encoding a

16.8 kD protein (106). This gene resides in a fragile site, FRA3B, which is a common target for cancer cell-specific homozygous deletions (106). FHIT protein shows 69% similarity to a Schizosaccharomyces pombe enzyme, diadenosine tetraphosphate hydrolase, which cleaves the ApsA substrate asymrnetricaily into ATP and AMP. The accumulation of diadenosine tetraphosphate could lead to the stimulation of DNA synthesis and proliferation (1 06). Human FHlT gene encodes a diadenosine triphosphate

(AP3A) hydrolase, in which the biological fbnction is not well known. Loss of the FHIT

gene results in 3p14.2 allele loss is fiequently observed in 38%-54% of NSCLC (107,

108). Allelic deletion at FHIT correlated with tumor histology with 20% of

adenocarcinornas displaying LOH compared to 55% in nonadenocarcinomas (1 07). In

Sozzi et al study, they have showed that lacking Fhit protein expression was higher in the

squamous type compared to adenocarcinoma (87% versus 57%) (1 10). It has been show that the fiequency of LOH at FHIT locus in heavy smoking subjects was significantly higher than non-smoking subjects (109).

Structural abnormalities of the FHIT gene in NSCLC are mainly in the form of homozygous deletions, which may include or exclude coding exons (1 1 1). Northem blot analysis showed reduced or absence of FHlT expression in most lung cancer ce11 lines and tumors, whereas RT-PCR was able to detect the aberrant FHIT transcripts lacking various exons (1 12). Immunohistochernistry showed lack of Fhit protein expression in lung turnors (1 10). There was a concordance between mRNA abnormalities and lack of

Fhit protein expression in lung hunors and ce11 lines (1 13). In Otterson et al study, they obsetved that stable overexpression of pFHIT in HeLa cells with undetectable pFHIT did not alter ce11 morphology, inhibit colony fornation, or inhibit ce11 proliferation in vitro.

Also, overexpression of pFHIT did not lead to altered ce11 cycle kinetics in dividing cells.

The in vivo turnorigenicity of a tumor ce11 line that expressed high levels of recombinant pFHlT was equivalent to that of control transfectants and of parental cells (1 14).

Therefore the role of FHIT as a classic tumor suppressor gene is unclear. Loss of chromosome 9t3

LOH on 4 chromosome occurs in 60%-90% of NSCLC and it is equally common in both SQCC and ADC (82,83,84). Deletion mapping has identified a cntical region on 9p21 where a tumor suppressor gene may be present. nie putative tumor suppressor genes located at 9p2 1 included pl 6 (CDKN~/MTSI/~~6'1'~~~) and p 15

(MTS~(~I5INKdB) (85,86). p 1 6 proteins are cyclin-dependent kinase inhibitors (CDIs) which block the action of cyclin D-dependent kinase phosphorylation of the retinoblastoma gene (Rb) product to induce ce11 cycle arrest. pl6 gene encodes a 16 kD protein which binds to cyclin dependent kinase 4 (CDM)and CDK6 in cornpetition with cyclin D to block CDK activity, and thereby prevents progression fkom G1 to S phase

(85).

INK4B is a gene adjacent to NK4A. They are 44% identical in the first 50 amino acids: 97% in the next 81. INMB is activated by TGFP. The level of mRNA increases

30-fold in cells treated with TGFP and this is reflected by a corresponding decrease in

CDK6-associated kinase activity (87).

Homozygous deletion, mutation, promoter methylaîion in p 16 gene are cornrnonly detected in NSCLC ce11 lines and tumor specimens (88,89). pl6 gene inactivation events that result in loss of protein expression cm be detected by imrnunohistochemistry (89). In

Gazzeri et al study, they showed that 90% of the tumors with p16M"A protein negative expression displayed one of these three alternative genetic alterations (1. Mutations, 2. Hypemethylation, 3. Homozygous deletion) (89). The commonest way for inactivation of pl6 is by homoygous deletion (89). Co-deletion of pl6 and pl5 also occurs in

NSCLC (88). However, mutation and methylation in the pl5 were infrequent.

Therefore, pl6 rather than pl5 plays a critical role as a tumor suppressor gene in NSCLC

(88). Recently another minimal region of loss at 9p21 proximal to the pl6 has been identified in lung cancer, suggesting that tumor suppressor genes other than pl6 are present on 9p (88,90). It has also been reported that there is an inverse correlation in protein expression between Rb and pl6 in NSCLCs (9 1). This would indicate that pathogenesis of some lung cancers could occur by either mutational disruption of Rb protein or by the absence of pl6 inhibitor that functions to keep the Rb hypophosphorylated. Study from Tanaka et al showed that the disruption of the RB pathway plays an important role in tumongenesis in NSCLCs and the increase in cyclin-

Dl expression leads to strong proliferative activity which may over-ride the suppressive effect of pl6 and Rb (92).

Loss of chromosome 17~

Many snidies have demonstrated that allelic loss of 17p is cornmon in NSCLCs.

About 60% of NSCLCs show loss of heterozygosity on chromosome 17p (65, 66, 67).

Allelic loss of 17p is more fkquently observed in squarnous ce11 carcinoma than adenocarchoma subtype (65, 67). One candidate tumor suppressor gene located at l7pl3.l is TPS3. Another independent, commonly deleted region(s) at 17p 13.3 was recently identified (68). LOH at 1îpl3.3 was shown to be even more fiequent than that at 17p 13.1 and it appeared to occur in the absence of p53 mutation ador 17~13.1

deletion (68).

TP53 gene encodes a 53 kD nuclear protein that acts as a transcription factor,

which blocks the progression of cells through the ce11 cycle in late GI phase, and can

trigger apoptosis (69). The genomic sequence is compnsed of 1 1 exons, which spanning

over 20 kb. The p53 protein contains: (1) an acidic domain at its N-terminal, which is

important for trans-activation of genes regulated by p53. (2) n central core domain,

which interacts with DNA. (3) a nuclear localization and tetramerkation dornain at the

C-terminal(69).

When normal cells is subjected to DNA-damaging doses of radiation, they will

arrest at the Gl/S-phase in the ce11 cycle. However, cells with mutant p53 fail to arrest

and proceed through S-phase. Noxmally, wild type p53 cm induce CDK inhibitor p2~WAF' gene. p2 1 WAF1 inhibits cyclin/CDK complex which leaves Rb in its

hypophosphorylation state. Hypophosphorylated Rb thereafter binds E2F and prevents

WAFI the DNA synthesis. Therefore, the ce11 cycle is blocked before S phase (25). p2 1 can also suppress DNA synthesis by binding directly to proliferating ce11 nuclear antigen

(PCNA is needed to activate DNA polymerase 6, the principal replicative DNA

polymerase involved in DNA synthesis) (70). It has also been shown that p2 1WA" can be

elevated by p53-independent mechanisms (71). DNA synthesis can also be blocked by p53 protein upregulation of the transcription of GADD45 (growth mest DNA damage

protein). Gadd45 binds to PCNA (similar to p2 1WAF') and inhibits DNA synthesis. (72). TP.53 can also induce apoptosis in response to DNA damage. In a normal cell,

there is a dynamic equilibrium between the opposite acting proteins in apoptosis, bcl-2

and bax. When there is a DNA damage, p53 levels rise and enhance the transcription of

BRY (induce apoptosis) and repressing the transcription of BCL-2 (inhibit apoptosis), and

drive cells toward apoptosis (25).

According to vanous studies, TP53 mutation occurs in > 50% of NSCLC (30,49).

Frequent mechanism of inactivation of the TP53 gene has been shown to occur by loss of

one copy of the gene and mutations in the remaining allele. In lung cancer, the majority

of mutations are missense mutations, which generally leads to mutant p53 protein

formation. Normal p53 has a short half-life but mutant p53 are much more stable making

them detectable in imrnunohistochemical assays (48). These proteins have transforming

properties by binding and inactivating available wild-type p53 protein. Other mutations

such as deletions, fiameshifis, insertions and splicing mutations have also been reported

(63,73,74). Missense mutations occur throughout the open reading fiame, although they

are most fiequent in the central exon 5 through 8 (3 1). There is a difference between the

TP53 mutational spectra between smokers and non-smokers. The prevalent type of point

mutation in smokers is G:C to T:A transverîons and it is thought to be related to adducts of benzo(a)pyrene from cigarette smoking (76,77). TP53 mutations in a particular series of non-smoking lung cancer subjects (fkom atornic-bomb survivors) were mostly G:C to

A:T transitions (75). The prognostic significance of Tl33 mutations in lung cancer is controvenial.

