Cytogenetic and molecular analysis of complex chromosome ......Cytogenetic and molecular analysis of complex chromosome rearrangements in human cancer: Application of chromosome microdissection

Cytogenetic and molecular analysis of complexchromosome rearrangements in human cancer:

Application of chromosome microdissection.

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Authors Guan, Xin-Yuan.

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Cytogenetic and molecular analysis of complex chromosome rearrangements in human cancer: Application of chromosome microdissection

Guan, Xin-Yuan, Ph.D.

The University of Arizona, 1993

V·M·I 300 N. Zeeb Rd. Ann Arbor, MI 48106

CYTOGENETIC AND MOLECULAR ANALYSIS OF COMPLEX

CHROMOSOME REARRANGEMENTS IN HUMAN CANCER:

APPLICATION OF CHROMOSOME MICRODISSECTION

by

Xin-Yuan Guan

A Dissertation Submitted to the Faculty of the

COMMITTEE ON GENETICS (GRADUATE)

In partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1993

1

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have

read the dissertation prepared by __ ~X~i~n~-_y~u~a~n~G~u~a~n~ ____________________ _

entitled Cytogenetic and Molecular Analysis of Complex

Chromosome Rearrangements in Human Cancer:

Application of Chromosome Microdissection.

and recommend that it be accepted as fulfilling the dissertation

requirement for the Degree of Doctor of Philosophy

1/12/93

Date

1/12/93 Date

1/12/93

Date

1/12/93

Date

1/12/93

Mary (ykowSki Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

2

I hereby certify that I have read this dissertation prepared unrier my direction and recommend that it be accepted as fulfilling the dissertation requ~rement.

1/12/93 Dissertatl.on D Date

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of

requirements for an advanced degree at The University of Arizona and

deposited in the University Library to be made available to borrowers

under rules of the Library.

Brief quotations from this dissertation are allowable without special

permission, provided that accurate acknowledgment of source is made.

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," \. ' .. SIGNED: ,l:1 l :/~0l'1\, ~;,.!¥ .. /L

,J

3

ACKNOWLEDGEMENTS

I am very grateful to Dr. Jeffrey M. Trent for his constant guidence, training, encouragement, patience, and friendship throughout my graduate work. As my academic and dissertation advisor, Dr. Trent provided an invaluable source of inspiration and direction which served as a catalyst for my maturation as a scientist.

I would like to give special thanks to Dr. Paul Meltzer who brought me into the molecular biology field by helping me to design experiments and to work through problems during the second part of my dissertation work. Without his continuous efforts, the completion of this dissertation would have been impossible.

I would like to thank my dissertation committee members: Drs. Harris Bernstein, Tim Bowden, William Dalton, and Mary Rykowski for their critical suggestions during my dissertation work and for their careful reviewing my dissertation.

I would like to thank Floyd Thompson, Jin-ming Yang, Ann Burgess for their excellent technical training in cytogenetics, as well as the time they spent reviewing karyotypes.

I would also like to thank Jun Cao, Tom Dennis, and Elizabeth Thompson for their technical assistance.

Finally, I want to thank my parents for their continuous support throughout my graduate work.

4

5

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS ............................. 7

LIST OF TABLES .................................... 9

ABSTRACT •.••..................................... 10

CHAPTER 1. INTRODUCTION .......................... 12

CHAPTER 2. CYTOGENETIC ANALYSIS OF HUMAN OVARIAN CARCINOMA ..................... 14

Introduction ........... ................ 14 Materials and Methods ................... 28

Materials ............................. 28 Short Term in vitro Culture of Tumors and Effusions ............... 29 Chromosome Harvesting ................. 30 Slide Preparation ..................... 32 Chromosome Banding .................... 32 Microscopy and Photography············ 33 Karyotyping ........................... 34

Results ................................. 35 Numerical Chromosome Alterations ...... 35 Structural Chromosome Alterations ..... 37

Discussion .............................. 62

CHAPTER 3. APPLICATION OF CHROMOSOME MICRODISSECTION ....................... 71

Introdution ............................. 71 Materials and Methods ................... 79

Chromosome Microdissection ............ 79 Preparation of Metaphase Spreads .... 79 GTG Banding Procedure ............... 80 Preparation of Glass needles ........ 82 Microdissection Procedure ........... 82

Amplification of Dissected DNA' ....... 83 Primers Used in Polymerase Chain Reaction (PCR) ................ 83 PCR Conditions ...................... 83 Testing PCR Results by Electrophoresis ..................... 84

Microcloning .......................... 85

Purifying PCR Products by Centricon ........................ 85 Precipitation ....................... 85 Ligation of PCR Products with A T-tailed Vector .............. 86 Transformation by Electroporation ... 87 PCR Mini-preparation ................ 88 Electrophoresis and Preparation of Probes ............... 88 Regional Chromosomal Assignment of Probes ................ 90 Autoradiography············ ......... 93

Fluorescent in situ Hybridization ..... 93 Labelling of Probes with Biotin-11-dUTP by Nick Translation ............ 93 Labelling of Probes with Biotin-11-dUDP by PCR ............... 94 Slide Preparation ................... 94 Probe Preparation ................... 95 RNAse Treatment ..................... 95 Denaturation ........................ 96 Hybridization ....................... 96 Washing ............................. 97 Staining with Fluorescent Conjugated Avidin and Amplification with Anti-avidin .................... 98 Counterstain ........................ 99 Microscopy and Photography·········· 99

Re s u 1 t s ................................ 1 0 0 Preparation of Chromosome Region-specific Libraries ............ 100 Preparation of Whole Chromosome Painting Probes ...................... 102 Preparation of Chromosome Region-specific Painting Probes ...... 103 Detection of Nonreciprocal Translocations and Breakpoints ....... 110 Detection of Homogeneously Staining Regions (HSRs) .............. 113

Discussion ............................. 114

CHAPTER 4. SUMMARy····························· 129

REFERENCE S ...................................... 131

6

LIST OF ILLUSTRATIONS

Figure Page 1. Histogram of chromosome numerical changes

in 50 cases of ovarian carcinoma ............... 46

2. G-banded karyotype from the ovarian carcinoma T89-232 .............................. 47

3. Histogram of chromosome structural alterations in 50 cases of ovarian carcinoma ... 56


5. Summary of all breakpoints involved in clonal structural abnormalities from 50 cases of ovarian carcinoma .............................. 58

6. Examples of hsr in ovarian carcinoma ........... 59


8. Examples of complex structural rearrangements observed in ovarian carcinoma .................. 61

9. A general outline of chromosome microdissection and microcloning ............... 81

10. FISH result of chromosome microdissection of 6q21 ....................... 104

11. Gel result of PCR products from microdissection of 6q21 ....................... 105

12. Example of 12 microclones from 6q21 library··· 106

13. Regional assignment of microclones from 6q21 library ............................. 107

14. FISH results using WCPs of chromos omes 6 and X ........................... 108

7

8

Figure Page

15. Example of FISH results using region-specific painting probes from chromosome 6 ............. 111

16. Mapping of a translocation and deletion chromosomes with Micro-FISH probes ............ 112

17. Microdissection of an unidentifiable translocation from UACC-383 ................... 115

18. Microdissection of an unidentifiable translocation from UACC-837 ................... 116

19. Microdissection of an hsr from NGP-127 ........ 117

9

LIST OF TABLE

Table Page

1. Histologic diagnosis and specimen source from 50 cases of ovarian carcinoma ............. 39

2. Clonal structural abnormalities, mode, and profile from 50 cases of ovarian carcinoma ..... 41

3. Structural abnormalities of chromosome 1 from 50 cases of ovarian carcinoma ............. 51




7. Structural abnormalities of chromosome 3q and 6q from 50 cases of ovarian carcinoma ...... 55

8. Summary of 8 hybrid used for chromosome 6 mapping panel .................................. 92

ABSTRACT

Cytogenetic analysis was performed on 50 short-tenn c;ultures

of human ovarian carcinoma from 48 patients. AlI 50 cases evidenced

clonal karyotypic abnormalities with 32/50 displaying numeric changes,

and 49/50 displaying structural alterations. The most notable numeric

abnormalities were loss of the X chromosome and gain of chromosome

7. Structural alterations appeared to be nonrandom. Chromosomes

most frequently involved in structural alterations included chromo

somes 1>7>11>12>3>6. When the chromosomal breakpoints were

analyzed they were shown to cluster to several chromosomal band

regions including: Ip31-p36, Ip13-qIl, 6q22-q25, 7p15-p22, 7q31-34,

I1p15, and 12pll-p13.

In addition, a novel procedure for chromosome microdissection

and in vitro amplification of dissected DNA was developed and applied

to several areas. Microdissection and in vitro amplification of the

dissected chromosomal fragments were perfonned, followed by

labeling for fluorescent in situ hybridization (FISH) to normal

metaphase chromosomes (Micro-FISH). Micro-FISH probes have been

used successfully to determine the derivation of chromosome segments

unidentifiable by conventional cytogenetic analysis. Micro-FISH

probes were also generated following microdissection of unidentifiable

pOltions of nonreciprocal translocations, allowing determination of the

derivation of these unknown chromosome segments. Another

application of this microdissection procedure has been to generate

10

whole chromosome "painting" probes by microdissection of an entire

chromosome.

Finally, a chromosome microdissection combining micro cloning

technique has been developed and used to generate chromosomal band

specific DNA libraries for physical mapping. A genomic library of

>20,000 clones, which is highly enriched for sequences encompassing

6q21, was constructed. Clones from this library have been charaterized

and localized to the target region by hybridization to a chromosome-6

mapping panel.

In summary, chromosome banding analysis of human ovarian

carcinoma has been performed, identifying regions of consistent

chromosome change. In addition a novel approach for chromosome

microdissection has been developed and applied to determine

previously intractable problems in cancer cytogenetics.

11

CHAPTER 1

INTRODUCTION

This dissertation includes two major sections: cytogenetic

analysis of human ovarian carcinoma for determination of recurring

sites of chromosomal change and application of chromosome

microdissection to complex chromosome rearrangements. Ovarian

carcinoma is the fourth leading cause of cancer death in women. The

histogenesis and biological charateristics of these tumors are not well

understood. To date, cytogenetic analysis of ovarian carcinoma is

limited. This study describes the results of the detailed chromosome

banding analysis of 50 samples from 48 patients with ovarian

carcinoma. Results indicate that recurring nonrandom karyotypic

alterations can be recognized in this cancer.

Chromosome microdissection is a new technique which has been

used to construct chromosomal band-specific DNA library for physical

mapping. Earlier microdissection protocols were too complex and

limited to normal metaphase chromosomes. This dissertation will

describe in detail about how I simplified previous microdissection

procedures and applied this new procedure to: 1) construct

chromosomal band-specific genomic library for physical mapping, 2)

generate Micro-FISH probes for cytogenetic analysis, and 3) generate

whole chromosome "painting" probes for cytogenetic analysis.

Because of the dualistic nature of this dissertation, there will be

two major sections for clarity of presentation. The first section will

12

present the results of detailed karyotypic analysis of human ovarian

carcinomas. The second section will present the application of

chromosome microdissection to both cytogenetic analysis and

molecular study.

13

CHAPTER 2

CYTOGENETIC ANALYSIS OF HUMAN OVARIAN

CARCINOMA

Introduction

Chromosomal Basis of Malignancy

As early as 1912, Theodore Boveri proposed that unequal

distribution of genetic material to daughter cells was responsible for

abnormal growth of malignant cells (1). In 1954, Armitage theorized

that cancer is the result of the accumulation of multiple genetic changes

(2). Each change, whether an initiation or a progression-associated

event, may be mediated through chromosome rearrangements and

therefore has the potential to be cytogenetically visible (3). A corollary

of this idea is that the cellular and molecular charaterization of

chromosomal rearrangements will lead to the identification of genes

involved in the development of cancer.

Methodologic difficulties in preparing metaphase chromosomes

hampered cytogenetic study during the early portion of the 20th

century. These technical shortcomings were overcome in the 1950's

with the discovering that colchicine could arrest cells in mitosis, and the

application of hypotonic techniques to effectively spread the chromo

somes allowed for the identification of and correct enumeration of

human chromosomes (4). Another technical limitation was the inability

to unequivocably identify all normal chromosomes and most

14

structurally altered chromosomes. This technical limit was overcome in

the late 1960's with the development of new staining techniques which

resulted in each individual chromosome displaying a specific banding

pattern, allowing for the first time unequivocable identification of all

individual chromosomes (5, 6). The application of chromosome

banding techniques to human neoplasia has resulted in the identification

of specific numerical and structural chromosome rearrangements.

The identification of recurring chromosome aberrations in solid

tumors (especially carcinomas) has been hampered by several difficul

ties including obtaining sufficient numbers of metaphase spreads, poor

morphology of chromosomes, and the complexity of chromosome

rearragements often observed. For these reasons, solid tumors have in

the past been more difficult to analyze and, as a result, their analysis

represents only 20% of the tumor cytogenetics literature although these

tumors represent almost 80% of the cancers of humans (7).

Chromosome Translocations

The first consistent chromosomal alteration observed in a human

neoplasia was a "minute chromosome", (termed the Philadephia

chromosome) associated with chronic myelogenous leukemia (CML) in

1960 (8). The proof that the Philadelphia chromosome represented a

translocation, rather than a deletion, would wait until improved

chromosome banding techniques became available in 1973 (9).

During the last decade, there have been considerable methodologic

advances in the cytogenetic analysis of human solid tumors (10). The

15

development of high-resolution chromosome banding techniques in

1980's have enormously enhanced the cytogeneticists ability to

interprete subtle chromosome aberrations in cancer. As a result, prior

to 1985 only a restricted number of chromosomal regions had been

implicated in cancer-associated aberrations (11-14). However, today at

least 149 nonrandom chromosome rearrangements have been identified

in at least 43 different malignancies (15). Identification of a consistent

chromosomal region alteration associated with a specific histologic type

of malignancy by cytogenetic analysis provides the basis to determine

whether an underlying molecular change may have taken place at the

corresponding locus. Several genes, including oncogenes and

suppressor genes, have already been identified through molecular

charaterization of nonrandom chromosomal rearrangements. For

example, molecular charaterization of the reciprocal translocation

t(9;22) (q34;qll) associated with CML led to the identification of the

proto-oncogene abl on 9q, and the ber gene on 22q II (16), with the

resulting ber/abl fusion (17) and the production of a chimeric bcr-abl

fusion protein (18, 19).