Several reports demonstrated adverse prognostic impact (49,80), but other studies found

no association (78) or even a su~valadvantage in those patients with high p53

oncoprotein expression (8 1).

RAS Familv

The MSgene family consists of three genes, NRAS, KRAS, and HRPS, which encode related 21 kD membrane bound proteins. Ras proteins have a high homology to large G proteins. Ras is a small GDP/GTP binding protein. Growth stimuli cause the substitution of GTP for GDP (activated Ras) and the intrinsic GTPase activity (with GTPase activating protein, GAP) which catalyzes the conversion of active Ras (Ras-GTP) back to the inactive form (Ras-GDP)(25). Ras proteins have a key role in signal transduction by linking tyrosine kinases to downstrearn senne/thereonine kinases, such as RAF, and mitogen-activated protein kinases. RAS gene activation usually occurs by point mutations at codon 12, 1 3, and 6 1 : the Ras mutants are defective in GTPase activity and thus are "locked" into the growth stimulatory GTP-bound form.

In human tumors HRAS mutations are rare, NRAS mutations are primaily associated with hemopoietic malignancies, and KRAS mutations are most frequently found in adenocarcinornas, particularly those of the pancreas and colon (32). In ce11 lines and primary resected NSCLCs, majonty of the mutations is restncted to the KRAS gene, especially at codon 12 (27,28). G:C to T:A transversions were the predominant mutation at codon 12 of KRAS in himors caused by smoking. (27,46, 109) Strong association is

found between smoking and the presence of KRAS mutation (47, 109). The presence of

KRAS codon 12 mutation is associated with shortened disease-fiee and overall survival in

resected adenocarcinorna of the lung (29, 93). A separate repoit demonstrated that

overexpression of the Ras protein has a similar effect on prognosis as KRAS mutations

(50)-

MYC Familv

The MYC family of cellular oncogenes includes three highly related genes (MYC,

LMYC and NMYC) which encode cell cycle specific DNA-binding proteins for transcriptional regulation. MYC was identified as a transfoning gene in the

=eloc -ytomatosis viruses (retrovituses that cause myeloid leukemias and O ther neoplasms in infected chickens). NMYC and LMYC were first detected in neuroblastomas and small ce11 lung carcinomas, respectively. The general structure for the MYC protein contains a trans-activating domain at the N-terminus, which mediates transcriptional activity for certain genes. At the C-terminus, there is a nuclear localization signal, helix-loop-helix domain and leucine zipper motif (69). MYC proteins fonn homodimers or heterodimers using the helix-loophelix and leucine zipper domains, and thereafter bind DNA and regulate transcription (69). MYC can heterodimerize with proteins such as MAX, MAD, and MX1 1. MAX can bind MYC to repress the transcriptional activation of MYC gene, whereas MAD and MX1 1 can bind with MAX and thereby release MYC to function as a transcnptional activator. The general mechanism of activation of MYC in lung cancer is by gene amplification.

Approximately 10% of the tumors fiom patients with non-small ce11 lung cancer have

MYC family DNA amplification (94). About 8% of the NSCLC patients have MYC gene

amplification. In contrast, MYC mRNA expression is detected more commonly than

MYC farnily DNA amplification in NSCLC patients (94,95). About 50% of the patients

had detectable MYC protein in their non-small ce11 lung cancer (94, 96). In Volm et al

study, they have showed that MYC is not a prognostic indicator for the su~valtime of

patients with squamous ce11 lung carcinomas (96). In this study, MYC FISH analysis is used to check whether MYC is frequently amplified in NSCLC and the possible mechanism(s) for increase in MYC copy number in this hunor.

Microsatellite instability in non small cell lung carcincoma

There are eight studies al1 together on microsatellite instability in 540 non-small ce11 lung carcinoma patients analyzing 92 loci (51). In non-mal1 ce11 lung carcinomas, microsatellite instability only affects few loci in contrast to widespread changes in

HNPCC and some sporadic colorectal carcinomas (5 1, 52). Loci at chromosome 3p and

5q are more susceptible to alterations (review in 5 1). There was no significant difference in the frequency of RER' between the adenocarcinorna and the squarnous-ce11 carcinoma subtypes (53). In these 540 patients, 12% (641540) of the patients showing a single unstable locus. ûnly eight percent (45/540) of the patients was displaying microsatellite instability in more than one locus. A recent study has investigated for both LOH and microsatellite instability with 34 marken at or linked to hMM (3p2 l), hMSHi (2p16),

hMSH3 (5q 1 1q 13) LMSH6 (2p 16), hPMSl(2q23), and hPMS2 (7~22)loci (5 1). LOH of

hMLHi and IiMSH3 was observed in 54.8% and 41.9%, respectively. There was only

low level of LOH detected in other chromosomes 2p 16, 2q3 Lq33 and 7p 15-pter (1 0-

11%). Only one tumor (3.2%, 1/31) was found unstable at more than one locus.

Mutational analysis was done for patients showing LOH of hMLHl and hMSH3 loci.

Four DNA variants (by SSCP and direct sequencing) were observed. However, al1 these

variant sequences were present in both tumoral and normal DNA fiom more than one

NSCLC patients and also in blood DNA fkom normal controls suggesting that these mutations represent common polymorphisms rather than pathogenic changes. From the low average frequency of microsatellite instability, it seems that the coirection of replication mors plays only a moderate role in NSCLC hunorigenesis (review in 5 1). Conventional and molecular cytogenetic analyses have dernonstrated that genetic alterations are commonly occurring in lung carcinoma. Alterations include both oncogenic activation and the inactivation of tumor suppressor genes by mutation, allelic deletion, and amplification. Studies of genetic alterations of various types of tumors in different organs suggest that different oncogenes and tumor suppressor genes may be associated with specific tumor types and these changes may have direct relationship with the pathogenesis of the tumon.

Objective:

To clariQ and confirm the genetic alterations in non-small ceil lung carcinoma by comparative genomic hybridization (CGH), spectral karyotyping (SKY) and FISH. Materials and Methods

A. Cell lines and tumor tissues

Twenty-two non-small ce11 lung carcinoma ce11 lines and primary turnors were analyzed by CGH. Samples including 9 squarnous cell carcinomas, 9 adenocarcinornas,

2 adenosquamous carcinomas and 2 large ce11 undi fférentiated carcinomas. Ce11 lines

Hl 57, H226, H520, H 1264, H358, H 125 and H661 were obtained fiom Amencan Type

Culture Collection (Rockville, MD). Other cell lines were established in Our lab.

Primary lung tumor and normal lung tissue specimens were collected fiom patients undergoing surgical resection for primary NSCLC at Montreal General Hospital between 1989 and 1995. Tissues were snap fiozen in liquid nitrogen and stored at -80'~ for subsequent isolation of DNA. Histological classifications of ce11 lines and primary tumors are summarized in Table 4.

B. Isolation of genomic DNA Rom tissues and cell lines

Lung cancer ce11 lines were cultured in 100-mm dishes until they becarne confluent. Two plates of cells were trypsinized and digested ovemight at 55'~with lOOul of 2Omg/ml proteinase K in 500 ul RSB buffer and 1% sodium dodecyl sulfate

(SDS). DNA extraction was done by adding an equal volume of salt-saturated phenol and 1/10 volume of SM NaCl. Samples were gently rnixed and centrifûged at 10000 rpm for 5 minutes. Afier centrifugation, Save the upper aqueous layer (containing DNA) by transfdng to a new tube. Extraction was repeated once by using equal volume of SS- phenoVchlorofonn (1 :1) and once using chlorofonn only. Purified DNA was precipitated using an equal volume of isopropyl alcohol. The DNA was spooled and air dry in a new eppendorf tube. DNA was resuspended in TE buffer ovemight at 4'~.The amount of

DNA was estimated by rneasuring the absorbance at 260x1111wavelength. The purified

DNA was stored at - 20'~ before labeling.