A second example followed the description of a translocation t(8; 14 )

(q24;q32) in Burkitt's lymphoma by Zech et al. in 1976 (20). Variant

translocations, t(8;22) (q24;qll)and t(2;8) (pll;q24), were reported

later (21). The detailed molecular analysis of these translocations have

revealed that the involvement of the proto-oncogene c-mye on

chromosome 8, and immunoglobulin loci on chromosomes 2, 14, and

22 is germinal in the development of Burkitt's lymphoma (for reviews,

16

see references 22-24). Other examples of oncogene activation

associated with specific trans locations include t(11;14) (q13;q32) in B

CLL affecting the BeLl oncogene (25-27) and t(7;19) (q35;p13) in T

ALL affecting the LYL1 oncogene (28).

Cytologic Evidence of Gene Amplification

Amplification of cellular oncogenes is an important mechanism of

altered gene expression in tumors such as neuroblastoma, breast cancer,

small cell lung cancer, and brain tumors (29). In 1965, Cox et al. (30)

first described the presence of double minutes (DMs) in

neuroblastomas, and Biedler et ai. (31, 32) identified chromosomally

integrated homogeneously staining regions (HSRs) in neuroblastomas

11 years later. Both DMs and HSRs are considered cytogenetic

manifestation of gene amplification. Based on the fact that about a third

of primary neuroblastomas and 90% of established cell lines had DMs

and/or HSRs (33), Schwab et al. (34) found the amplification of an

oncogene N-myc in neuroblastoma. Amplification of an oncogene

ERBB2 on 17qll-q12 has been found in more advanced stages of

adenocarcinoma of the breast and ovary (35).

Chromosome Deletions

Cytogenetic analysis has also provided the basis for identification of

several tumor suppressor genes whose inactivation plays an important

role in tumor development. A recurring site of chromosome deletion is

frequently suggestive of a suppressor gene localized within the deleted

17

chromosomal region. For example, the Rb suppressor gene found in

retinoblastoma was initially identified based on the cytogenetic

identification of a deletion of chromosome 13 including 13q14 (36, 37).

Restriction fragment length polymorphism (RFLP) studies on

chromosome 13 demonstrated loss of heterozygosity (LOH) of

chromosome 13 corroborating the cytogenetic information (38, 39).

The definitive proof that Rb had tumor suppressing activity was found

when concomitant loss of tumorigenicity of human prostate carcinoma

cells occurred following replacement of a normal RB gene (40).

Loss of tumorigenicity has been demonstrated in malignant

melanoma when a whole chromosome 6 was introduced into a

melanoma cell line (41). This study was based on the cytogenetic

analysis of melanoma cells demonstrating the simple deletion of 6q

occurring in excess of 60% of melanomas (42-44). Other examples of

the identification of tumor supressor genes based on the consistent

deletion of specific chromosome regions include: identification of the

WT-1 gene in Wilms' tumor (45, 46) based upon the cytogenetic

demonstration of the deletion of chromosome 11 p 13 in Wilms' tumor

(47-49); the identification of p53 as a tumor suppressor gene (50) based

on chromosome 17p loss in colorectal carcinomas often being a late

event associated with the transition from benign to the malignant state

in colorectal tumor (51); identification of the NFl gene (52-54) again,

based on the cytogenetic analysis showing that rearrangements of

chromosome 17q11.2 were involved in the development of

neurofibromatosis (55, 56).

18

Cytogenetic Analysis of Ovarian Carcinoma

Although ovarian tumors are the fourth leading cause of cancer

death in women and the leading cause of death due to gynecologic

cancer (57), the histogenesis and biological characteristics of these

tumors are not well understood. The data of cytogenetic analysis of

ovarian carcinoma are limited, both in terms of the number of tumor

specimens analyzed and in the quality of metaphases karyotyped.

Cytogenetic analysis of ovarian carcinoma has in general found that the

tumor cells were aneuploid, often with extensive and complex chromo

some rearrangements. According to the Catalog of Chromosome

Aberrations in Cancer (58), to date a total of 159 published cases of

ovarian carcinomas with chromosome abnormalities has been reported.

Nonrandom structural or numerical aberrations have been reported for

several different chromosomes such as chromosomes 1,3,6, 14, and

19. The following section will review the aforementioned literature on

cytogenetic evaluation of human ovarian carcinomas.

In 1974, Atkin et al. (59) reported on chromosome studies on 14

near-diploid carcinomas of the ovary. Chromosome analysis of 26

samples from 14 near-diploid ovarian carcinomas indicated that in 13

carcinomas a single aneuploid clone were present. Their attempts to get

chromosome banding were unsuccessful presumably due to the age of

the material. Their conclusions were that ovarian carcinomas were

commonly but not invariably monoclonal. In 1977, Atkin and Pickthall

(60) presented their results from cytogenetic analysis of 14 ovarian

19

cancers. Nine out of 14 ovarian carcinomas they studied had structural

rearrangement of chromosome 1 including: Ip+, Ip-, lq+, and lq-.

In 1980, Wake et al. (61,62) reported their results of cytogenetic

analysis of 12 papillary serous adenocarcinomas of the ovary with G

and Q- banding techniques. In their study, 6q- and 14q+ were found to

be the most frequent structural abnormalities. Eight out of 14 cases had

both 6q- and 14q+ markers, and 4 cases had either 6q- or 14q+ marker.

They suggested that the 6q- and 14q+ markers were derived from a

reciprocal translocation t(6;14) (q21;q24). In 1981, Atkin and Baker

(63) supported the finding of Wake et al. by reviewing their cases of

ovarian carcinoma including cases dating from the prebanding era.

They found a "Dq+" chromosome in the majority of tumor cells from

most of their 43 cases. Five of the tumors were G-banded and the Dq+

chromosome were suggested to be a 14q+.

Also in 1981, Trent and Salmon (64) reported a detail chromosome

banding analysis (G- and C- banding) of 6 ovarian carcinomas,

summarizing their previous results (65-67). Their results showed that a

variety of chromosome changes including 6q-(qI5-21) in 4 of 6

patients. Comparing with previously published studies on ovarian

carcinomas, they suggested that the deletion of 6q may represent a

characteristic chromosomal rearrangement in ovarian carcinoma.

In 1984, Whang-Peng et al. (68) reported on cytogenetic studies

of 72 ovarian carcinomas. Forty-four of their patients were

successfully analyzed with G- and C- banding techniques, 39 of this 44

cases had structural chromosome alterations. The most frequent

20

chromosomes involved in structural alterations were chromosomes 1, 3,

2,4,9, 10, 15, 19,6, and 11. The most frequent abnormalities of

chromosome 1 included Ip+ and lq-, while another frequent change

was 3p-. Of interest, reciprocal translocations between chromosomes 6

and 14 were not observed in their study. They also found that the

number of chromosome structural alteration increased with duration of

the disease, and chromosomal changes were more extensive in patients

previously treated with chemotherapy.

In 1985, Trent et al. (69, 70) reported on cytogenetic analysis of

two fresh malignant ascites obtained over a nine-month interval from an

untreated patient with ovarian cancer. Both ascites specimens were

analyzed with G- and Q-banding techniques and they compared the

difference in the karyotype of both specimens. The karyotype of first

sample was: 50,XX, -3,+8, +12, +20, and an inversion marker

inv(3)(pI3q23). Progression of this tumor in vivo for 9 months resulted

in two previously unrecognized chromosome alterations; 1) loss of one

copy of chromosome X, 2) an increase in DM containing cells. Also in

1985, Panani and Ferti-Passantonopoulor (71) presented a cytogenetic

analysis results of 6 ovarian adenocarcinomas following G-banding.

The chromosomes most commonly involved in structural alterations

were chromosmes 1, 3, 6, 7, and 17. In all 6 tumors chromosome 3p

was observed, with 4/6 having a breakpoint at band 3p21.

In 1986, Crickard et al (72) reported their cytogenetic analysis

with G-banding technique of 5 cases of borderline malignant serous

cystadenocarcinomas of ovary. All of the tumors had a diploid or near-

21

diploid modal chromosome number. Numerical chcmges included

trisomy 2, 7, and 12. No structural rearrangements were observed.

Also in 1986, Augustus (73) reported on cytogenetic analysis of

metastatic cells in 34 malignant effusions from 24 patients with ovarian

carcinomas. Chromosome number varied from the hypodiploid «45

chromosomes/cell) to hyperdiploid (47-59 chromosomes /cell) with

most cases hyperdiploid in nature. Chromosomes most frequently

involved in the structural rearrangements included l(q21), 5, 9, 3p, 13,

14, 15, and 18.

In 1987, several reports about cytogenetic analysis of ovarian

carcinoma were published. Smith et al.(74) reported the cytogenetic

fmdings in cell lines derived from four ovarian carcinomas. Six

different cell lines were established from four primary ovarian

carcinomas and were examined cytogenetically with G- and C-banding

techniques. The most frequent numerical alteration was the loss of

acrocentric chromosomes, as well as the gain of chromosomes 1,5, 7,

19, and 20. The most frequent structural rearrangements in this study

were deletions of chromosome 1. Deletions of chromosomes 3p, 4p,

7p, and 7q were also noted. Jenkyn and McCartney (75) reported the

cytogenetic results of three different types of malignant ovarian tumors.

Consistent chromosome rearrangements of trisomy 2, del(3)(p 14), and

der(5)t(5;8)(q33;qll) were detected in a case of immature teratoma.

An isochromosome i(12p) was identified in a case of dysgerminoma. A

clone of pseudodiploid cells was found in a treated case of serous

cystadenocarcinoma. Atkin and Baker (76) studied 14 ovarian

22

carcinomas including 11 primary tumors in direct preparations. The

most frequently involved in structural rearrangements were

chromosomes 1 (12 tumors), 3 (12 tumors, including 5 tumors with 3q

), 6 (8 tumors, including 6 tumors with 6q- and 2 with isochromosome

6p), 11 (7 tumors with IIp+), and 14 (7 tumors with 14q+). They also

observed abnonnal small metacentrics in 11 tumors. Ten of these

metacentic chromosomes appeared to be an i(4p) or i(15p) and the other

one interpreted to be an i(12p). Sheer (77) reported on the cytogenetic

analysis of four cell lines derived from adenocarcinomas of the ovary.

Chromosomes 1,3, and 6 were identified to be frequently involved in

translocations with various other chromosomes. Chromosome band

Ip36 was involved in trans locations in 2 cell lines. Chromosome bands

3q13-21 was also involved in translocations in 2 cell lines. Bands

3p 11-13 were rearranged in 3/4 cell lines.

In 1988, Zhou et al. (78) analyzed 12 cases of ovarian

adenocarcinoma for alterations in proto-oncogenes. Six of 12 tumors

had detectable proto-oncogene abnonnalities, and 2 of them had

multiple proto-oncogene alterations. Frequently observed alterations

included amplification of c-myc on chromosome 8q24 (3/12 tumors)

and c-ras-Ki on chromosome 12p12.1 (3n patients). Less frequent

alterations included amplification of c-ras-Ha on chromosome Ilp15.5

(l/12 tumors) and c-ERBB-2 on chromosome 17q11-12 (1/12 tumors).

Also in 1988, Samuelson et al. (79) reported a cytogenetic study on one

benign ovarian fibroma and one Grade I, well differentiated ovarian

23

adenocarcinoma. The only chromosomal abnormality observed in both

tumors was trisomy 12.

In 1989, Tanaka et al. (80) reported a cytogenetic analysis of nine

patients with ovarian carcinomas. Rearrangements of chromosomes 1,

3,6, 7, 10, and 12 were each observed in five or more patients.

Although no specific recurring translocations were identified in their

study, deletions of 3p, 6q, 8p, and 10q were each observed in three

patients. The most frequently affected bands were 3p21, 6q1S, and

8p21, each of them was involved in rearrangements in four tumors.

Other frequently affected bands were 1p36, 3p2S, 6p23, 6q21, 7p13 and

12q24, with each of them involved in rearrangements in three patients.

Also in 1989, Guan et al. (81) presented a chromosomal analysis of 90

ovarian carcinomas. The chromosomes most frequently involved in

structural rearrangements were 1, 7, 11,3, 12, and 6, with a majority of

breakpoints on chromosome bands or regions 1p22-1q21, 3p13-3q13,

6q1S-27, 7p22-q21, 7q31-36, 11p1S, 11p23, 12p13-cen, and 12q24. A

distinguishing feature of ovarian carcinomas in their study was the

finding of cells which displayed chromosome fragmentation,

quadriradials, and complex structural rearrangements. Another report

in 1989 was presented by Pejovic et al. (82) about their cytogenetic

analysis of 11 moderately to poorly differentiated ovarian seropapillary

cystadenocarcinomas. Nine of 11 tumors had clonal chromosomal

abnormalities. The most frequent aberration was a 19p+ marker

involved in band 19p 13 which was observed in 7 patients. Four of the

19p+ markers appeared to be identical. Another frequent

24

rearrangement, deletion of 11 p (p 13-pter) was observed in 6 tumors.

Later, Pejovic et ai. (83) reported on cytogenetic analysis of 42 benign

ovarian tumors. Clonal chromosomal rearrangements were detected in

7 tumors. Trisomy 12 was observed in 5 tumors. Among them, trisomy

12 was the only aberration in 2 fibromas and 1 serous cystadenoma. A

mucinous cystadenoma had + 12 and + 10, and a fibrothecoma had +4,

+9, and + 12. The chromosome aberration in the remaining 2 tumors

included loss of one X chromosome in a adenofibroma and a

translocation t(1;II)(q25;q23) in a mucinous cystadenoma.