Similarly, primary tumor DNA was extracted fiom 0.5 g of normal and tumor tissues. Tissues were immersed in liquid nitrogen and cut into small pieces with a scisson. DNA fiom tissues was extracted as described above for cultured cells except twice amount of proteinase K was used.

C. Normal Metaphase Preparations

1. Normal Lymphocytes Isolation and Culture

Thirty milliliter of blood was collected by a sodium heparin vacutainer fiom a healthy donor. Whole blood was diluted with lXPBS in a 3:8 ratio (i.e. 3ml of blood mixed with 5 ml of PBS). Eight milliliter of diluted blood was layered over 3 ml of

Ficoll-Paque and was centrifbged at 1400 rpm for 20 minutes at room temperature.

Differential migration during centrifugation results in the formation of layers containing different ce11 types. After centrifugation, the erythrocytes and granulocytes sedimenteci to the bottom of the tube and the purified lymphocytes could be collected fkom the interface beîween the plasma and the Ficoll-Paque. Purified lymphocytes were subjected to a lXPBS wash and centrifuged at 1 lOûrpm for 10 minutes at 4'~. After the rernoval of PBS, lymphocytes were seeded at 4 x 104 cells/ml in the supplemented RPMIRO%

FBS medium. Cells were allowed to grow for 60-70 hours in a 37'~,5% COz incubator before they were harvested.

2. Preparation of metaphase chromosome spreads frorn lymphocytes

Metaphase cells were obtained by treating cultures with colcemid. Colcemid is a colchicine analog that dimpts the spinde-fiber complex by interfaing with microtubule formation. A final concentration of O.lug/ml colcemid (GibcoBRL) was added to lymphocyte culture and incubated at 37'~for 30 minutes. Then cells were collecied by centrifugation at 1500 rpm for 10 minutes. After the mitotic arrest, cells were treated with wami (37'~)0.065M KCl solution for 10-20 minutes in a 37'~incubator. This hypotonie "shock" increases the cellular volume. Just before centrifugation at 1 500 rpm for 10 minutes, 5 drops of fixative was added. This treatment serves to reduce the pH of the cells gradually to precondition them for the following fixation steps. Mer centrifugation, cells were fixed by dropwise addition of 10 ml of freshly prepared ice- cold fixative and incubated for 10 minutes. The fixation procedure were repeated two more times and cells were stored ovemight at 4'~before preparation of the metaphase slides. Lymphocytes suspension was dropped ont0 a precleaned microscope slide (with

ethanol and then distilled water). The slides were dehydrated, 5 minutes each, through

ethanol series of 70%, 90%, and 100%. Air-dry slides cm be stored for a few days to

several weeks at room temperature. For long term storage, slides should be stored in a

clean, dry container in dark fiozen at -80°c.

D. Comparative Genomic Hybridization

1. DNA Labeline bv Nick Translation

Two microgram of normal and tumor/cell line DNA was labeled with digoxigenin

and biotin, respectively. Tumor DNA was mixed with 10 ul of 1OX Biotin dNTPs, 10

units of E.coli DNA polymerase 1 (Pharmacia Biotech) and 0.075U of DNaseI

(Pharmacia Biotech). Stock DNase I was diluted with DNase I dilution buffer.

Sirnilarly, normal reference DNA was labeled with 5 ul of 20X digoxigenin dNTP, 1Oul of 10 X dipiogenin incubation buffer, 10 units of E.coli DNA polymerase I (Pharmacia

Biotech) and 0.075U of DNaseI (Phmacia Biotech). Final reaction volume was adjusted to 100 ul by autoclaved distilled water. The reaction was carrieci out in 16'~ waterbath for one to one and a haif-hour. The reaction time and the amount of DNA polymerase I/DNase 1 used were adjusted to obtain a probe fiagrnent size ranging from

500-2000 bp. To check the fragment size, 10 ul of the labeled DNA was subjected to a

1% nondenaturing agarose gel electrophoresis stained with ethidium bromide in 1X TAE buffer. Reaction was stopped by adding 1/10 volume of 300 mM EDTA, pH 8.0. Labeled probes were precipitated for 20 minutes at -80'~ by adding somicated salmon spm(50 ug of spem per ug of labeled probe); 15 ul of 3 M sodium acetate and 350 ul of 100% ethanol. Labeled probe was centrifuged at 15000 rpm for 20 minutes and washed with 350 ul of 70% ethanol. Afier washing, probe was allowed to air dry and resuspended in autoclaved distilled water to give a final concentration of 10 ng/ul.

2. Metaphase slides pretreatment

Two to three weeks aged slides were denatured in 70% fonnamide/ZXSCC, pH

7.0 for 2.5 minutes at 75'~. After denaturation, slides were dehydrated in a graded series of (70%, 90%, 100%) ethanol and air dry at room temperature. Air-dried slides were digested with O. 1ug/ml proteinase K in 1 Oûml proteinase K buffet at room temperature for 7.5 minutes, and slides were dehydrated again through ethanol senes.

3. Hvbridization

Genomic DNA probes were hybridized ont0 the pretreated normal lymphocyte metaphase slide. One hundred fi@ nanogram digoixgenin labeled normal DNA, 150 ng of biotin labeled tumor DNA and 10 ug of unlabeled human Cot-1 DNA (GIBCO)were precipitated for 20 minutes at -80'~with the addition of 15 ul of 3M sodium acetate and

350 ul of 100% ethanol. Probe was centrifùged at 15000 rpm for 20 minutes and washed with 350 ul of 70% ethanol. Afier washing, probe was dissolved in 30 ul of Hybrisol VI1

(Oncor Inc., Gaithersburg, MD). The probe mixture was heat denatured for 10 minutes at 75'~and pre-annealed for an hout at 37'~incubator. The pre-annealecl probe was applied ont0 a pretreated nomal metaphase slide using a 22 X 40 mm2glas coverslip.

Rubber cement was applied to seal the coverslip onto the slide. Slides were incubated in a humidified chamber at 37'~for three days.

4. Post hybridization wash and detection

Afkr hybridization, slides were washed three times, 5 minutes each, in 50 ml of wash A solution following by three washes in wash B solution at 45'~. Slides were blocked with 40ul of Block 1 solution and 40 ul of TNB solution for 30 minutes at 37'~.

After blocking, signals were detected by 40ul of fluorescein-labeled avidin (ONCOR) and anti-dig-rhodamine, fab fragments (Boehnnger) mixture (10% of the total volume of the detection mixture should be made of rhodamine anti-dig diluted in fluorescein-labeled avidin) and incubated at 37'~for 30-45 minutes. Slides were washed three times, 5 minutes each, at 45'~wash C solution and counterstained with 20 ul DAPVAntifade

(ONCOR).

5. Digital Image Analysis for CGH

At lest seven images per case were captured using a Nikon Microphot microscope connected to a Photometrics (Tucson, AZ) SenSys KAF 1400 charge-coupled device (CCD)camera. The QuipXL Genetics Workstation (Vysis Inc., Downers Grove, IL) was used for the image anaiysis. The image analyzing software calculated an average

ratio of FITCxhodarnine and expressed it as a green:red ratio for each metaphase. A 95%

confidence level was also caiculated. Gains or losses of chromosomal regions were

detected when the fluorescent intensity ratio was deviated from 1. A ratio greater than

1.2 was considered a gain of chromosomal material whereas ratios les then 0.8

constituted a loss. When the profile exceeded a ratio of 1.5, the region was considered to

be highly amplified. Analysis was excluded fiom the following regions: centromere,

acrocentric p-anns, teleomere and heterochromatic nch areas including lq 12, 9q 12,

16q12, 19 cen (1 7). Those regions cannot be reliably evaluated by CGH as they are

being blocked to various extents by the unlabeled Cot- 1 DNA in the hybridization (1 8).

E. MYC Fluorescence In Siru Hybridiraion

The procedure for FISH is very similar to CGH. Twelve ce11 lines were subjected

to direct MYC FISH analysis. Cell lines were grew to 70% confluency before harvesting

the interphase nuclei/metaphases. Metaphase slides obtained fkom ce11 lines were used

for FISH analysis. Ce11 lines were harvested the same way as lymphocytes except a

longer incubation penod (1 hr-2hr) was needed for the colcemid treatment. Cells were

trypsinized and washed with 1X PBS before performing the hypotonie and fixation steps.