Several reports about cytogenetic analysis and proto-oncogene

alterations in ovarian carcinomas appeared in 1990 and are reviewed

below. Samuelson et al. (84) recognized trisomy 12 again as a frequent

change in numerous ovarian tumors of benign to low malignancy. In

their cytogenetic analysis, trisomy 12 was detected in 5 patients

including 2 benign fibromas, a low malignant potential epithelial tumor,

a well-differentiated serous adenocarcinoma, and a granulosa cell

tumor. In this report, trisomy 12 was also observed to be the only

alteration in 4/5 tumors. Monosomy 22 and a translocation t (3;9;22)

were also observed in a granulosa cell tumor. The patient with the low

malignant potential epithelial tumor had tumors in both right and left

ovaries. Trisomy 12 was found consistently in tumor cells from both

right and left ovarian tumor.

Bello and Rey (85) reported on the cytogenetic analysis of 20

malignant effusions from ovarian carcinomas. Chromosomes 1 and 3

were the most frequently rearranged. There were 14 markers from 9

25

tumors which involved chromosome 1. Nine out of 14 markers were

deletions of lq, the most frequent breakpoint was located at lq32.

ll1ere were 10 markers from 8 patients involving chromosome 3, the

most frequent abnomality was loss of a region of 3q by deletion or

translocation with the breakpoints at q21-23 or q27 -28. Another report

presented by Bello et al. (86) included the cytogenetic analysis of 5

metastatic ovarian adenocarcinomas. All cases had complex

chromosomal rearrangements with alterations of chromosome 9p (with

breakpoints at p13 or p22-23) observed in all tumors. Other structural

abnormalities included rearrangements of chromosomes 1, 3, and 6.

Sasano et al. (87) examined DNA for proto-oncogene amplification

in 24 patients with ovarian tumors, including 16 carcinomas and 8

benign tumors. All cases were examined for amplification of the proto

oncogenes c-myc (on chromosome 8q24), int-2 (on 1IqI3), and

c-erbB-2 (on 17 q 11-12). Amplification of c-myc was identified in 6 of

12 patients with invasive carcinoma, int-2 amplification was detected in

1 case, and c-erbB-2 amplification was not detected in any case. Proto

oncogenes amplification was not detected in any benign ovarian tumors

or in any ovarian carcinomas with low malignant potential. In another

study, Roberts and Tallersall (88) karyotyped 27 solid ovarian

specimens from 22 patients. Eighteen specimens were from serous

carcinomas. Clonal changes were detected in 21 specimens.

Chromosomes 1, 6q, 7, 4, and 3 were most frequently involved. The

most frequent structural changes were deletion of Ip and 6q.

26

There were at least two articles about loss of heterozygosity (LOH)

in ovarian carcinomas in 1990. Lee et al. (89) analyzed normal and

tumor DNA samples from 19 patients with ovarian carcinomas using a

series of polymorphic DNA probes that map to chromosome loci along

6q, IIp, and 17p. The results showed that LOH was observed in 9 of

14 cases (64%) at the estrogen receptor (ESR) gene locus on the

terminal region of the long arm of chromosome 6. LOH was detected

in 6 of 8 cases (75%) and 9/14 cases (64%) respectively at the D17S28

and D17S30 loci on chromosome 17p. LOH at the HRASI gene locus

on chromosome IIp was detected in 5/11 cases (46%). The authors

suggested that loss of heterozygosity at high frequency in the terminal

region of the long ann of chromosome 6 appears to be a genetic change

specific to ovarian carcinoma. LOB on IIp and 17p may reflect the

presence of tumor- or growth-suppressor genes on these chromosomes

that are important in the genesis of many tumor types including ovarian

carcinoma. Ehlen and Dubeau (90) used polymorphic probes on

chromosomal segments 3p, 5p, 6q, IIp, and 21q to examine the DNA

of normal and neoplastic tissues from 12 different patients with ovarian

carcinoma. LOH at loci on 3p21-25, 6q21-qter, and llp15 were found

in high frequency. In contrast, LOH at loci on 5p and 21q were not

found in any case. Thus, these authors also suggested that LOH at loci

on 3p, 6q, and 11 p were non-random, and inactivation of genes located

on these chromosmes played a role in the development of ovarian

carCInoma.

27

In 1992, Pejovic et al. (91) reported a cytogenetic analysis of 62

primary ovarian carcinomas. Clonal chromosome changes were

observed in 35 tumors. Five tumors had only numerical changes or a

single structural change. Trisomy 12 was observed in two tumors as

the sole aberration and as one of the clonal numerical changes in

another tumor. Of the remaining two tumors with a single structural

change, one had a balanced t(I;5), and the other had an unbalanced

t(8;15). The majority of the cytogenetically abnormal tumors had

complex rearrangements. The most frequent structural abnormalities

were deletions and unbalanced translocations involving chromosome

arms 19p, 1 p, 3p, 6q, and 11 p with the cluster of breakpoints on

chromosome bands 19p13, lq21-23, Ip36, 3pI2-13, 6q21-23, and

llpI3-15. The most consistent change was a 19p+ marker which was

observed in 16 tumors.

In summarizing the published reports concerning the cytogenetic

analysis of human ovarian tumors, a nonrandom pattern of chromosome

rearrangement is beginning to be uncoverd. The potential significance

of these chromosomal rearrangements in ovarian carcinomas will be

discussed in detail later.

Materials and Methods

Materials

Fifty specimens were obtained from 48 previously untreated

patients with ovarian carcinomas during the period from 1988 to 1990.

28

All these specimens were obtained at the time of initial exploratory

laparotomy. Among them, 18 specimens were derived from primary

ovarian carcinomas, and the remaining 32 were derived from metastatic

tumors. In one patient, specimens from both primary tumor and

omentum metastasis were obtained. Specimens from the second patient

were obtained from two metastatic sites, an omentum metastasis and

peritoneal fluid. The histologic diagnosis and tissue source of all cases

are listed in Table 1.

Short Term in vitro Culture of Tumors and Effusions

Solid tumor samples were obtained immediately after surgery and

were mechanically dissociated under aseptic condition in a laminar

flow hood. Tumors were brought into a single cell suspension by

mincing the specimen with fine sterile scissors and forceps in the

presence of McCoy's-plus medium. Clumps of cells were then removed

by passage through sterile gauze (8ply). The single cells were washed

and resuspended in McCoy's-plus medium and then plated in Falcon

T25 culture flasks using 5 ml complete McCoy's medium. Cell

concentration needed were approximately 1-1.5 X 106 cells per flask.

The effusions cell cultures were treated just like solid tissue samples

except no mechanical dissociation was required. Also, the cells in

malignant effusions were only washed one time in McCoy's-plus

medium. All flasks were incubated horizontally at 37°C in a

humidified, 5% C02 incubator with caps loosened 1/4 turn to permit

29

C02 exchange. The culture medium was changed after 5 days and then

changed every 3-4 days until cytogenetic harvest.

Biologicals and Reagents:

1. McCoy's-plus Medium (100 ml)

McCoy's serum-free medium

Fetal calf serum

Preservative-free heparin

89ml

10ml

final concentration

10 units/ml

Pen/Strep 1 ml (10,000 units/ml)

2. Complete McCoy's Medium (100 ml)

McCoy's serum-free medium 80 ml

Fetal calf serum 17 ml

Glutamine 2 ml (200 mM)

Pen/Strep 1 ml (10,000 units/ml)

Chromosome Harvesting

The chromosome harvesting procedure is based upon that described

by Trent and Thompson (92). Briefly, when examination of flasks

showed high mitotic activity and near confluency of cells, specimen

was treated to arrest mitotic figures by the addition of colcemid

(Gibco) to the culture medium at a final concentration of 0.05 Jlg/ml for

1.5-2 hours. Cells were then detached from the bottom of the flask by

trypsinization with trypsin-EDTA(Gibco). The cells were incubated at

37°C until inspection with an Olympus inverted microscope revealed

the detachment of all cells (usually 3-8 minutes). The cells were then

30

transferred to centrifuge tubes and centrifuged at 1,000 rpm for 5

minutes. The supernatant was discarded and the remaining cell pellet

was resuspended in 8 ml of 0.075 M KCI prewarmed to 37°C. Cells

were then incubated in a 37°C waterbath for 18-20 minutes, followed

by centrifugation at 1,000 rpm for 5 minutes. The supernatant (except

for 0.2-0.5 ml) was discarded and the remaining cell pellet was

resuspended thoroughly but gently in 8 ml of freshly prepared Carnoy's

fixative, and placed at _20°C for a minimum of 30 minutes. Cells were

then centrifuged at 1,000 rpm for 5 minutes and the supernatant was

discarded. The cells were then washed in 8 ml of fresh cold fixative

and centrifuged at 1,000 rpm for 5 minutes. Finally, the cells were

resuspended in 0.5 to 1.5 ml of fresh cold fixative to obtain a slightly

opague cell suspension before preparing slides.


1. PBS-Phosphate Bufferred Saline

NaCI 8.00 g

KCI 0.20 g

1.15 g

0.20 g

Dissolved in one liter distilled water.

2. Hypotonic Solution (0.075 M KCI)

KCI 0.54 g

Dissolved in 100 ml distilled water.

31

3. Camoy's Fixative

3 parts absolute methanol

1 part glacial acetic acid

Slide Preparation

Microscope slides were immersed in absolute ethanol and stored at

_200

C. Slides were then wiped with lint free tissue to clean and dry the

slides. Three to five drops of cell suspension were placed onto a slide

held at 450

angle, allowing the cell suspension to run down the length

of the slide. The fixative was then ignited by passing the slide through

a flame and forcibly blowing on the slide to extinguish the flame and to

help spread the cells. Examination of the slides by phase microscopy

(200X) was performed to evaluate cell density, and to the frequency

and spreading of mitotic figures.

Chromosome Banding

Standard G-banding was performed by utilizing the procedure of

Yunis et at. (93). The slides were placed in a horizontal position on a

staining rack and flooded with fresh Wright stain for approximately 2

minutes, followed by a brief rinse with distilled water and air drying. If

the banding was not well differentiated, slides were destained and

restained as follows: the slides were dipped in 95% ethanol for 1-2

minutes, followed by 0.5-2 minutes in 95% ethanol+ 1 % HC}; and then

1-2 minutes in absolute methanol. The slides were air dried and

restained as above.

32


1. Wright Stock Stain (0.25%)

Wright stain

absolute methanol

1.25 g

500ml

2. Wright Working Stain Solution

1 part Wright stock

3 parts Working buffer

This working solution should be made just prior to use.

3. Working Buffer

0.06M Na2HP04 buffer

0.06M KH2P04 buffer

Microscopy and Photography

49 parts

51 parts

G-banded slides were scanned using a lOX objective and lOX

oculars on a Zeiss Universal II photomicroscope. Chromosome counts

and initial chromosome analysis was performed using an 80X dry

objective and lOX oculars. Selected G-banded mitotic figures were

photographed at a magnification of 630X, using Zeiss II

photomicroscope. Kodak Technical Pan 2415 was utilized because of

its highly degree of contrast. G-banded chromosomes were

photographed at a reciprocity setting of 3, film speed (DIN) varying

between 13-16 depending upon the staining intensity and automatic

exposure setting. A green filter was used to enhance the contrast of the

chromosomal image and banding resolution. Negatives were developed

using Kodak HCllO developer (dilution D) at 20°C for 6 minutes.

33

Following development, the film was rinsed three times with distilled

water and then fixed for 3 minutes using Kodak fixer. The film was

then rinsed one time with distilled water, followed by two minutes in

Kodak hypoclear. The hypoclear was decanted and the film was rinsed

for 5 minutes in distilled water and air dry.

G-banded chromosomes were printed on Kodak polycontrast rapid II

RCB paper. A Bessler automatic printer and enlarger was used for all

the printing. The appropriate exposure time, diaphragm opening, and

contrast filter were selected to expose the print paper. The paper was

then developed by an automatic print processor using Kodak Dektol

developer and Kodak stabilizer. The prints were then placed in a tray

with Kodak fixer for 2 minutes, followed by a 20 minutes rinse in

distilled water. The prints were then allowed to air dry.

Reagent:

1. HC-IIO/dilution D (lliter)

HC-IIO stock solution

distilled water

Cooled to 20°C before use.

Karyotyping

IOOml

900ml

All of the karyotypes were prepared and discribed according to the

International System for Human Cytogenetic Nomenclature (94). A

chromosome abnormality was considered as clonal in origin when at

least 2 cells from a given tumor had the identical structural

chromosomal rearrangement, or when 3 or more cells demonstrated

34

gain or loss of the same chromosomes. When it was impossible to

accurately identify the origin of rearranged chromosomes, they were

referred to as unidentified markers.

Results

Cytogenetic studies by G-banding were performed on ovarian

carcinomas from 48 patients in order to determine the frequency and

specificity of chromosome abnormalities. No patients had received

chemotherapy prior to the chromosome examination. All cases

described here were successfully analyzed by chromosome banding

analysis.

Numerical Chromosome Alterations

Chromosome number varied greatly in this series of ovarian

carcinomas, from hypodiploid to tetraploid. The chromosome range in

each case also varied considerably. The modal chromosome numbers

discribed in Table 2 represent the most frequently observed

chromosome number in each tumor sample.

The modal chromosome number of these tumors varied from 38 to

95 with a chromosome range of 32->300. Based upon modal

chromosome numbers, three major ploidy populations could be

identified in this study; near triploid mode (59-82 chromosomes/cell)

was the most frequent mode (14/50 tumors, 28%), followed by

hypodiploid «46 chromo-somes/cell) (9/50 tumors, 18%) and

3S

hyperdiploid (47-58 chromosomes/cell) (5/50 tumors, 10%). Tumors

with a near-tetraploid mode (83-102 chromosomes/cell) were

uncommon in this study (2/50 tumors, 4%), as were pseudo diploid

tumors (46 chromosomes/cell) (3/50 tumors, 6%). In this later case, all

tumors contained at least one rearranged chromosome. Nine tumors

(18%) had mixture of normal and abnormal metaphases with the

majority of cells normal, so their mode was 46. Eight cases (16%) had

no mode because the chromosome number were too variable among

different cells to identify a clear mode.

Numerical chromosome changes were analyzed in detail in 32

cases. All chromosomes (except the Y) were involved one or more

times in numerical changes. Figure 1 shows a summary of the

numerical changes in this study. The most frequent chromosome lost

was chromosomes 13 (13/32 tumors), 19 (12 tumors), 22 (12 tumors),

and X (10 tumors). The most frequent chromosomes gain was +1

(13/32 tumors), +3 (12 tumors), +2 (11 tumors), +20 (10 tumors), and

+7 (9 tumors). The loss of the X chromosome involved whole

chromosome loss in 10/10 tumors. Loss of chromosomes 19 and 22

also involved whole chromosome loss in many cases. Interestingly,

gain of chromosome 7 was found in two tumors with simple

chromosome rearragements. In one case (T89-232), the only

chromosome changes were gain of one copy of both chromosomes 7

and 8 (Figure 2). In another case (T89-321), the only chromosme

changes were gain of two copies of an apparently novel chromosome 7

and one copy of terminal deletion on the long arm chromosome 3.

36

Trisomy 12 was found in six cases in this study. However, all of these

cancers also often had complex chromosome rearrangements.

Structural Chromosome Alterations

All chromosomes were involved at least once in structural

rearrangements except chromosomes 4 and 20. The frequency of

sturctural alterations of each chromosome is shown in Figure 3.

Structural chromosome alterations appeared to be nonrandom with

chromosome 1 the most commonly involved, followed by

chromosomes 7, 11, 12,3, and 6. Figure 4 shows a representative

karyotype from one case (T88-286). Several chromosomal bands or

regions were found most often to be affected by structural

rearrangements including: Ip31-p36, Ip13-qll, 6q22-q25, 7p15-p22,

7q31-q34, I1p15, and 12pll-pI3. Figure 5 shows all identified

breakpoints involved in clonal structural abnormalities in this study.

Chromosome 1

Twenty-eight of 48 patients (58%) had structural abnormalities

of chromosome 1 (40 different clonal structural alterations). Of 40

structural aberrations of chromosome 1, 27 involved the short arm.

Deletions, iso(1q), and unbalanced translocations in 19 tumors resulted

in visible loss of the chromosome material of Ip (Table 3).

37

Chromosome 7

Chromosome 7 rearrangements were present in 22 patients (45.8%).

The most frequent alteration in the short arm was the terminal deletion.

Four patients had deletions involving 7p15, while deletions involving

7p 11, 7p 12, 7p 14, and 7p22 were also noted (Table 4). The addition of

unidentifiable material onto the distal short arm of chromosome 7 was

also seen in 3 patients. Translocations involving the short arm were

rare, only one patient had a translocation which involved 7p22. In

contrast, translocations involving the long arm were more frequent. Six

patients had translocations of 7q involving unknown chromosomal

segments with 3 of them involving 7ql1. Deletions and 7q+ were

each observed in 3 patients. One patient had a duplication between

bands 7q22-31.

Chromosome 11

Chromosome 11 abnormalities were seen in 22 patients (25 clonal

aberrations). The most frequent abnormalities were in the short arm

with the extra material of unknown origin observed in 10 tumors (Table

5). Other rearrangements commonly involving chromosome 11 were

translocations involving the long arm (4 cases) and the short arm (1

patient) and llq+ (3 patients). Deletions of chromosome 11 were only

seen in 3 patients; 2 involving the long arm and 1 involving the short

ann.

38

Table 1. Histologic Diagnosis and Specimen Source from 50 Cases of

Ovarian Carcinomas.

Case ID Sample Tumor Type

T88-028 omentum undifferentiated T88-157 abdomen serous T88-163 primary undifferentiated T88-173 primary serous T88-183 omentum serous T88-184 peritoneal fluid serous T88-207 primary serous T88-267 omentum endometrioid T88-275 ascites fluid serous T88-286 primary undifferntiated T88-297 mets* serous T88-301 omentum serous T88-304 omentum serous T88-313 ascites fluid endometrioid T88-320 omentum serous T88-339 pnmary serous T88-372 omentum serous T88-411 pnmary serous T89-003 abdominal wall undifferentiated T89-011 ascites fluid adenocarcinoma T89-018 pnmary serous T89-026 pnmary undifferentiated T89-029 ascites fluid serous T89-032 pnmary serous T89-052 omentum serous T89-059 omentum serous T89-060 pnmary serous T89-069 omentum serous T89-070 omentum undifferentiated T89-097 mets* serous T89-098 primary unknown T89-107 omentum clear cell

39

T89-119 mets* clear cell T89-126 primary endometrioid T89-133 omentum serous T89-134 intraperitoneal serous T89-143 mets* serous T89-154 jejunum serous T89-166 left adnexa unknown T89-187 omentum serous T89-224 omentum undifferentiated T89-232 primary serous T89-243 primary undifferentiated T89-266 primary unknown T89-277 omentum unknown T89-321 adnexa endometrioid T89-348 adnexa serous T90-031 pnmary mUCInOUS T90-062 primary serous T90-063 primary serous

* unspecified regional metastatic sample from the lower abdominal cavity.

40

41

Table 2. Clonal Structural Rearrangements, Mode, and Profile from 50 Cases of Ovarian Carcinoma.

Case ID Mode Profile* Clonal Structural Rearrangements

T88-028 no AA inv(1)(pllp36), i(I)(ql0),

add(2)(q37).

T88-157 62 AA add(1)(p36), del(6)(q23), del(lO)

(q24), add(12)(q24).

T88-163 64-66 AA add(l2)(p 13)

T88-173 no AA del(l)(q21), add(6)(q25), der(12)

t(12; 14 )(p 13;q 13)

T88-183 56-63 AA dup(12)(q24q21),

der(12)dup(12)(q24qll),

dup(12)(q21qll)

T88-184 56-57 AA t(7;?)(q32;?), add(11)(p15), add(12)

(q24), add(17)(q22).

T88-207 43-44 AX del(1)(q25), add(5)(pI5).

T88-267 46 AN del(1)(p21), del(1)(q25).

T88-275 60-69 AA t(1;1)(1p36;lq21), add(3)(q29),

del(7)(pI5), del(l1)(p13),

int del(11)(q21q23).

T88-286 45 AA add(1)(p36), i(1)(qlO),

add(5)(pI5), t(6;13)(q25;q34),

t(18;22)(pl1.1;q 11.2).

T88-297 no AA del(1)(pll), del(7)(q32), der(13)

42

t(11;13)(qI2;q22), add(16)(qI3),

del(X)( q23).

T88-301 41-45 AX add(1)(q44), add(7)(q36).

T88-304 no AA del(2 )(p 15), hsr(7)(p21),

add(11)(p 15).

T88-313 no AX del(7)(pl1).

T88-320 no AA del(1)(p34), add(3)(p25),

hsr(6)(p22), add(6)(q27), del(7)

(q31), add(7)(p15), add(lI)(q25),

ider(12)(ql0q22).

T88-339 53-58 AA add(2)(q34), add(3)(pll),

add(12)(pl1), add(16)(q24),

add(19)(q 13.5).

T88-372 80-88 AA add(l)(pll), add(l)(p33),

del(1)(q23), add(3)(q27),

del(7)(pI4), del(7)(q34).

T88-411 46 AA hsr(l7)(q25).

T89-003 no AX del(l)(p31), add(II)(q24).

T89-011 no AX add(ll )(pI5).

T89-018 78 AA del(1)(q25), i(3)(ql0),

i(13)(ql0).

T89-026 41 AX del(6)(qI6), der(7)add(7)(p22)

add(7)(q34)

T89-029 39 AX der(1)t(1;3)(q21;pI4), del(2)

(q31), der(5)t(5;9)(q31;qI2),

43

dup(7)(q22q31), t(1;9)(pI2;p24),

der(11 )add(11 )(p 14 )del(11)( q23),

del(12)(pI2), der(1l)t(11;14)

(pll;qll), add(16)(pI3).

T89-032 38 AX add(12)(q24).

T89-052 67 AX add(1)(p34), add(1)(P32),

del(3)(qI3), add(6)(p22),

add(11 )(q23).

T89-059 70 AA der(9)t(1;9)(pll;ql1).

T89-060 45 AA der(9)t(1 ;9)(p II;q 11)

T89-069 46 NX t(1; 1)(p36q32), dup(5)(qI2q35),

add(7)(ql1), add(12)(pll),

t[del(12)(q24); 14 ](p 12;p 13),

deli(17q)(ql0q22).

T89-070 46 AN add(1)(p36), add(6)(q22),

add(7)(p22),der(11 )t(11; 11)

(pI5;qI3) dup(11)(qI3q25).

T89-097 64 AA hsr(1 )(p33), del(1 )(p35), add(2)

(q37), der(6)t(5;6)(q 11 ;q24),

add(7)( q 11), der(7)t(7; 11)

(qll;qI3), del(11)(q21),

add(15)(pl1).

T89-098 46 NX hsr(11)(q?)

T89-107 46 NX add(1l)(pI4), del(12)(pI2.1).

T89-119 46 AN del(1)(pI3), del(1)(q32), del(6)

44

(q 13), del(7)(p 11), add(12)(p 13),

add(12)(pll).

T89-126 38 AA add(1)(p33), del(7)(pI5), der(9)

t(6;9)(pll ;pl1), add(9)(q34),

der(1l)t(1l;14)(pI4;q23), der(12)

hsr(12)(pl1), add(12)(qI5),

add(13)(p 11), add(16)(pll).

T89-133 46 AN del(7)(p 12).

T89-134 43 AA add(1)(p36), add(3)(q29),

hsr(6)(p21), add(7)(q31),

add(11)(qI3), del(l2)(pI2),

der(14 )t(13; 14 )(q 11 ;pI3).

T89-143 46 AN add(5)(q35), add(9)(p24).

T89-154 46 AA i(1)(ql0).

T89-166 46 AA dele 3)(q21).

T89-187 59-60 AA add(2)(q37), del(6)(q 15), add(7)

(p22), add(8)(p23), add(7)(ql1.2),

add(12)(pI2), add(12)(pI3).

T89-224 46 NX add(3)( q29), del(7)(p 15),

add(11)(pI5), del(l2)(pll).

T89-232 47 AA +7, +8.

T89-243 70 AX add(1)(p32), del(6)(q23),

del(7)(p 12), add(1l)(p 15).

T89-266 93-95 AA del(l)(p31), del(3)(q21),

add(13)(pll), add(l6)(Q24),

T89-277

T89-321

T89-348

T90-031

T90-062

T90-063

74-76

49

56-57

70

65

68-72

AA

AA

AX

AA

AX

NX

add(17)(q25).

del(3)(p22), del(3)(qI2),

add(11)(pI5), del(12)(pll).

del(3)(q21).

del(1)(pI2), add(I)(pll),

add(6)(qI5), hsr(11)(pI4),

add(12)( q24 ).

der(7)t(7;9)(p22;q 12).

i(1)(ql0), der(7)t(3;7)

(q 13 ;q21), der(17)t(8; 17)

(qll;pI3), i(21)(ql0).

add(1)(p36), add(12)(pI3).

4S

* A=abnonnal metaphase; N=nonnal metaphase; X=metaphase which was too complex and/or contains fragmentations, quadriradials so that it cannot be fully analyzed. AA=all abnonnal metaphases; AN=a mixture of nonnal and abnonnal metaphases; AX=a mixture of abnonnal and complex metaphases; NX=a mixture of nonnal and complex metaphases.

14

12

10

8

Gain 6

4

2

2

4

6

Loss 8

10

12

14

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

Chromosome Number

Figure 1. Histogram summarizing the numerical chromosome alterations observed in 50 cases of human ovarian carcinomas. Above the X-axis is the distribution of gained single chromosomes, and below the X-axis is the distribution of lost single chromosomes.

46

;} II )) ), \\ 1 2 3

it nl I

):1 ( 6 7 8

It t t I' 13 14 15

-, 19

Ii 9

iI 10

.{ 16

•• 21

iii 4

f ,

\It 5

,: 11

~, 17

U 22

Figure 2. Example of only numerical chromosome change in one case of ovarian carcinoma. G-banding karyotype from case T-89-232. Gain of chromasoes 7 and 8 is the only change in this case.

47

i~ 12

ia 18

~( XIV

Chromosome 12

Chromosome 12 abnormalities were seen in 20 patients with 28

clonal markers. Six patients had a 12p+, which was the most frequent

abnormality involving the short arm of chromosome 12. Another

common abnormality involving the short arm was deletion of bands

12p12 (4 tumors) and 12pl1 (2 tumors). Eight patients had

rearrangements involving the long arm, half of them had a 12q+.

Iso(12q), invertion, deletion, and duplication of 12 were each observed

in one patient (Table 6).

Chromosome 3 and 6

Rearrangements involving the long arm of both chromosomes 3 and

6 were each seen in 11 patients (Table 7). Deletion of 3q was seen in 5

patients, with three of them involving 3q21. The other rearrangements

involving 3q included 3q+ in 3 tumors, an unbalanced translocation in 1

tumor, and an iso(3q) in 1 tumor. Deletion of 6q was also the most

frequent aberration involving chromosome 6q (Table 7). Breakage of

bands 6q15-25 were observed in 7 tumors,S of them caused by

apparent terminal deletions and 2 by translocations.

Other Chromosome Alterations

Cytological evidence for gene amplification was observed in 9

patients as HSRs. They were localized to Ip33, 6p22 (two tumors),

7p21, llpll, llpl4, llq, 12pll, and 17q25. Figure 6 displays

representative examples of HSRs from 5 cases of ovarian carcinomas.