Specimen slides were denatured at 73'~for 2 min in 70% fomamide/2XSCC, pH 7.0 and proteinase K digested before hybridization. Ten microliter of digoxigenin labeled

MYC DNA probe (ONCOR)was denatured at 73'~ for 5 minutes and then incubated at

37'~for an hou. before hybndization. Hybrîdization was done under sealed 22 X 22 mm2 coverslip ovemight at 37'~in a moist chamber. Wash A and wash B were

performed following hybridization as mentioned in the CGH procedure. Slide was

blocked by 40 ul of TNB buffer for 30 minutes at 37'~.A three-layer detection system

was used for signal amplification. For the first layer detection, 40ul of 1 ug/ml mouse

anti-digoxigenin antibody was incubated with the slide at 37'~ for 30 minutes. Excess

antibody was nnsed off by immersing the slide into 50 ml wash C solution three times

(five minutes each) at 45'~. Just before incubation with 40 ul of 2ug/ml Dig anti-mouse

IgG, the slide was incubated with 40 ul of MB at room temperature for 5 minutes.

Excess antibody was again rinsed off by immersing the slide into 50 ml wash C solution

three times (five minutes each) at 45'~. After washing, slide was blocked with 40 ul of

TNB at room temperature for 10 minutes and then detected by 2ug/ml anti-dig

rhodamine. AAer detection, slides were washed three times (5 minutes each) in wash C

solution and counterstained with 20 ul DAPVAntifade (ONCOR). Same CGH digital analysis system was used to capture FISH images. At least 200 cells were scored for each ce11 lines.

F. Spectral Karyotyping

Slides for SKY were fkeshly prepared using chromosome suspensions fiom MGH-7 ce11 line that was stored in fixative at -20'~. On average 5 metaphases were analyzed by

SKY. The commercially available SKY TM Kit (ASI, Carlsbad, CA) was used for spectral karyotyping. The slide treatment, posthybridization detection and washes were done according to the manufacturer's instruction. 1. Metaphase slides pretreatment for SKY

Metaphase slides were pretreated in 37'~pepsin solution for 5 min and then washed

two times in lXPBS at room temperature and once in 0.05M MgClZ/PBS at room

temperature. Following the washes, slides were incubated in solution of 1 %

fonnaldehyde in PBS/MgC12 for 10 minutes following a 5 minutes wash in PBS/MgC12 at

room temperature. Slides were dehydrated in a graded senes of (7056, 90%, 100%)

ethanol and air dry at room temperature.

2. Chromosome and probe denaturation

Specimen slides were denatured at 72'~for 2 min in 70% fomamide/2XSCC, pH

7.0. Slides were dehydrated in a graded series of (70%, 90%, 100%) ethanol and air dry at room temperature. Ten microliter of SKY probe was denatured at 75'~for 7 minutes and then incubated at 37'~for 1 hour before hybridization. Hybndization was done under sealed 30 mm coverslip for two nights in a 37'~moist chamber.

3. Detection

Slides were washed three times in 50% formamide in ZXSCC, pH 7.0 and two times in IXSCC,pH 7.0 at 45'~.Slides were blocked with 80 ul of blocking reagent (supplied by ASI) for 30 minutes at 37'~incubator. Signals were detected by adding 80 ul of buffer 1 (supplied by ASI) and incubated at 37'~for 45 minutes. Slides were washed three times (5 minutes each) in 4XSSClO.l% Tween 20 at 45'~. Then eighty microliter of bufk II (supplied by ASI) was added to the slides and incubated at 37'~ for 45 minutes. The 4XSSC/O.l% Tween 20 wash was repeated once more. After washing, slides were counterstained with 20 ul D AP IlAnti fade (supplied by ASI).

4. Digital Image Anaiysis for SKY

Image acquisition was performed using a SD 200 spectral bio-imaging system

(AS1 Ltd, Migdalhaemek, Israel) attached to a Zeiss microscope (Axioplan-2) using a custom designed filter set (Chroma technology, Brattleboro, VT). The samples were illuminated with a Xenon lamp (OptiQuip 77011600). The emitted light after passing through the filter set was sent into the Sagnac interferorneter in the optical head, and imaged with a cooled CCD camera. An interferognun was generated for each pixel based on the optical path difference of light, which depends on the light emitted fiom the chromosome painting probes. The spectral information was further analyzed by Fourier transformation and converted to display or classification color. The images were stored in a cornputer for Meranalysis using the SKYVIEW (ASI, Carlsbad CA) software. 4.1 CGH analysis for ceU lines and primary tumors

To evaluate the presence of genetic aberrations in non-small ce11 lung carcinoma,

CGH was performed on 14 ce11 lines and 8 primary tumors. Figure 5A and 5B represents an example of the fluorescence image of the MGH7 ce11 line. Figure 5C shows the cornparison of red-to-green fluorescence intensities dong the metaphase chromosomes in the MGH7 ce11 line.

CGH data for the MGH7 ce11 line shows an over-representation of whole or part of chromosome arm: 1 p 13, 1 q2 1.3, 1 q3 1-4 1, 2p 13-pter, 3q2 1-q28, 8q22.2-q24.2, 1 1 q23-q24, l2q23-q24.3, 15q2 1.3-q22, 1 7q2 1 -qter, 19q 13.3 and under-representation of 1 7p 1 1.2, 1 8q2 1 - q22. DNA amplification sites included 3q26-q28 and Sp. Figure SA and B. Digital image of a CGH-experimentfor MGH7. Hybridization with DNA from a squamous cell carcinoma cell line MGH7 (in green) and normal male reference DNA (in red) to normal metaphase chromosomes. Chromosomes were counterstained with DAPI. (in blue) C. Chromosornal regionr thut were over-represented in the tumor are visualized as a predominantly green color, whereas regions that were under-represented in the turnor are seen as predominantly red color. Small changes on chromosomes are denoted by arrows. The bfack line in the middle of the CGH ratio projies is the baseline ratio (1.0). The red lhe to the fefl and the green line to the right indicute the cut-of values of 0.8 and 1.2 respectively. men the profile exceeded o ratio 1.5, the region was considered to be highly amplified. DNA copy number changes were detected in al1 22 cases of non-small ceIl lung

cacinomas. The mean number of chromosornal abnomalities per specimen was 1 1.3 (range,

1-26). The total number of imbalances and DNA sequence copy number changes in each

specimen was summarized in Table 4. Gains of genetic material predominated over losses

with a ratio 3: 1. Al1 chromosomes were involved in imbalances. A schematic representation

of the genetic imbalances detected by CGH analysis in each case is shown in figure 6. Figure

7 shows genetic imbalances detected by CGH in al1 14 ce11 lines.

Figure 6. Gains und losses of DNA sequences in 14 non-small cell hgcarcinomas cell lines and 8 primary tirmors. Gains are shown as vertical line on the right side of the chrommorne and fosses are shown us vertical line on the Ieft. High Ievel gains/umplifcutions (gains h 1.5) are shown as empîy box on the right side of the ideogram. 8E%I - Ar-am@. mH - - Table 4: Histological Subtype and Copy Number imbalances Detected by Comparative Cenomic Hybridization in 22 Non-Small Cell Lung Carcinomas

Cell lines Gains (21.2) '

imbalances 17pll.2. 18q21q22 q24.2, I i q23-gi4'4,I 2q23'-q24:3, I sqiI Xq22, i 7q2i qt~t.I 9q I 3.3 2qUI2q32, Sp, 7q22q35.8q2 1. I qtcr. 1 I p.