48

Five of the 48 patients displayed simple karyotypic changes

(numerical change only or a single structural aberration). Among these

cases, one tumor (T89-232) displayed numerical change only involving

gain of one copy each of chromosome 7 and chromosome 8 (48, XX,

+7, +8). Two tumors had both numerical change as well as a single

structural chromosome aberration. The first case, T89-060 had a

reciprocal translocation t( 1;9) (p II ;q II) and lacked of one copy of the

X chromosome [45, XX, -X, t(1;9) (pll:qll)]. The second case, T89-

321, had a del(3)(q21) and two additional copies of chromosome 7 [49,

XX, +7, +7, +del(3)(q21)] (Figure 7). Two tumors had only a single

structural aberration. First, an iso(1)(qIO) was observed as the only

change in case T89-154 [46,XX, iso(I)(ql0)]. The second case (T89-

166) had only a deletion chromosome [46,XX, del(3)(q21)].

Specimens from one patient were obtained from both the primary

tumor (T89-160) and an omentum metastasis (T89-159). The primary

tumor, as mentioned above, had a mode of 45 chromosomes (range of

44-46). The chromosomal aberrations included a reciprocal

translocation t(1 ;9) (pI 1 ;qll) and loss of one copy of the X

chromosomes. In contrast, the omentum metastasis had a mode of 70

chromosomes (range of 64-75) and evidence numerous complex

structural rearrangements. The t(I;9) was a consistant finding in both

the primary and matastatic tumors.

Specimens from a second patient were obtained from an

omentum metastasis (T88-183) and a peritoneal fluid metastasis (T88-

184). Consistent rearrangements including chromosome 12 alterations

49

were observed in both specimens (Table 2). In general, the peritoneal

fluid metastasis displayeda much more complex karyotype than the

omentum metastasis.

Normal metaphases were observed in 10 cases, likely representing

the mixture of tumor and nontumor cells of which these samples were

comprised.

Complex Chromosome Breakage

A distinguishing feature of ovarian carcinomas in this study

was that 17/48 tumors (35.4%) contained cells which displayed

significant chromosome fragmentation, triradials, quadraradials, and

very complex structural rearrangements. Because of the extraordinary

complex karyotype of these tumors the complete descriptions were not

possible. Figure 8 shows representative example of these complex

changes.

50

Sl

Table 3. Structural Abnormalities of Chromosome 1 (28/48 patients)

Short arm (p) Long arm (~)

Abnormalties # of l2atients Abnormalities # of l2atients

I. Deletions I. Deletions

Ip13 1 lq21 1

Ip21 1 lq23 1

Ip31 1 lq25 1

Ip34 1 Iq32 1

Ip35 1

II. Additions II. Additions

Ipll 1 Iq44 1

Ip31 1

Ip32 1

Ip33 2 III. Translocations

Ip36 6 t(1; I)(p3 2;q21) 1

ffi. Translocations t(1; 1 )(p34;q32) 1

t(1 ;I)(p32;q21) 1 t(1; 1)(p36;q32) 1

t(1; 1 )(p34;q32) 1 t(1;3)(q21;p14) 1

t(1;1)(p36;q32) 1

t(1;9)(pll;qll) 1

t(1;9)(p12;p24) 1 IV. iso(1q) 4

N. inv(1)(pllp36) 1

V. HSR(1)(p33) 1

S2

Table 4. Structural Abnormalties of Chromosome 7 (22/48 patients)

Short arm (n) Long arm (g)

Abnormalities # of Patients Abnormalities # of Patients


pll 2 q3I I

pI2 2 q32 I

pI4 I q34 I

pIS 4

p22 I II. Additions

II. Addition qll 3

p22 3 q3I I

q32 I

m. Translocation q36 3

t(7;II)(p22;qI3) III. Translocation

t(3;7)(qI3;q2I) I

IV. HSRs

p2I I IV. Dunlication

q22q3I 1

Table 5. Structural Abnormalities of Chromosomell (22/48 patients)

Short arm (p) Long arm (q)


I. Deletion

p13

II. Addition

p15.3

ill. Translocations

t(11;14)(pl1;qll)

t(11;14)(pI4;q23)

IV. HSRs

pll

p14

1

10

1

1

1

1

I. Deletions

q21

q23

Int del(11)(q21q23) 1

II. Additions

q13

q21

q23

q24

q25

III. Translocations

t(7; 11 )(p22;q21)

t(11; 13)(q22;q22)

1

1

1

1

1

2

1

1

1

S3

S4

Table 6. Structural Abnormalities of Chromosome 12 (20/48 patients)

Short arm (n) Long arm (Q)

Abnormalities # of natients Abnormalities # of Patients

I. Deletions I. Deletion

pll 2 q22 1

p12 4

II. Addition

II. Additions q24 1

pH 2

p13 6 III. Inversion

qllq24 1

III. HSRs

pH 1 IV. Dunlication

qlIqIS 1

V. iso(12)(qIO) 1

Table 7. Structrual Abnormalities of Chromosome 3q and 6q (11148

patient each)

3q 6q



q12 1 q13 1

q13 1 q15 2

q21 3 q16 1

q23 1

II. Additions q25 1

q27 1

q29 1 II. Additions

q22 1

ID. iso(3q)(ql0) 1 q27.3 1

III.t(5;6)(qll;q24) 1

55

P

q

24

22

20

18

16

14

12

10

8

6

4

2

2

4

6

8

10

12

14 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 14 1 5 1 6 1 7 1 8 1 9 20 21 22 X

chromosome number

Figure 3. Histogram summarizing the structural chromosome alterations observed in 50 cases of human ovarian carcinomas. Distribution of alterations involving the short arm (p) of sing I e chromosomes is above the X-axis, and the distribution of alterations involving the long arm (q) of single chromosomes is below the X-axis.

56

':.I:

" , J. ,. J. ~~."- ,,! /1 t( :J .) . ' , \ ,

1 2 3 4 5

J. • j"

..... ... l Ii ... ,

'''' ~\ 11 , f H •• 6 7 8 9 10 11 12

J. J.

., , It f • , , . j;

4 ' , .

• 13 14 15 16 17 18

i . " •• i • •• 19 20 21 22 XN

J \ Umara

Figure 4. G-banded karyotype from case T88-286. Solid arrows indicate clonal structural abnonnalities given in Table 2. Clonal unidentified marker chromosomes (Umars) are shown at the bottom of the karyotype.

57

1 2 3 4 5 6

7 8 9 10 11 12

13 15 16 17 18

P I

B mn q I ~ ~

19 20 21 22 Y X

Figure 5. Summary of all breakpoints involved in clonal structural abnormalities (dots) from 50 cases of ovarian carcinomas.

58

Ip hsr T89-097

.1 6p hsr

T88-320

12p hsr T89-126

17q hsr T88-411

" •• IIp hsr T89-348

Figure 6. Examples of homogeneously stained region (hsr) markers from five different cases of ovarian carcinomas. Right chromosome of each pair has an hsr. Left one of each pair is corresponding normal chromosome from the same metaphase.

59

\, h 1,

t· I, fA .;. fl (. It

\' , X ~~ ~ tJ .;.. u \\. A .~ .. , 14 ~ • .. '"

2 3 4 5

l,l, . "il " ? fl ~J ,4 .' n ft h ~ f: ~. ~ " 'J '- ,,, ~tlD;-'

, " •

CI " t Of " 6 7 8 9 10 11 12

, . ~ M q ;1 t' .;. ~ . • ~ • Jf ~ '" t"lo

13 14 15 16 17 18

ph

• , , .. AI. . I b ~" . 19 20 21 22 XIY

Figure 7. G-banded karyotype from case T88-321. Arrows indicate clonal abnonnalities, including del(3)(q21).

60

Figure 8. Example of highly abnonnal, unanalyzable spread from ovarian carcinoma. The presence of this kind of cell helped define the AX

61

Discussion

As discussed in the Introduction, the data of cytogenetic analysis

on ovarian carcinoma is currently limited both in terms of the number

of tumor specimens analyzed and in the quality of metaphases

karyotyped. In general, the karyotypic data to date has shown that

ovarian carcinomas commonly exhibit extensive and complex

chromosomal rearrangements, including both nonrandom numerical

and structural abnormalities. Recurring sites of chromosome change

has been suggested for several chromosomes including: 1, 3, 6, 11, 14,

and 19. The most frequent clonal numerical and structural changes

described by previous reports were in general also noted in this study.

The most common numerical chromosomal changes in my study

were loss of chromosomes 13> 19>22 and X; and gain of chromosomes

1>3>2>20>7 (Figure 1). Loss of chromosomes 13 and 22 was also

described by Smith et al. (74), who described loss of acrocentric

chromosomes as the most frequent numerical chromosomal alteration.

Loss of a whole X chromosome was observed in 10 patients in this

study, which also noted in several previous reports (64, 80, 83, 91).

The biologic significance of loss of the X chromosome is uncertain.

However, it does appear that loss of an X chromosome may playa role

in the development of ovarian malignancies.

In my study, gain of chromosome 7 was observed in 10/48

patients. Although this number of affected patients with chromosome 7

alterations is less than gain of chromosomes 1 (13 patients) and 3 (12

62

patients), it is of interest to note that in my study three cases have

chromosome 7 alterations as one of very few main changes.

Specifically, clonal gain of one copy of chromosomes 7 and 8 was the

only abnormality in case T89-232. In T89-321, the karyotype was

49,XX, +7, +7, +del(3)(q2l). The karyotype of T89-154 was 45,XX,

+7, -22, iso(1q). An HSR involving the short arm of chromosome

7(p2l) was also observed in one patient. Therefore, my results may

suggest that over expression of a gene(s) on 7 may be an early

chromosomal changes which may playa role in the development of . .

ovanan carCInoma.

The analysis of chromosome mode has demonstrated that near

triploid chromosome numbers (59-82 chromosomes/cell) were the most

frequently observed, followed by hypodiploid modes «46

chromosomes/cell) and hyperdiploid modes (47-58 chromosomes/cell).

One interesting finding is that 7/9 (77.8%) cases with a hypodiploid

chromosome number had very complex karyotypes, with five of them

containing fragmented chromosomes including triradials and/or

quadriradials. The percentage of complex karyotype was much lower

in hyperdiploid (20%) and near-triploid (28.6%) tumors. The

mechanism causing this increase in chromosome rearrangements and its

apparent association with a decrease in chromosome number is not

understand. However, it is of interest to note that a potentially similar

phenomenon is operative in normal cell populations (95).

In regards to structural alterations, all chromosomes were

involved except chromosomes 4 and 20 in my study of 48 ovarian

63

carcinomas. Chromosomal structural rearrangements were clearly

nonrandom, with chromosome 1 the most frequently affected

chromosome, followed by chromosomes 7, 11, 12, 6, and 3. Structural

alterations of chromosome 1 have been very frequently reported in

variety human malignancies including: breast cancer (96), colon cancer

(97), retinoblastoma (98), uterine cancer (99), testicular cancer (100),

and ovarian cancer (59,68, 76,80,85,91). Visible loss of all or part

of the short arm of chromosome 1 resulted from deletions, iso(lq), and

unbalanced translocations which were observed in 19 tumors (-40%).

In one case (T89-154), iso (1q) was the only chromosomal change

observed. The most commonly involved regions on Ip were lcen-p13

(13 clonal structural changes) and Ip31-p35 (10 clonal changes).

Considering that chromosome 1 abnormalities are observed in a variety

of human malignancies, loss of material on chromosome 1 appears

most likely to be a secondary chromosomal change, perhaps playing a

role in tumor progression.

Rearrangements involving chromosome 7 were present in 22

patients with 27 clonal changes. Deletions of the short arm were

observed in 10 cases, four of them involving the same band (7p15).

Deletions involving bands 7pll, 7p12, 7p14, and 7p22 were also

observed (Table 4). Since visible deletion of 7p was observed in 20.8%

patients in this study, it is reasonable to believe that loss of activity of a

gene or genes on 7p may playa role in the development or progression

of ovarian cancer.

64

Chromosome 11 abnormalities were observed in 22 patients with

25 clonal changes observed. The most frequent site of abnormality

involved the short arm and particularly band 11 pIS .3 (observed in 10

patients). Additional material on IIp (11 p+) has been previously noted

by Atkin and Baker (76) as a frequent change (-50% of cases) in

ovarian cancer. Pejovic et al. (91) found that Ilpl3-pl5 was

commonly involved in the structural rearrangements. Ehlen and

Dubeau (90) used polymorphic probes on IIp 15 to examine the DNA

of normal and neoplastic tissues from 12 patients with ovarian

carcinoma. Loss of heterozygosity (LOH) was also frequently found

(4/9 patients). Chromosome 11 rearrangements involving loss of the

short arm are also frequently observed in other types of tumor,

including cervix carcinoma (101) and bladder cancer (102). All these

fmdings suggest that a gene or genes on chromosome 11 p 15 playa role

in the development or progression of ovarian cancer. Other structural

rearrangements involving IIp included translocations, homogeneously

staining regions (hsr), and deletion (Table 5). None of them involved

the band pIS. Deletions, additions, and translocations were also

involved in llq in my study. However, no specific band was clearly

identified.

Structural abnormalities involving chromosome 12 were observed

in 20 patients with 28 clonal markers. Six patients had additional

material on 12 involving band p13, which was the most frequent

abnormality involving the short arm of chromosome 12. In additional

to unidentified translocations, an HSR on 12p 11 was also observed in

65

one patient. Also, loss of a portion of material of 12p was seen in 6

patients, with four of them involving 12p. Zhou et al. (78) has reported

that the proto-oncogene c-ras-Ki which is localized to 12p12.1 was

overexpressed in 3n patients with ovarian carcinoma. Several other

reports (72, 79, 83, 84, 91) have presented that trisomy 12 is observed

(frequently as the sole karyotypic change) in benign or low grade

malignant ovarian tumors. Although trisomy 12 was not found in a

significant portion of cases in this study, structural rearrangements

involving 12p were very common. As discussed previously, the more

accurate localization of these rearrangements may result in the

identification of a gene(s) which would playa role in the development

and progression of ovarian cancer.

Rearrangements involving the long arm of chromosome 3 were

seen in 11 patients (Table7). Five of these 11 patients had apparent

terminal deletions of 3q, three of them involving the same band 3q21.

The remaining two were involved in 3q12 and 3q13 respectively.

Interestingly, 3q- (q21) was observed in two patients with simple

karyotypes. Case T89-166 had a karyotype of 46, XX, 3q-(q21), and

the karyotype of case T89-321 was 49, XX, +7, +7, +3q-(q21). The

abnormality of chromosome 3 clearly appears to be a common site of

chromosome change in ovarian carcinoma (68, 69, 71, 73, 74, 76, 77,

80,81,85,86,91). Historically, most of these abnormalities have

involved chromosome 3p which was not the most common site

observed in this study. Translocations involving 3q13-21 was observed

by Sheer (77) in two of four cell lines derived from ovarian cancer.

66

Bello and Rey (85) reported a cytogenetic study of 20 malignant

effusions from ovarian carcinomas with loss of 3q21-q23 or q27 -q28

observed in eight patients. Loss of material on 3q seems one of the

early, if not primary, changes and playa role in the development of

ovarian carcinoma.

Rearrangements involving the long arm of chromosome 6 were

observed in 11 patients (Table 7). Loss of bands q 15-qter were

observed in 7 tumors; 5 of these were caused by deletion, the remaining

two by translocation. Loss of chromosomal material from 6q has also

been commonly reported by many investigators (61, 62,64, 76, 77, 80,

81, 88, 91). In addition, Lee et al. (89) detected loss of heterozygosity

(LOH) in 9/14 cases with ovarian carcinomas at the estrogen receptor

gene locus at 6q27. Ehlen and Dubeau (90) also detected a high

frequency of LOH at loci on 6q in ovarian carcinoma. Deletion of

chromosome 6q may have a significance in ovarian carcinoma;

however, 6 deletions are also commonly seen in other types of

malignancies (e.g. malignant melanoma) (42,44). All these findings

suggest that there may be a gene(s) whose inactivation playa role in the

development of ovarian carcinoma which is resident on the long arm of

chromosome 6.

Cytogenetic manifestation of gene amplification was observed in 9

patients as HSRs with no evidence of DMs observed. These HSRs

were localized to Ip33, 6p22 (2 patients), 7p21, llpll, llpl4, llq,

12p 11, and 17 q25. Several oncogenes have been mapped within or

near these HSR regions. These oncogenes include JUN on Ip32, PIM

67

on 6p21, RAL on 7p15-p22, HRASI on llp15.5, KRAS2 on 12p12.1,

and ERBA2L on 17q25. It is not known whether the HSRs obselVed in

this study in fact relate to the amplification of these genes or other

unknown oncogenes. HSRs in ovarian cancer have been frequently

reported in several studies and have been localized to several regions

including: 3q13-q23 (64), 7p (68), 7qll and 17q12-q21 (73), 4q, 7p,

9p13, 12p12, and 16pll (91). The biologic significance of HSR in

ovarian carcinoma is uncertain. Further, considering the HSRs in my

study with those previously published, no consistent chromosomal site

for HSRs has been identified. For this reason HSRs in ovarian

carcinoma may be considered secondary changes, and therefore would

playa role in the progression (rather than genesis) of ovarian cancer.

Considered HSRs obselVed in my study appear not to be related to drug

resistance sequences (e.g. P-glycoprotein) because no patients in my

study had received chemotherapy prior to chromosome analysis.

As described previously, five of the 48 patients in this study

displayed simple karyotypic change with either numerical change only,

or a single structural change. These cases are of interest because they

may be providing information concerning the primary chromosomal

changes important in the initiation, promotion, and early development

of ovarian cancer. These findings, although limited, suggest that gain

of chromosome 7, and structural abnormalities of 3q may be early, or

primary changes in at least a subset of patients with ovarian carcinoma.

A further distinguishing feature of ovarian carcinoma in this study

was the finding that 17/48 patients (35.4%) contained cells with

68

complexity rearranged chromosome alterations. These cells typically

contained fragmented chromosomes, triradials and/or quadriradials, and

other kind of complex structural rearrangements, such that complete

karyotypic descriptions were not possible. This finding is remarkably

charateristic of ovarian carcinoma and exceedingly rare in other tumor

types (81). Whang-Peng et al. (68) noted an increase in the number of

structural rearranged chromosomes in patients who had received

chemotherapy before the cytogenetic analysis. In their study, the

median number of structurally rearranged chromsome was 3 in 13

untreated patients, while the median number increased to 9 in 29 treated

patients. The increase in the number of structural rearranged

chromosome was significant between the patients with or without

previous chemotherapy. However, no patients with ovarian carcinoma

in this study had received chemotherapy before the cytogenetic

analysis. Therefore, chemotherapy damage can not explain this finding

in my study. Very complex karyotypes in ovarian carcinoma have been

commonly seen by other investigators (71, 73,86,91). One possible

explanation of this finding is that some unknown mechanisms influence

specifically mitosis of ovarian cancer cells and results in a tremendous

increase in the breakage of chromosomes.

In conclusion, there is no single recurring clonal chromosome

change that charaterizes ovarian carcinoma. However, several

chromosomal regions unquestionably show nonrandom involvement.

Chromosome 1 was the most commonly involved in structural changes,

as it is equally in the majority of solid tumors. The most commonly

69

involved regions on chromosome 1 were 1 p31-p36 and 1 p 13-q 11.

Chromosome 3 structural abnormalities appeared to be particularly

common in my study of ovarian carcinoma. This study found deletions

of chromosome 3 in 5 patients involving the long arm, which is in

contrast to earlier reports which involved the short arm of chromosome

3. The other chromosomal bands and regions which were commonly

involved in strucatural abnormalities in this study included: 6q22-q25,

7pI5-22, 7q31-q34, Ilpl5, and 12pll-pI3. Molecular analysis may be

appropriate to further characterize these regions in search of genes

which may be directly affected by these rearrangements. It is hoped

that this detailed characterization of ovarian carcinoma may help

pinpoint the chromosomal locus of a gene(s) which plays a role in the

development and progression of ovarian carcinoma. The second

section of this thesis will define a novel stratagy to commence the

molecular analysis of chromosome rearrangements.

70

CHAPTER 3

APPLICATION OF CHROMOSOME MICRODISSECTION

Introduction

Although more than 2,000 genetic diseases have been recognized in

man, only a small number of them is understood at the molecular level

(103). Without knowledge of the underlying biochemical defects, the

genetic lesions can be identified most readily on the basis of their

chromosomal localization ("reverse genetics "). For the most part, these

genes must be cloned by first determining their position in the genome

and then successively narrowing the candidate region until a small

number of genes can be found and tested for their specific involvement.

With the development and application of several new techniques,

including the use of DNA polymorphisms as linkage markers (104),

pulsed field gel eletrophoresis (105), polymerase chain reaction (PCR)

(106), yeast artificial chromosome (YAC) (107), PI bacteriophage

vector (108), and flow-sorted chromosome (109), a number of disease

loci have been successfully mapped to specific chromosome regions.

These diseases include retinoblastoma (110), Duchenne muscular

dystrophy (111), cystic fibrosis (112), neurofibromatosis (52,53, 54),

Wilms' tumor (45), and familial polyposis coli (113, 114). However,

the process from linkage to the cloning of a gene is tedious and has not

yet been accomplished for most diseases. This is in part due to the lack

of narrowly spaced DNA markers for defined regions of the human

genome. Therefore, the isolation of chromosome region-specific

71

probes is an essential initial step in either the cloning of a hereditary

disease gene or the characterization of a cancer associated chromosome

alteration. Single chromosome-enriched libraries have been prepared

by using fluorescence-activated chromosome sorting techniques (115).

However, these chromosome-specific libraries are prepared from

human/rodent hybrids, and thus during the sorting process,

contaminating DNA from non-human regions abound in these libraries.

Thus, to use this kind of library requires the mapping of a large number

of clones in order to find probes which are located in the particular

subchromosomal region of interest. With this limitation in mind,

physical dissection of metaphase chromosomes is currently a

straightforward approach to solve this difficult problem.

Historical Perspectives on Chromosome Microdissection

Chromosome microdissection was first applied to DNA from

individual bands of Drosophila melanogaster polytene chromosomes

by Scalenghe et al. (116) in 1981. This approach was then extended to

mammalian chromosomes with the isolation of clones mapping to the

mouse t complex (117), and to a proximal region of the mouse xchromosome (118). In 1986, Bates et al. (119) successfully applied this

technique to isolate clones from the distal region of the short arm of

human chromosome 2. In 1987, Kauser et al. (120) applied

chromosome microdissection and micro cloning techniques to clone

DNA from distal7q. The methods used by all the aforementioned

groups were similar and involved the dissection of the specific

72

chromosome region with a glass needle, and the pooling of this material

into a nanoliter collection drop containing proteinase-K. This step was

repeated until the desired number of DNA copies had been collected

(usually 100-200 dissections). After inactivation of proteinase-K and

phenol extraction, the dissected DNA was then digested with a

restriction endonuclease followed by ligation of digested DNA with

corresponding vector. All these microdissection and micro cloning

procedures were performed in a nanoliters volumn under a microscope

with a micromanipulator and microinjector. The use of these

microdissection and microcloning techniques resulted in significant

DNA loss and as such were inefficient to saturate specific chromosome

regions with DNA markers. Also, this approach had limited value

because microdissection was only performed on unbanded

chromosomes. Accordingly, only the cell lines in which the

chromosome of interest can be identified without banding could be

used. Unfortunately, most human chromosomes cannot be identified in

unstained preparations severely limiting this approach. Another

technical limitation is that the size of the libraries derived from the

dissected DNA was severely limited due to DNA loss associated with

the multiple steps in the cloning process, and because dissected DNA

was microcloned directly without peR amplification.

In 1989, Liidecke et al. (121) successfully isolated probes for human

chromosome region 8q23-q24 by microdissection and microcloning.

They dissected chromosome region 8q23-q24 from GTG-banded

metaphase chromosomes (G-banding with trypsin-Giemsa) and then

73

amplified the dissected DNA by polymerase chain reaction (peR).

Microdissection of banded chromosome made it theoretically possible

to dissect any region on any human chromosome. Another technical

improvement, amplification of dissected DNA by peR, decreased the

required number of dissected DNA copies «50) and tremendously

enhanced the efficiency of microcloning. Since then, microdissection

and micro cloning with similar methods has been reported for human

chromosomes 11 (122-125),2 (126), and X (127,128).

In 1991, Trautmann et al. (129) used a microdissection library of

human chromosome 5q22 as a probe to detect the region encompassed

by the gene adenomatous polyposis using nonisotopic chromosomal in

situ suppression hybridization. Also in 1991, Lengauer et al. (130)

used chromosome painting of defined regions by in situ suppression

hybridization using libraries from laser-microdissected chromosomes.

In 1992, Deng et al. (131) reported the painting of specific bands on

chromosomes by chromosome in situ suppression hybridization using

peR products from a microdissected chromosome band as a probe

pool.

In summary, the technique used for chromosome microdissection

and microcloning have been improved significantly by Liidecke et al.

(121), resulting in the selection of dissected chromosome regions from

GTG-banded chromosomes and amplification of the dissected DNA by

peR. However, this method still uses restriction endonulease digestion

and DNA ligation steps, which, because of the exceedingly small

amount of microdissected DNA, must be performed using

74

microchemical techniques in a nanoliter microdrop contained in an oil

chamber. This method remains so labor-intensive that only a few

probes could realistically be prepared. Furthermore, this tedious

process of microdissection was restricted to normal metaphases

prepared from peripheral blood lymphocytes and had not been applied

to abnormal human chromosomes.

As mentioned above, chromosome microdissection and micro

cloning is the most straightforward approach to isolate chromosome

region-specific probes. The focus of this dissertation was then to

examine the feasibility of chromosome microdissection in the

examination of chromosome rearrangements in malignancy.

Unfortunately, the previous microdissection and microcloning

procedures required microchemical techniques in a nanoliter microdrop,

with the procedure being so labor-intensive that only a few probes

could realistically be prepared. It was therefore highly desirable to

simplify the microdissection and microcloning procedures. At least

three steps of the aforementioned procedures were targeted for

improvement. First, we proposed to process the dissected DNA

fragments by transferring them directly into a collecting solution in a

microcentrifuge tube, eliminating the transfer of fragments into a

nanoliter micro drop with a manipulator under the microscope. Second,

we proposed to use oligonucleotides from either Alu sequence (132-

134) or random sequence (135, 136) as primers to amplify the dissected

DNA fragments directly, instead of amplifying the dissected DNA after

restriction endonuclease digestion and ligation of the digested DNA

7S

requiring microchemical techniques. Third, ligation of PCR amplified

DNA fragments was proposed to occur directly with a T-tailed vector

(137) without any restriction endonulease digestion. The T-tailed

vector provided a single 3' T-overhang at the insertion site which can be

ligated with the 3' A-overhangs characteristically remaining on most

Taq polymerase PCR products.

Applications of Microdissection to the Analysis

of Complex Chromosome Rearrangements

Cytogenetic analysis of recurring chromosome abnormalities in

malignant cells has become an integral part of the diagnostic and

prognostic workup of many human cancers (14,138), and their

molecular analysis has facilitated the identification of genes related to

the pathogenesis of both hereditary diseases and cancer (139-143). One

significant technical limitation of conventional cytogenetic techniques

is the inability to characterize unequivocally all cytologically

recognizable chromosome rearrangements. For example, amniocentesis

may reveal unidentifiable de novo unbalanced translocation or unknown

supernumerary marker chromosomes (144, 145). Similarly, in many

human cancers (particularly solid tumors such as ovarian carcinoma

and malignant melanoma), the presence of unidentifiable marker

chromosomes or unidentifiable unbalanced translocations frequently

prevents complete karyotypic analysis (146). Recently, whole

chromosome composite "painting" probes (WCPs), constructed from

chromosome-specific DNA libraries, have been successfully used to

76

visualize specific chromosomes and their derivatives (147-151).