14q24.2qter 4q33qter. 9q3 1 qtcr, 19p 13.2 9~23-p24,9q21,Xp 12; ~OC&~I~.U~O~I~~~~J- MGH-8 ADC 16 8p, 9p 1 2,9q2 I 322, l q2343,2p/2p23, 3q 13.1 413.3, Sp, Sq3 1,8q 13qter/8q23-24.2, I3q. 19q13.1 IOq21.3qter. 12q 14-24.3, I Sq26, tOq 12413.2, Xq22q27 MGH-13 ADC 13 6qlSq24,7qI 1.2, 11p11.2. lq2243,4p14-16, Sp, 7qI I .2,8ql3qter. 1 lq23.3, IJq13qter, 9p 12-p2I t 5q2 1qtcr, 17q25 MGH-24 ADC 13 6q, 13q, 18q, XP Iq23-24,2p22-23,3q24q28, 4q3 1.3-32, Sp/Spl3-pier. 7/7p21,7q31.1-31.2. Bp21,8q22.2qtcr, I lq23 H-358 ADC I6 6q 15q24, I Ocenq22, Iq2 I q25,4p13-ptcr, Sp, 8q2 1. I qtcr, 9q22q34.2, I l q23, 14q2 1 - 1 lp12-pl3, 18q12, 19pl3.1 qter, 15q24-25, 16q2Iqtcr. 20p12,2Oq12q13.3 RVH6849 ADC --9 1 3p12.5ql2.2q 14.6q. I p36. I, lqt I-22.4~15.I-pter. 5p. 6p2 I. I -p21.3,7pq 1 1.2.7q34- 9p, 9q2 1422, I l p 13-ptcr, q36,8qAq2l.lq24.2, I Iq/l lq13q24, l4q I 2q24. 13q2lqU. 18 lSq21.3q24, 17q, 19q13.l,2Oql 1.2-q13.1 H-125 ADSQ 15 lp32.2-pter, 9p. IOccn-pl3, 1 p2 I -p22,3p, ken-pl4.5~.6p2 I .3q26/6cen-p21.1. 1 7q. 18q. 19~.22q 6q25q26,7p 12-15,7q2 I q31.2, Xq2 1 qter MGH-30 A DSQ 9 6cenq24,18q2 1-22 5p/SplS, 8p, 8q21.3q24.4, 12p, 13q3 1433, Xp I1.4-ptcr, Xq MGH-4 LCC 17 IqZ, 2p12-pter. 5p, 7p2 1 q34.8q23-24.28q24.1, IOcen-pl3, I Iq, 12~12-pter,l3q32, 15q22.2qtcr, 16ccn-PI 3. I, 164 12,2q23,17q21 qtcr, 18q. 12422, - 19~I 3.i. 20ccnq 13.2, ~ccn-~22.I - H661 LCC I I 4q21 qter, 5q 14, 2q3 1-33,5p, &cn-p22.8q24,2Oq I Zqter. Xq22q27 Primary tumor

lp13-p31, 3p, lp36, lq21q23, lq32,2pql3,3q13.2q26.3, Scen-pl4, 4p 1 SSqttr, 5q l2q14,6ccn- 5q3 1.2-q35. 6p, 14q2l qter, 15q22,16p, 1 7q, lap 1 1.2,19p, 19cm- q22, 13q14q33, 18q21q22 q13.2,2OpqI3.1, 2lq2I. 22q, Xp22.I 5p-q I lA2,8q212q24.2, 12pq 1 2 pFpp= 8q22q24.2, 15q22q25, l6q2 1qter, 19q, 20q, 22q 13 SQCC, squnmous cell cminoma; ADC, Adenoc minoma; ADSQ, Adenosquamsous cell carcinoma; LCC, Large cell undifferentiated carcinoma. b High-level amplifications are bold. Chromosomal regions involved in gains and amplifications

The most frequently gained chromosomal arm was 5p (72.7%; 16/22). In four cases

(MGH8, MGH24, MGH30, MGH'I), 5p gain was present as hi&-level amplification, with

the minimal common region mapped to 5p 15. 0thFrequently gained chromosomes

included 8q (68.1 %; 15/22), 1Sq22-qter (50%; 1 1/22); lq2 1.3-4 1,20q (45.5%; 10122); 3q,

1 lq and 19q (36.4%; 8/22). DNA amplifications were detected in ten cases at eight different chromosomes. Subchromosomal regions amplification were detected in 8q24 (4 cases), 5p (4 cases), 3q24-27 (3 cases), 2p23-24,6cen-p2 1.1,6q26, 7p2 1,7q3 1, 1 1q 1 3-qter, 20q 12- 13.2 ( 1 case),

Chromosomal regions involved in losses

The most fiequent loss involved chromosome 9 and 18q (36.4%; 8/22). The minimal deleted region on 1 8q was 18q2 1-22 except for one case (8q 12). Other common losses were found in 6q 16-22 (27.3%; 6/22), 5q 12-32, 13q and 19p (1 8.1%; 4/22).

Chromosomal changes detected in primary tumors

Among prirnary tumors, the most cornmon gains were at 5p and 8q, both seen in five cases (63%). DNA gain was also detected in 19cen-q13.3 in four cases (50%).

Amplification was only found in one primary -or (L45) in which 8q24 region was involved. Figure 8 is a schematic representation of the genetic imbalances detected by CGH analysis in 8 primary tumors. Figure 8. Gains and iosses of DNA sequenees in 8 nonmal1 celi Iung carcinoma primary tumors. Gains are shown as vertical line on the right side of the chromosome and losses are shown as vertical iine on the lef High level gains/amplijcations (gains 2 1.5) are shown as empty box on the right side of the ideogram.

Cornparison of the CGH results for squarnous cell carcinoma and adenocarcinoma

The two major histological subtypes of NSCLC are squarnous ce11 carcinoma (SQCC) and adenocarcinoma (ADC) each representing approximatel y 40% of NSCLC. A surnmary of genetic changes of the adenocarcinornas and squamous ce11 carcinomas is displayed in figure 9. In squamous ce11 carcinoma, high-level amplifications were detected three times in

3q24q27 and once in 5p, 8q24 and 20q12-13.2. The most huent gain involved

chromosome 3q (55%), with the minimal region at 3q24-27. Other common gains involved

5p and 8q23-24 (44%). The most common underrepresented site was the short am of

chromosome 9 (33%).

In adenocarcinorna, high-level amplifications were detected two times in 5p, two

times in 8q23-qter and once in 2p23-24,7p2 1,7q3 1 and 1 Iq. The most frequently occumng aberrations were gained in 5p and 8q (88%). Other recurrent gains of genetic material were detected in lq22-q3 1, 15q23-25 (66%) and 20q (55%). The most frequent losses, detected in

5 out of 9 tumors, involved 6q 16-22 region. Other recurrent losses involved 13q2 1, 1 8q

(44%) and 9q (33%).

Adenocarcinornas carried more DNA overrepresentation at 1 q22-32.2, 1Sq, 20q and deletions on chromosome 6q, 13q and 18q. In contrast, squamous ce11 carcinomas showed more frequent gain in chromosome 3q as compare to adenocarcinornas. Figure 9. Summary of gains and fosses of DNA sequences in 9 squamous cefl carcinomas (5 ceil Iines and 4 primary tumors) and 9 adenocarcinornas (5 celi lines and 4 primary tumors). Gains are shown as vertical line on the righr side of the chromosome and losses are shown as vertical line on the leji. High level gains/amplifcations (gains 2 1.5) are shown as empty box on the right side of the ideogrnm. The first chromosome in pair represents squamous ce1l carcinoma (SQCC)and the second adenocarcinorna (ADC).

4.2 MCanalysis for NSCLC ce11 iines

Since overrepresentation of 8q was fhquently detected in these sarnples, MYC FISH analysis was done to venfy how MYC (mapped to 8q24) contributing to the gain of chromosome 8q. MYC aneuploidy was observed in al1 tested cell lines. At least 200 cells were scored in each ce11 line. Summary of MYC FISH result is shown in Table 5. Table 5 Summary of MYC FISH results in 12 NSCLC ceii lines