However, using these reagents to identify unknown chromosomal

segments requires appropriate painting probes for all 24 different

human chromosomes, and no regional sublocalization information is

obtained. Although cosmid clones have also been used for FISH

analysis of disease-specific loci (for example, the Philadelphia

chromosome in CML), this procedure is limited to those

rearrangements for which defined probes are available (152, 153).

Unfortunately, specific-probes are not available for the majority of

disease-associated chromosome rearrangements.

Tumor suppressor genes (whose inactivation results in the release of

a cell from normal growth control) have now been clearly documented

to play an important role in the multistep process of malignant

transformation (154-157). Evidence suggesting the presence of tumor

suppressor genes comes from several sources including: 1)

cytologically recognizable deletion of genomic material (157); 2)

evidence of allelic loss (assessed by restriction fragment length

polymorphism [RFLP] analysis) (158, 159); and 3) somatic cell

hybridization experiments documenting reversion of tumorigenicity

(41, 154). Nonrandom rearrangements of the long arm of chromosome

6 occur in several malignancies, including ovarian carcinoma and

malignant melanoma (160). The most frequent alteration of

chromosome 6 in these cancers is the simple deletion of the long arm of

chromosome 6 occurring in excess of 50% of both ovarian carcinoma

and malignant melanomas examined by banding analysis (42,43,44,

77

62, 65). In these cancers, the single most frequent region of cytologic

loss encompasses 6q16-q21. Loss of heterozygosity (LOH) for

chromosome 6q has also been documented in both ovarian carcinoma

and malignant melanoma (89, 90, 159), and recently microcell

mediated chromosome transfer has provided biologic evidence for a

tumor suppressor gene on 6q (41). To define more accurately this

deletion region, the production of a complete physical map of this

region of chromosome 6 would be invaluable. Unfortunately, only a

limited number of probes for the long arm of chromosome 6 are

presently available, and numerous additional probes will be require to

generate a physical map.

This problem has been the focus of this dissertation and the problem

was circumvented by chromosome microdissection of the chromosome

region of interest. Combining application of chromosome

microdissection and fluorescent in situ hybridization (we term Micro

FISH) can provide a useful method to produce painting probes for

whole chromosome, or chromosome-specific band regions. These

Micro-FISH probes have made it possible to identify explicitly the

chromosome constitution of virtually all cytologically visible

chromosome rearrangements. Coupled to the application of

microdissection, FISH reagents can be combined with band-specific

microclone libraries to rapidly generate an unlimited number of band

specific probes.

This section of my dissertation will describe in detail 1) how to

simplify the previously used chromosome microdissection and

78

micro cloning procedures, 2) application of this new procedure to the

isolation of chromosome region-specific DNA microclones, and 3)

combined application of chromosome microdissection and FISH to

approach previously intractable cytogenetic problems, such as the

identification of unrecognizable unbalanced translocations and

unidentifiable marker chromosomes.

Materials and Methods

The materials and methods used in this project are described in

more detailed in publications stemming from this dissertation (161,

162). A general outline of the strategy is shown in the flow diagram

illustrated in Figure 9. Briefly, the microdissected DNA fragments are

pooled into a 5 III collection solution in a 0.5 ml micro centrifuge tube,

followed by PCR amplification. The amplified DNA can be either used

as a painting probe to perform fluorescent in situ hybridization (FISH)

or immortalized as a chromosome band-specific library.

Chromosome Microdissection

Preparation of Metaphase Spreads

Whole blood (0.5 ml) from karyotypic ally normal donors was

cultured in a sterile flask containing 4.5 ml RPMI complete medium

with 0.1 ml Phytohemaglutinin (GIBCO, PHA, purified form). The

blood was incubated at 37°C for 65-68 hours and then cells were

79

arrested in metaphase by adding colcemid into the culture medium to a

fmal concentration of 0.05 Jlg/ml for 20 minutes (92). The blood

culture was then transferred to a 15 ml conical centrifuge tube and

centrifuged at 1,000 rpm for five minutes. The supernatant was

decanted and the remaining cells were then resuspended in 8 ml of

0.075 M KCI prewarmed to 37°C and incubated in a 37°C waterbath

for 20 minutes, followed by centrifugation at 1,000 rpm for 5 minutes.

The supernatant was removed and the remaining cells were fixed in

fresh fixative [3:1 (vol/vol) absolute methanol and glacial acetic acid]

for less than 2 hours before making slides. The cells were centrifuged

again at 1,000 rpm for 5 minutes, and the supernatant was decanted.

The cells were resuspended in 0.5-1.5 ml of fresh fixative for preparing

coverslips. Three drops of the cell suspension were then dropped onto

a clean coverslip (22X60 mm, No.1112) held at 45° angle and air dried.

The prepared coverslips were stored at 45°C for 2-3 days. G-banding

with trypsin-Giemsa (GTG) was performed prior to microdissection.

GTG-banding Procedure

The prepared covers lips were dipped in 50 ml HBSS with Trypsin

for about 1 minute at room temperature, followed by successive rinsing

in HBSS, 70% ethanol, and 95% ethanol. The coverslips were air dried

and then stained in a Giemsa Working Solution for 5-6 minutes,

followed by a brief rinse in distilled water.

80

6

Chromosome microdissection

(20 to 40 copies)

Direct ligation of PCR products with T-tailed vector.

Band-specific library.

Pool dissected DNA fragments into a S-ul collecting drop in a 0.5 ml centrifuge tube.

Proteinase K treatment (37°C for 60 minutes) and Inactivation of Proteinase K

(900C for 10 minutes).

PCR amplification.

Preparation of Micro-FISH probe.

Figure 9. A general outline of chromosome microdissection and microcloning procedure used in present study.

81


1. Trypsin Working Solution

Preparing trypsin stock (lOX) first by dissolving Trypsin 2.5%

(Gibco No. 610-5095AE) into 20 ml distilled water.

Trypsin Working Solution is prepared by adding 5 ml trypsin

stock in 45 ml HBSS.

2. Giemsa Working Solution

2% Giemsa's stain (Gurr's improved R66) in 0.01 M Phosphate

Buffer.

Giemsa's stain 1 ml

0.01 M Na2HP04 25 ml

0.01 M NaH2P04 25 ml

Preparation of Glass Needles

Glass needles used for microdissection were prepared by

extending glass micropipettes (1.2 mm X 4 inch, World precision

instruments, Inc.) with a vertical pipette puller (Model 720, David

KOPF Instruments). Prior to use in microdissection, microneedles were

treated with ultraviolet light for 5 minutes (Stratalinker, Stratagene).

Microdissection Process

Microdissection was performed with glass micro needles controlled

by a Narashige micromanipulator (Model MO-302) attached to an

inverted microscope (Zeiss, magnification 1250X) as described

elsewhere (161, 162). The dissected chromosome fragments (which

82

adhere to the microneedle) are transferred to a 5-10 III collection drop

(containing proteinase-K, 50 Ilg/ml) in a 0.5 ml microcentrifuge tube.

A fresh microneedle is used for each fragment dissected. In general,

20-40 copies are dissected from each chromosome band-specific

region. After dissection, the collection drop was incubated first at 37°C

for one hour and then at 90 ° C for 10 minutes to inactivate the

proteinase-K.

Amplification of Dissected DNA

Primers Used in Polimerase Chain Reaction (PCR)

a universal primer (UNl, 5'-CCGACTCGAGNNNNNNATGTGG-3')

(135, 136) has been used for generating region-specific Micro-FISH

probes and for generation of micro clone libraries. An Alu primer

(PDJ34, 5'-TGAGCYRWGATYRYRCCAYTGCACTCCAG CCT

GGG-3') (R=A/G, y=crr, W=Arr) has also been used for stratigies

where less complex probes are desirable.

PCR Conditions

The following components of the PCR reaction were added to

the tube containing dissected DNA fragments to a final volume of 50

Ill: [1.0 IlM UNI primer or 0.25 IlM Alu primer, 200 IlM each of

dNTP, 2 mM MgC12, 50 mM KCI, 10 mM Tris HCI, pH 8.4,0.1 mg/ml

gelatin and 2 U Taq DNA polymerase (Perkin-Elmer Cetus)]. The

reaction was heated to 94°C for 4 minutes, then cycled for 8 cycles of 1

minute at 94°C for denaturing the dissected DNA, 1 minute at 30°C for

83

annealling, and 3 minutes at 72 ° C for extension. This was followed by

another 30 cycles of 1 minute at 94°C, 1 minute at 56°C, 3 minutes at

72 ° C with a 10 minutes final extension at 72 ° C.

As described below, first round PCR worked well, a second round

PCR would be carried out. Usually, 2 -5 /-LI of first round PCR product

were added to a PCR reaction solution ( exactly the same as first round

PCR) at a final volume of 50 /-Ll. The reaction was heated to 94°C for 4

minutes, followed by 15-20 cycles of 1 minute at 94°C, 1 minute at

56°C, and 3 minutes at 72°C with a 10 minutes final extension at 72°C.

Testing PCR Result by Electrophoresis

Ten /-LI of the 50 /-LI PCR products was examined on 1 % Agarose

gel in 0.5X TBE buffer to check the microdissection and PCR results.

Following eletrophoresis, the gel was stained with a 0.5 /-Lg/ml ethidium

bromide solution for 20 minutes. The DNA on the agarose gel was

visualized using UV fluorescence transilluminator (Ultra-Violet

Products, Inc.) and photographed (Polaroid, Folodyne, Inc.).


1. TBE buffer (pH 8.25)

Tris-borate 0.089 M

Bori-borate

EDTA

0.089 M

0.002 M

84

Microc1oning

Purifying PCR Products by Centric on

The generation of DNA microc1one libraries was only

performed if the blank reaction was completely negative as determined

by running the PCR product on an agarose gel. Unincorporated

nuc1eotides and primers were removed using a Centricon-30

concentrator. Second round PCR products were pooled into the sample

reservior of the Centricon-30 concentrator (30,000 MW cutoff,

Amicon). A total of I ml of IX TE buffer was added into the sample

reservior of the concentrator, and centrifuged at 6,000 rpm for 10

minutes. At this time, another 1 ml of 1 X TE buffer was added into the

sample reservior and spined at 6,000 rpm for 18 minutes. Finally, the

concentrated sample was transferred into the retentate cup by inverting

the unit and centrifuged at 1,000 rpm for 2 minutes. The collected

sample was then transferred into a 1.5 ml centrifuge tube for further

use.

Biological and Reagent:

1. TE Buffer (IX)

Tris·CI (pH 7.4)

EDTA (pH 8.0)

Precipitation

10mM

1mM

Purified PCR products were concentrated by ethanol precipitation.

A total of 1/10 volume 3M sodium acetate and 2 volume cold absolute

ethanol were added into a 1.5 ml Eppendoff tube containing the purified

85

PCR products and mixed by inverting the tube. The sample was then

frozen at -20°C for 2 hours, followed by centrifugation at 14,000 rpm

for IS minutes at 4°C in a microfuge. The supernatant was removed

with an automatic micropipetter. The tube was then half filled with

70% ethanol and centrifuged at 14,000 rpm for 2 minutes at 4°C. The

supernatant was recovered with an automatic micropipetter and the

pellet was dryed. The DNA pellet was then dissolved in the desired

volume of IX TE buffer.

Ligation ofPCR Products with A T-tailed Vector

The PCR products were directly inserted into a T-tailed derivative

(pGEM) [pGEMSfZ(+), kindly provided by Weisblum (137)]. This T

tailed vector provides a single 3' T-overhang at the insertion site which

can be ligated with the 3' A-overhangs characteristically remaining on

most Taq polymerase PCR products (137). Ligation reactions are

perfonned under the following conditions. First, the concentration ratio

ofPCR products of the microdissected DNA to pGEM5fZ(+) vector

(cut with XcmI) is 2:1. The reaction is performed in 10 Jll of reaction

solution (200 mM Tris·Cl pH 7.6, 50 mM MgCh, SO mM dithoitheritol,

SOO Jlg/ml bovine serum albumin, 1 U T4 DNA ligase) at 12°C

overnight. After ligation, an ethanol precipitation was performed. The

pellet was dissolved in 20 Jll of O.SX TE buffer.

86

Transformation by Electroporation

Transformation of E. coli cells was perfonned by electroporation

(163, 164). A total of 1,.d of ligation product was mixed with 20 f..ll of

competent E. coli cells (XLI-blue). The electroporation was performed

with the BRL Cell-Porator™ electroporation system. E. coli cells were

introduced into polypropylene electroporation chambers containing two

flat aluminum electrodes, 0.4 cm apart. The cell suspension in the

electroporation chamber was chilled on ice for 3 minutes prior to

electroporation. The electroporation chamber was then transferred to

the chamber safe and cooled in ice water during electroporation. Cells

were electroporated at 2.4 kV and 2 kQ. After pulse the cells were

transferred to 2 ml Luria-Bertani (LB) medium (165) and incubated at

37°C with shaking at 225 rpm for 1 hour. A total of 50 f..ll of this

transformation solution was then spread on LB plates containing 100

f..lg/ml ampicillin, 50 f..lg/ml IPTG, 50 f..lg/ml X-gal. The plates were

incubated at 37 ° C overnight.


1. LB Medium (1 liter)

bacto-tryptone

bacto-yeast extract

NaCI

dH20

10 g

5g

10 g

to 1000 ml

Shake until the solutes have dissolved. Adjust the pH to 7.0

with 5 N NaOH. Sterilize by autoc1aving for 20 minutes at

15 lb/sq.in. on liquid cycle.

87

2. LB Plates

Prepare liquid medium as described above and add 15 g/liter

bacto-agar in the medium before autoclaving. Sterilize by

autoclaving for 20 minutes at 15Ib/sq.in. on liquid cycle.