Ccll Histologic Sigmls per nucleus (%) lines types 2 3 4 5 6 r6 Modal numbcr Ploidy Chromosome Modal CGH profile MYC signals index count b number on 8q MGH7+ SQCC O O O 93 7 O 5 1.62 73 3n+ +8q22.2q24.2 MGH8 ADC 6 6 78 8 2 O 4 1.74 ND 31t+ +8q 13-tat8q24.2 MGH13' A-ûC O O 2.5 5 89 3.5 6 1.94 ND 4n- +8q 13-qter MGH2QC ADC O 5 8 79.5 7.5 O 5 1.77 ND 4n- +8q22.2-qter MGWO* ADSQ O O 7 83 IO O 5 1.77 ND 4n- +8q2 1.3-q24.4 MGH4* LCC LOO High level 1 A0 ND 3n- +8q23-4.218q24.1 amplification H226 SQCC 3 94.5 2.5 O O O 3 ND 73 3n+ No change H520 SQCC 9 78 8 5 O O 3 ND 58 3n- Nochangc HI57 SQCC 3 87.5 7 2 0.5 O 3 ND 54 2n- +8q2 1.1 -qtcr H358 ADC O O 2 5 88 5 6 ND 70 3n+ +8qZ 1. l -qtcr Hl25 ADSQ 10 85 5 O O O 3 ND 65 3n- No change H66 1 LCC O O 0.5 0.5 95.5 3.5 6 ND 60 3n- +8q24 a (+)asterisk denoted ce11 line which has increase copy of MYC signals relative to its ploidy index. modal nurnber is the most common chromosome number in a tumor ce11 population. (Zn-): hypodiploid, 35-45; (3~):hypotriploid, 58-68; (3n+): hypertriploid, 70-80; (4n-): hypotetrztploid, 8 1-9 1. C High-level amplifications are bold. ND,not done. When considering ploidy level together with the MYC result, five out of six ce11 lines showed an increase in copy number of MYC signals relative to the ploidy level of the ce11 line

(indicated by asterisk * in table 5). Only one ce11 line (MGH4) showed highly amplified

MYC signals, shown in figure IOA. In the metaphase spread of the MGH4 ce11 line (figure

1OB), a small marker chromosome that contained MYC signals was identified. This small chromosome may represent an early stage of the homogenous staining region (hsr). Although only low level of MYC amplification was detected in most of the ce11 lines, MYC signals were not only localized to normal chromosome 8, but also translocated to various marker chromosomes (fig 1OC, 1OD, 1OE). FISH identified formation of isochromosome 8q in two ce11 lines MGH 13 (fig 1OF) and H358. Figure 10. Fluorescence in situ hybriditarion of MYC probe to interphase/metaphase(s). The Dig-labeled probe is fluoresceimted and appears red under microscope; the chromosomes are counterstained with DAPI. Only representative area of metaphase is shown. Arrows denoted normal chromosome 8 and arrowheads mark the marker chromosome(s) where MYC is tronslocated (A) MGH4 nucleus (B) MGH4 (C)MGH7 (D) MGH30 (E) MGH8 (F) MGH13 metaphase. 4.3 Conventional G-banding anafysis

Conventional G-banded analysis of MGH7 (SQCC) revealed a very complex pattern of chromosomal aberration involving many numerical and structural abnomalities with 19 different unidentified marker chromosomes. The chromosome number in ranged fiom 70-73 in 10 counted cells. The karyotype was hypertriploid. G-banding could not identifi a majority of these changes. Figure 1 1 shows representative karyotype fiom MGH-7.

Markers

Figure II. Representative karyotype of a G-bunded metaphase ceil jiom MGH-7 ce11 fine. Structural reurrangements are denoted by arrows. A549 (ADC) ce11 line showed less structural and numerical changes as compare to

MGH7 ce11 line. The karyotype was hypotriploid. The chromosome number varied in range

from 60-63 in 10 counted cells. Four consistent markers and a dicentric chromosome 6 were

observed. The origins of these marker chromosomes were undetemined. G-banding could

not identiQ a majority of these changes. Figure 12 shows representative karyotype from

A549.

Figure 12. Representutive karyotype of a G-banded metaphase cellfiom A549 ceil làne. Structural rearrangements are denoted by arrows. 4.4 SKY analysis for MGH7 and A549 cell lines

SKY analysis is able to resolve areas of uncertainty of interpretation when cornparhg to G-banding. Figure 13 shows an example of the SKY (RGB display) for the MGH7 cell l ine.

Figure 13. An example of SKY image for MGH7 celf Iine (RGB dispfuy)

SKY for the MGH7 ce11 line detected structural rearrangements in chromosomes 1,3,

Il, 15, 16, 17, 18 and 19. Eleven undetemined markers including a large marker chromosome with complex rearrangements were also identified by SKY. Aberrations that were identified by G-banding were modifed afier SKY analysis and are shown in Figure 14. Figure 14. Summary of aberrant chromosomes afrer G-banding and SKY in MGH7 ceIl line. Both normal and aberrant chromosomes are displayed. The arrows denoted the structural rearrangements in the aberrant chromosomes while a number is placed below the normal chromosome. The DAPI, RGB and spectrully classijied images are also shown (See Fig I4(iJ) The numbers on the side of the classifed image indicate the chromosomal origin of the translocoted material. Some abnormal chromosomes identified by G-banding include a del(I)@2I), del(3) (p l4)x2, der(] 1)t(11 ;?)x2, der(lS)t(lS;?), odd(19p) which are shown to be der(l)t(l;l6) mg. 14((i)), der(3)t(.3;21)x2 (Fig.ll(ii)), der(ll)t(ll;l6)x2 (Fig. 14(iii)), der(1 'i)t(I'i;1 7) (Fig. 14(iv)) and der (19)t(3; 19) (Fig. 14 (v)) by SKY analysis. Undetected translocations were shown by SKY in chromosome 16, 17 and 18, which are thought to be normal when studied by G-banding analysis. The "normal" 16q terminal was replaced by material derivedfiom chromosome 17 (Fig. 14(vi)) whereas the 17p was shown to contain chromosome 16 material (Fig. I4(vii)). Chromosome 18 was found to be a der(18)(12;18) where the 18q2I-ter was replaced by chromosome 12 material (Fig. 14(viii)). Eleven undetenined markers includhg a large marker chromosome with complex rearrangements were also identifed by SKY (Fig.14 0).Several markers and aberrant chromosomes were found in duplicates. Three undetermined markers in A549 were identified by SKY. SKY showed that the dicentric chromosome 6 contained chromosome 1 and 6 materials. Surnmary of the aberrations detected by SKY for the A549 ce11 line is shown in tigure 1 5.

Figure 15. Surnmary of aberrant chromosomes aftr G-banding and SKY in A549 cell he. Borh normal and aberrant chromosomes are displayed. The DAPI, RGB and spectrah'y classifed images are ulso shown (See Fig I5(i)l The numbers on the side of the classifed image indicate the chrornosomal origin of the translocuted material. The long arm of the dicentric chromosome contained chromosome I material (Fig. 15.Three undetermined markers were ahidentified by SKY (Fig.15 (ii) and (iii)). 4.5 Comparison of SKY and MYC FISH analysis in MGH7 cell line

MYC FlSH analysis for MGH7 ce11 line showed that there were on average five signals per interphase nucleus (data not show). Metaphase FISH for MGH7 showed that three signals were localized to the trisomy chromosome 8, and the other two signals came to two identical markers with a bright centromeric region (Figure IOC). These findings were supported by the results of SKY analysis, which showed that extra chromosome 8 materials were presented in two unidentified chromosomes (Figure 16)

Figure 16. Comparison between SKY and MYC FManalysis of MGH7 ce[[ line. The identifiesof the two novel markers with WCsignals are clurifed by SKY. These markers contain chromosome 1, 8 and 12 material.

1.6 Comparison between G banding, CGH and SKY analysis in MCH-7

A cornparison of the results between CGH and SKY is illustrated in figure 17 (i-vi).

Extra chromosome 1 material was found in three markers. Two were identical with a remangement involving chromosomes 1,8 and 12 and the other involving chromosomes 1,8 and 19. This is depicted by a gain in the chromosomal regions of lq, 8q and 19q in the CGH katyogram (fig.17 i). CGH identified 3q gain in this cell line and fiom SKY extra chromosome 3 material was detected in one marker t(3; 14) and a der( 19)t(3;19) (fig. 17 ii).

High level amplification was detected in the Sp region for this ce11 line by CGH (fig. 17 iii).

SKY identified three small markers as isochromosome 5p. This accounts for the gain in the

5p region. As a result, nine copies of the 5p were presented in this ce11 line with three copies coming fiom the trisomy 5 and six copies coming from three isochromosomes of the short arm of chromosome 5. SKY showed that the distal region of chromosome 18 was replaced by material derived from chromosome 12 leading to a loss of the 18q2 1-ter region and a gain in the distal region of 12q. Extra chromosome 12 matenal was also detected in two markers

(fig. 17 iv). Although no rearrangement was observed in chromosome 17 by G-banding,

SKY indicated that 17p was replaced by material derived from chromosome 16. This stmcturai rearrangement resuited in a loss of chromosome 17p. The l7q gain is contributed by the extra matenal found in a der(16)t( 16,17) and a der(15)t(15; 1 7) (fig. l7 v). CGH detected a gain in the 1 Sq2 1 region. The rearranged chromosome 15 in the SKY showed that

17q has translocated and replaced the small acrocentric p-arm of chromosome 15. An extra band, denved fiom chromosome 15 was juxtaposed to this 17q material on this marker.

Since the CGH profile indicated a gain in the 15q2 1 region, it is possible that this extra band also derives ûom the same region (fig. 17 vi). ii gb pa 3 der(3)t(3;2l) marker der(19)t(3;19)

3 identical markers 5

1 2 identical markers

Figure 17. Cornparison between G-banding, CGH and SKY in MGH7 ceIl Iine. Discussion:

CGH was first developed in 1992, since then the number of CGH publications

have nearly doubled every year and are now covering over 1500 tumors cases (1 25). It is

a valuable tool for detection of chromosomal imbalances. Although CGH is very helpful

in mapping unbalanced aberrations, it cannot detect balanced rearrangements including

inversions and translocations. If metaphase preparation from tumors is available, this

drawback cm be overcome by a recently developed technique: spectral karyotyping

(SKY). SKY pennits identification of structurai aberrations where there is sufficient

chromosomal material present on the aberrant chromosome to generate a hybndization

signal (1 19). The only current disadvantage of SKY is that structural changes within a

chromosome cannot be detected easily since rearrangement does not generate color

changes. However, conventional G-banding can still provide information on

intrachromosomal changes.

In this study, a number of nonrandom chromosomal aberrations in non-small ce11

lung carcinoma were identified by CGH, which agree with previously published CGH reports in this tumor type (126, 127, 128). Chromosomal regions that were fiequently

affected by DNA gains included lq, 3q, Sp, Sq, 1 lq, 1Sq, 19q and 20q. DNA losses were

found in chromosome 9, 18q, 6q, 5q12-32, 13q and 19p. Table 6 shows a list of candidate genes mapped to the regions of chromosornai imbalance identified by CGH. Table 6: Candidate genes mappd to the regions of chromosornal irnbalance identified by CGH

Chromosomal changes Candidate genes Gene location detected by CGH Gain of 5p SU2 5p13

Gain of 8q24 WC 8q24

Gain of 3q24-q27 BCHE (butyry lcholinesterase), 3q26.1 -q26, SLC2A2 (solute carrier family 2) 3q26.2-q27 Loss of 9p p161"'A. pljmD 9p2 1

1 Loss of 139 1 RB 1 13q14.2

Loss of 18q2 1 -qter DCC, DPC4 18q2 1

The most frequently gained chromosome am in this series was 5p (72.7%), with

three cases showing amplification of the whole p-arm and one case (MGH30) showing

amplification in the 5p15 region. Amplification of 5p has been detected in many

different tumors including small ce11 lung cancer, squamous ce11 carcinoma of the head

and neck, carcinoma of the uterine ceMx. (1 29, 130, 13 1) A candidate gene mapped to

the 5p amplicon is SKP2 (5p13) (132). SKP2 is a protein associated with the cyclin A-

CDIU cornplex, which is essential for the cells to enter S-phase (1 33).

It is known that ce11 cycle progression is controlled by the positive and negative regulation of CDK activity via binding of a cyclin and of a CDK inhibitor, respectively.

In general, the steady-state levels of most CDK proteins remain relatively constant during the ce11 cycle, whereas the abundance of both cyclins and CDK inhibitors oscillates

(1 34). The half-lives of the mamrnalian cyclins (cyclin Dl and E) and some of the CDK inhibitors (p21, p27) are short. Together, rapid tunover of cyclin and CDK inhibitor

proteins provides cells with the ability to quickly respond to a variety of environmental

cues to drive unidirectional ce11 cycle progression. However, the mechanisms that

regulate the degradation of both cyclin and CDK inhibitor proteins are not clear at present

(134). Most intracellular proteins with a short half-life are degraded via the ubiquitin-

dependent proteolytic pathway. In bnef, this process begins with activation of ubiquitin

(a 76 amino acid protein expressed in al1 eukaryotic cells) hy an enzyme designated as

El. nie ubiquitin is then transferred to an ubiquitin-conjugating enzyme (E2). From here, the ubiquitin cm either be directly transferred to a side chain amino group of a lysine residue in the substrate or indirectly targeted through an additional step involving an ubiquitin ligase (E3). The tagged polyubiquitin proteins are then degraded by the 26s proteasorne ( 134).

A ubiquitination pathway has been recently discovered in yeast. The yeast SKP 1-

CDCS3-F-box (SCF) protein complex acts as a ubiquitin E3 ligase to regulate the Gl/S transition. It promotes DNA replication by catalyzîng ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1 (135). In human a similar SCF-like particle is identified. This complex consists of 3 units: pl 9(SKP 1), CUL-I (closest human homologue of CDC53) and p4S(SKP2) @45 is the human F-box protein) (135). A recent study has identified two potential substrates for the pl9/CUL- l/plS complex. Yu and colleagues have used specific antisense oligodeoxynucleotides against either SKPI,

SKP2. or CUL-I mRNA to inhibit their expression (136). Treatment of cells with these oligonucleotides caused the selective accumulation of p2 1 and cyclin D proteins. These data suggest that the human p l9lCUL- 1lp 15 complex is likely to function as an E3 ligase

to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation

(1 36). Therefore the aberrant expression of hurnan p 1 9KUL- l /p 15 complex may

contribute to tumongenesis by deregulating the protein levels of G 1 ce11 cycle regdaton.

The second most common chromosome gain was 8q. Sixty eight percent of the

sarnples showed gain of chromosome 8q, four cases showed amplification at 8q24. One of the candidate genes that maps to this region is MYC. MYC DNA amplification is detected in 8% of the primary non small ce11 lung carcinoma (94). Despite the low percentage of MYC gene amplification in NSCLC, MYC mRNA and protein expression is more cotnmonly detected (94). MYC mRNA expression in NSCLC ce11 lines and patient tumors was present in 67% (1 211 8 cases) and 33% (33169 cases), respectively (94). Volm and colleagues studied MYC protein expression in 202 human squamous ce11 lung carcinomas by irnmunohistochemistiy, they have found that 48% of the tumors had detectable MYC protein. (96, 137) MYC protein was more freguently expressed in lymph node metastases of patients with pnrnary NSCLCs (1 37) As a result, the increase in mRNA and protein expression of MYC may not directly relate to the MYC DNA amplification. An alternative mechanism for MYC activation is by translocation (1 38).

Our MYC FISH result indicated that high level of MYC DNA amplification was not a fiequent event occumng in NSCLC. Five out of six ce11 lines showed 1 to 2 copies of

MYC increase relative to theu ploidy level. Oniy the MGH4 ce11 line showed highly amplifiai MYC signals. The MGH4 metaphase contained a small marker chromosome which showed MYC signal amplification. This chromosome may represent an early stage of the homogenous staining region (150). Although most of the ce11 lines did not show high levels of MYC amplification, MYC signals were found translocated to various marker chromosomes. There are three studies showing the importance of translocation in activation of MYC in lung cancer (139, 140, 141). By FISH analysis, Lu and colleagues discovered MYC üanslocation in a lung adenocarcinorna ce11 line and a SV40T- transfomed human branchial epithelial ceIl line (1 4 1). In another study, they found two out of the 12 primary lung cancers showed MYC translocation, and the overexpression of the MYC protein was confixmed in these two cases (140). Therefore, translocation of the

MYC gene might play an important role in carcinogenesis of lung cancer.

Deletion or chromosomal loss is less fkquently found in this series.

Chromosome regions subjected to loss included 5q 12-32 (1 a%), 6q 16-22 (27%), 9

(36%), 13q (1 8%), 18q2 1-22 (36%) and 19p (1 8%). Some of those regions contained known twnor suppressor genes such as CDKNtA at 9p2 1, RB at 13q14.2, DCC and

DPC4 genes in 18q21. DCC and DPC4 are frequently inactivated in colorectal carcinoma and pancreatic carcinoma, respectively (142,143). Loss of heterozygosity of chromosome 18 was also observed in advanced stages of NSCLCs. LOH of DCC was found in 83% (10/12) of the brain metastases and 33% (3/9) of the stage 1 pnmw

NSCLCs (1 44). Therefore, the loss of 18q (DCC) might conûibute to the progression of

NSCLC. However, no DCC mutations have as yet been reported in lung cancer. In contrat, missense and frameshift somatic mutations in DPC4 have been identified in

NSCLC patients (145). Two missense and 2-bp fiameshifi somatic mutations in DPC4 were detected among 42 lung cancer. However, these mutations were not present in al1 lung cancers carrying 18q21 deletions. This suggested that there may be another tumor

suppressor gene exist in this chromosome region (145). A recent study has identified a

novel tumor suppressor locus on chromosome 18q, which may involved in lung cancer

development (146). The size of this region is less than 1 Mb, and the coding exons of

DPC4 and DCC were mapped outside this region (146).

LOH was frequently detected in chromosome 3p in non-small ce11 lung

carcinomas. Two CGH studies in non-small ce11 lung carcinomas also showed fiequent

3p loss in their samples (126, 128). Another snidy showed only 6%, (3/44 cases) loss in

chromosome 3p (127). Only 13 % of Our samples (3122 cases) showed deletion in

chromosome 3p. One possible explanation is that in our samples the chromosome 3 regions involved in loss are too small to be detected by CGH.

When comparing difference in changes in the two major histological subtypes: squamous ce11 carcinoma (SQCC) and adenocarcinorna (ADC), adenocarcinomas carried more DNA overrepresentation at 1q22-32.2, 15q, 20q and deletions on chromosome 6q,

13q and 18q. In contrast, squamous ce11 carcinomas showed more fiequent gainlamplification in chromosome 3q compare to adenocarcinomas. Since the sarnple size in this study was small, the difference between two groups may not be significant.

Two studies comparing chromosomal imbalances in SQCC and ADC by CGH found that chromosome (3q24-qterl3 q26.2q26.3) ovmepresentation/amplification were more cornmonly detected in SQCC than ADC (127, 128). In the Bjorkqvist et al study, they found 94% of the SQCC had gains in 3q material, including six high-level amplifications, whereas only 24% of ADC showed an increase in DNA sequences without hi&- level

amplifications (127). In our samples, overrepresentation of 3q24qter was detected in 2

samples of ADC and 5 samples of the SQCC. Three high-level amplifications in 3q26- q28 were detected in SQCC but none were observed in the adenocarcinoma samples.

This suggests that amplification of genes in 3q may be important in the development of squarnous ce11 carcinoma but not necessarily of adenocarcinoma.

Two candidate genes (BCHE and SLC2A2) are considered to be possible targets of the 3q amplification. In Brass et al study, they found bat 6/15 SQCC showed amplification of at least 1 of the two genes (BCHE and SLC2A2). In 4 cases, these two genes were coamplified. Therefore, about 40% of the squamous ce11 lung carcinomas showed an amplification of at least one gene at 3q26 (147). BCHE

(butyrylcholinesterase) is located on chromosome band 3q26.1 q26.2. Abnormal expression and amplification of this gene have been reported in leukaemia, brain and ovarian cancer (147). However, the true physiological fûnction of serum cholinesterase has not been identified (147). SLC2A2 (solute camer family 2) is located on chromosome band 3q26.2-q27. A high expression of SLC2A2 was observed in head and neck tumors, but not associated with amplifications or DNA rearrangement (147). This gene plays a role in the regulation of systemic blood glucose level. Again, how this gene is involved in the genesis of hurnan cancers is not known.

In this study, a squamous ce11 carcinoma ce11 line (MGH7) was used to demonstrate the complernentary role of CGH, SKY, FISH and G-banding analyses. With the help of SKY,some unidentifiable markers after G-banding were clarified. SKY and

FISH together were able to define the insertion site of MYC in the duplicated marker chromosomes. CGH is able to account for the whole gemme imbalance even when highiy complex aneuploid markers are present. Conclusion:

In the present study, CGH has been used to better characterize consistent chromosomal imbalances in squamous and adenocarcinorna subtypes of NSCLC. DNA overrepresentations at 1qZbq32.2, 1 Sq, 20q and deletions on 6q, 13q and 18q were more frequently detected in adenocarcinornas. In contrast, squamous ce11 carcinomas showed more muent gain in 3q24-q27. This set of aberrations can serve as the entry points for identifjhg oncogenes / tumor suppressor genes. The complementary role of CGH, SKY,

FISH and G-banding analyses has been demonstrated by analysis of a squamous ce11 carcinoma ce11 line. These approaches together provide a more comprehensive picture of chromosornal changes leading to an improved understanding of the complex karyotype of the lung cancers and provide insight into the molecular genetics of tumor progression in these tumors.

Future studies:

In this study, CGH showed that chromosome 5p is frequently gainedhnplified in

NSCLCs. SKY suggested that the 5p gain might be from the formation of an isochromosome of 5p. Therefore, it will be intmting to determine by FISH if the SKPI gene mapped to the 5p 13 region is amplified in NSCLCs References:

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Buffers and solutions For DNA extraction

RSB buffer

i O mM Tris, pH 7.4 10 mM NaCl 25 mM EDTA, pH 7.4

Salt-saturateci phenol

1-lb bottie phenol 100 ml 2M Tris, pH 7.4 130 ml H20 Heat at 37'~carefully until phenol is dissolved. Remove upper aqueous phase.

Add: 100 ml 2M Tris, pH 7.4 25 ml m-cresol 1 ml P-mercaptoethanol 500 mg 8-hydroxyquinoline

Store aqueous and phenol layers together in hood at 4'~.Use the yellow phenol solution for extraction. Note that the phenol will rernain as the top layer in the stock bonle for easy rernoval. When used to extract DNA, the SS-phenol will be typically the bottom layer.

TE buffer

10 mM Tris, pH 7.4 0.1 mM EDTA, pH 8.0

Culturing and hawesting lymphocytes

Supplemented RPMI 1640 medium for lymphocytes

77 ml of RPMI 1640 20 ml Fetal Bovine Senun 1 ml 200niM L-glutamine (GibcoBRL, reconstituted with autoclaved distilled water according to the manufacture's protocol) 1 ml Penicillin.Streptomycin (GibcoBRL, reconstituted with autoclaved distilled water according to the manufacture's protocol) 1 ml Phytohemagglutinin(GibcoBRL, reconstituted with autoclaved distilled water according to the manufacture's protocol)

Fixative

3: 1 methanol/acetic acid

CGH nick translation

10X Biotin dNTPs

1 00 uM Biotin 14-dATP (GIBCO) 200 uM dCTP 200 uM dGTP 200 UM dTTP 100 uM dATP 500 mM Tris, pH 7.4 50 mM MgCl2 1O0 mM p-mercaptoethanol 100ug/ml BSA

1 mMdATP 1 mMdCTP 1 mMdGTP 0.65 mM dTTP 0.32 mM Dig-1 1-dUTP (Boehringer)

1 0X Digoxigenin incubation buffer

2 mM Tris, pH 7.4 0.2 mM MgCl 0.4 mM 100 uglml BSA

DNase 1 dilution buffer

50 mM Tris, pH 7.4 5 mM MgCl2 1 mM P-mercaptoethanol 100 ug/d BSA 0.04 M Tris base 0.02 M Na acetate . 3H20 1 mM EDTA-Na2 .2H20 adjust pH to 7.2 with acetic acid

Metaphase slides pretreatment for CGH and FM

Proteinase K buffer

0.02 M Tris, pH 7.5 2 mM CaC12, pH 7.5 Post hybridization wash and detection for CGH and FISH

Wash A

50% formamide in 2XSCC,pH 7.0

Wash B

Block 1

3% BSA, 4XSSC 0.1% Tween 20

O. 1M Tris-HC1,pH 7.5 0.1 SM NaCl 0.5 % Boehringer blocking reagent (Boeh~ger)

Wash C

Metaphase slides pretreatnzent for SKY Pepsin solution

3ug of pepsin dissolved in 0.0 1 M HCI

1 % formaldehyde in PBS/MgC12

2.7 ml of 37% formaldehyde to 100 ml PBS/MgC12