Allow the medium to cool to 50°C and then can be poured into

plates.

PCR Mini-preparation

Individual white bacterial colony were picked and suspended in

an Eppendoff tube containing 50 JlI of 1 % Triton X-I00, 20 mM

Tris· HCl, pH 8.5, 2 mM EDT A (166). The tube was then placed in a

boiling water bath for 15 minutes for complete disruption of bacterial

membranes. T7 primer (5'-TAATACGACTCACTATAGGG-3') and

pUC/M13 reverse primer (5'-CAGGAAACAGC TATGAC-3') were

used to amplify cloned DNA segments. PCR was performed for 30

cycles at 94°C for 1 minute, 52°C for 2 minutes, and 72 ° C for 3

minutes. A final extension at 72°C for 5 minutes was then performed.

Electrophoresis and Preparation of Probe

The PCR minipreparation products were checked by agarose gel

electrophoresis. Usually 8 ul of PCR product were run on a 1 %

Agarose gel in 0.5X TBE buffer as mentioned before. Inserts amplified

by PCR with sizes of 200-600 bp were chosen as probes. The

remaining 42 JlI of PCR product was then precipitated with a 1/10

volumes of 3M sodium acetate and 2 volume of cold absolute ethanol

88

as described before. The DNA pellet was dissolved in 10 III of IX TE

buffer, followed by running on a 1 % low melting point agarose gel in

IX T AE buffer. After electrophoresis, the gel was stained for 20

minutes in a 0.5 J.1g/ml ethidium bromide solution. The desired band

was excised cleanly on an VItro-violet fluorescence transilluminator.

The cut band was put into a preweighted 1.5 ml micro centrifuge tube.

Distilled water was added at a ratio of 3 ml H20/g of gel. The tube was

then placed in a boiling water bath for 7 minutes to dissolve the gel and

denature the DNA. Radiolabeling of the probes were perfonned

following the technique of Feinberg and Vogel stein (167, 168).

Briefly, the DNA (20-30 ng) was labeled overnight at room temperature

by the addition of the following reagents: 1) H20 (to a total volume of

50 J.11); 2) 10 J.11 of oligo-labeling buffer; 3) 2 J.11 of bovine serum

albumin (10 mg/ml, Sigma); 4) DNA in agarose (up to 32.5 Ill); 5) 5 III

of 32P-dCTP (Amersham No. PB 10205,3,000-4,000 Ci/mmole, 10

IlCi/IlI); 6) 2 units of large fragment of E. Coli DNA polymerase I

(BRL). The reaction was stopped by addition of 200 III of stop buffer

(169).


1. TAE Buffer (IX)

Tris-acetate

EDTA

0.04M

0.001 M

89

2. Stop Buffer

NaCl

Tri-HCI (pH 7.5)

EDTA

Sodium dodecyl sulfate (SDS)

dCTP

20mM

20mM

2mM

0.25%

111M

Regional Chromosomal Assignment of Probes

Once DNA microclone libraries are established, it is important to

confirm the regional specificity of DNA microclones. For our

chromosome 6 libraries, a recently described series of somatic cell

hybrids which identifies 7 regions along the long arm of chromosome 6

(151) was utilized to confirm the chromosomal position of a series of

DNA microclones. A summary of the cell line designation, rodent

background, chromosome 6 segment, and notation of other human

chromosomes are provided in Table 8 (151). Ten microgram of human

placenta DNA, hamster DNA, mouse DNA, and each hybrid DNA from

this panel were digested with EcoRI (l00 Units each). The digested

DNAs were electrophoresed on a 1 % agarose gel and the DNAs were

then blotted onto a nylon filter (Schleicher & Schull) by vacuum

blotting. Prehybridization and hybridization were performed following

the procedures of Meese et al. (170). Nylon membranes were

prehybridized at 65°C overnight in sealed plastic bags containing

prehybridization mixture. After prehybridization, the labeled probes

were added, and hybridization was carried out for 12-18 hours at 65°C.

90

After hybridization membranes were washed 2 times with Wash

Solution I at 55°C for 10 minutes each, and 2 times with Wash Solution

IT at 55°C for 30 minutes each.

Biologicals and Reagents

1. Prehybridization Mixture (20 ml)

SET (20X) 4 ml

Dextrans (50%)

SDS (20%)

Napp (10%)

dH20

SSSS DNA

2. 20X SET

4ml

1 ml

0.2 ml

10.6ml

0.2ml

NaCI 3 M

Tris-HCI (pH 7.8) 0.4 M

EDTA 20mM

3. 20X SSC (pH 7.0) (1 liter)

NaCI

Sodium Citrate

dH20

175.3 g

88.2 g

to 1,000 ml

4. Wash Solution I (lliter)

20X SSC 100 ml

20% SDS

dH20

5ml

to 1,000 ml

91

5. Wash Solution II

20X SSC

20% SDS

dH20

6ml

5ml

to 1,000 ml

Table 8. Hybrid Mapping Panel for Chromosome 6*.

Other Huma

Cell line Background Chromosome 6 Chromosome(s}

Q998-lB Hamster Whole #6 None

MCH262

AID6 Mouse Whole #6 None

640-5A Hamster 6( cen-qter) 9,10, Y

Q836-3A Hamster 6(qI3-qter) Y

5184-4 Mouse 6(q21-qter) 3, der7, 10, 15,

17, 19, 21, 22

HAL26-12 Mouse 6(pter-qI3) 3,15, 17,21

R21-lB Hamster del(6)(q21q25) 6q-, Y

MCH381.2D Mouse del(6)(q16q24) None

* From Meese et al. (151).

92

Autoradiography

Membranes were autoradiographed with Kodak X-OMAT AR

film and DuPont Cronex intensifying screens at -70 ° C for 1-4 days. The

film was then processed using a Kodak X-OMAT developer and fixer.

Flourescent in situ Hybridization

Labeling of Probes with Biotin-ll-dUTP by Nick Translation

UNI-PCR amplification products were labeled with biotin-ll-dUTP

using nick translation procedures (171). First, pipetting of the

following components into a 1.5 ml microcentrifuge tube on ice was

performed:

5 JlI lOX dNTP Mix

5 JlI lOX Enzyme Mix

(1 Jlg) DNA (PCR product of microdissection)

d H20 to 50 JlI

The components were mixed well and centrifuged at 14,000 rpm for

5 seconds. The mixture was then incubated at 16°C for 1 hour and

stopped by addition of 5 JlI of Stop Buffer. Unincorporated nuc1eotides

can be seperated from the labeled DNA probes by ethanol precipitation.

1/10 volume 3M sodium acetate and 2 volumes of cold absolute ethanol

were added to the reaction tube. This was then mixed by inverting the

tube and freezen at _20°C for at least 1 hour. The tube was then

centrifuged at 14,000 rpm at 4°C for 15 minutes. The supernatant was

carefully removed with an automatic pipetter and the pellet was air

dried. The pellet was resuspended in 20 JlI 1 X TE buffer (pH 7.5).

93

Labeling of Probes with Biotin-ll-dUTP by PCR

PCR amplified microdissected DNA was labeled with biotin-ll

dUTP in a secondary round PCR reaction (1 JlM UN 1 primer, 200 JlM

each of dNTP, 2 mM MgC12, 50 mM KCI, 10 mM Tris·HCI, pH 8.4,

0.1 mg/ml gelatin and 2 U Taq DNA polymerase). Three reactions with

a total of 150 Jll of products were cycled 15-20 times for 1 minute at

94°C 1 minute at 56°C, and 3 minutes at 72°C (with a 10 minutes final

extension at 72 ° C). The products of these reactions were purified with

a Centricon-30 filter as described before. Purified PCR products were

then concentrated by ethanol precipitation. The pellet was resuspended

in 20 Jll 1 X TE buffer.

Slides Preparation

Peripheral blood lymphocytes from nonnal donors (46,XX or

46,XY) were used for FISH. Metaphase spreads were prepared on

clean slides as described by Trent and Thompson (92). The area of the

slide with the highest mitotic index was located using low power phase

contrast microscopy. The back of the slide in this area was marked

with a scribe to indicate roughly the boundaries of the FISH reaction.

The hybridiaztion and (multiple cytochemical reactions) were carried

out on the same area of the slide to minimize the amount of reagent

required.

94

Probe Preparation

in situ hybridization master mix MM2.1 was prewarmed at

room temperature so that its viscosity was decreased to the point that it

could be accurately pipetted. The composition of hybridization mix is

as follows:

7 J.lI MM2.1

2 J.lI biotin-labeled probe

1 J.lI human CotI blocking DNA (BRL)

The final concentration of the probe is used at 20 J.lg/ml. The

hybridization mix is vortexed for 5 seconds, followed by centrifugation

in a microfuge to collect the liquid on the bottom of the tube. The

hybridization mix was then heated to 70 ° C for 5 minutes to denature

the probe and prehybridized in a 37°C waterbath for 10-20 minutes.

Biological and Reagent:

I.MM2.1

formamide

Dextran sulfate

20X SSC

RNase Treatment

5.5 ml

I g

0.5 ml

The slides prepared for FISH were first treated with RNase. A

total of 200 J.lI of RNase (100 J.lg/ml in 2X SSC) was applied to each

slide and covered with a large coverslip (24 X 50). The slides were

incubated in a moist chamber at 37°C for 1 hour, followed by rinsing in

9S

4 changes of 2X SSC (pH 7.0) at room temperature for 2 minutes each.

The slides were then dehydrated in a graded ethanol series (70%, 85%

and 100%) and air dried.

Denaturation

Target DNA was denatured by immersing the slide into a 50 ml

coplin jar with denaturing solution (70% formamide, 2X SSC) for 2

minutes at 70°C. Because a room temperature slide put into 50 ml

coplin jar will cause the temperature to drop by about 1 ° C, the actual

temperature of the denaturing solution is maintained 1 ° C higher for

each slide. Following denaturation the slides were quickly transferred

to 70% ethanol for 1 minute and agitated to rinse off the denaturing

solution. This was followed by dehydration in 85% and 100% ethanol

and air drying.

Biologicals. and Reagents:

1. Denaturing Solution (for 50 ml of IX)

formamide 35 ml

20X SSC 5 ml

dH20 10 ml

Hybridization

Prewarmed denatured slides were maintained on a slide warmer at

45°C for five minutes. Prehybridized probes were centrifuged briefly

to collect all of the liquid to the bottom of the tube. Prehybridized

probe mix (10 Ill) is placed on the target area of the slide and then a

96

22X22 mm (No. 11/2) coverslip is placed over the probe mix. Air

bubbles will preclude hybridization, and should therefore be avoided as

far as possible. The edges of the coverslip were then sealed with rubber

cement to prevent evaporation of the hybridization mix. The slides

were then incubated overnight at 37°C in a moist chamber.

Washing

After hybridization, the rubber cement gasket was removed

from the slide with forceps and slides were washed 3X in 50%

formamide, 2X SSC at 42°C for 3 minutes with periodic agitation. This

was followed by a wash in 2X SSC at 42°C for 3 minutes. The slides

were then washed in PN buffer 3 times at room temperature for 2

minutes.


1. PN buffer (1 liter of IX)

0.1 M sodium phosphat, 0.1 %(v/v) NP 40, pH 8.0.

dH20 800 ml

Na2HP04·7H20 25.2 g

NaH2P04·H20 0.828 g

Dissolved the mix thoroughly, and added dH20 to 100 ml. The

solution was then filtered through a 0.45 micron filtration

apparatus and added with dH20 to 1 liter. Finally, 1 mlof

NP40 (Calbiochem) was added and mixed thoroughly.

97

Staining with Flourescein Conjugated Avidin and

Amplification with Anti-avidin

The washed slides were transferred into a foil wrapped Coplin jar

with fluorescein conjugated avidin (5 )lg/ml, Vector Laboratories) in

PNM buffer at room temperature for 20 minutes. After avidin

treatment, the slide was washed in 3X of PN buffer at room temperature

for 2 minutes with periodic agitation. Amplification of the fluorescent

signal was accomplished by applying a biotinylated goat-anti-avidin

antibody in PNM buffer (5 )lg/ml, Vector Laboratories) followed by

another layer of avidin. The time for anti-avidin treatment was also 20

minutes followed by a wash in 3X of PN buffer as above. The slide

was then incubated in fluorescein conjugated avidin as described above.


1. PNM Buffer (for 1 liter of IX)

PN buffer supplemented with 5% (w/v) nonfat dry milk and

0.02% (w/v) Sodium Azide.

Carnation nonfat dry milk

Sodium Azide

PNbuffer

25 g

O.l g

500ml

This mix was combined in a 1 liter bottle and shaken several

times over a 30 minutes period to allow all of the milk go into

the solution. The solution was then incubated at 37°C for 2

hours and left on lab bench for 1 day, followed by spinning in

a table top centrifuge to pellet the solids. The supernatant was

transferred to another bottle and stored at 4 ° C until to be used.

98

Counterstain

After the final washing, the slides were thoroughly drained and 40

J.d of Antifade solution with 0.5 Jlg/ml of propidium iodide was placed

on the target area of the slide followed by covering a 24X50 mm

coverslip on the area.


1. Antifade Solution

Dissolve 100 mg p-phenylenediamine dihydrochloride in 10 ml

of PBS and adjust pH to 8.0 with 0.5 M Carbonate-Bicarbonate

Buffer. Filter the solution through 0.22 Jl syringe filter. Add to

90 ml glycerol. Store in dark at _20°C.

2. Propidium Iodide Counterstain in Antifade Solution (0.5 Jlg/ml)

Propidium Iodide stock (1 mg/ml) 2.5 JlI

Antifade solution 5 ml

Mix well and store in the dark at _20°C.

3. Carbonat Bicarbonate Buffer (0.5 M NaHC03)

NaHC03 0.42 g

dH20 8 ml

Dissolve salts and adjust pH to 9 with NaOH.

Microscopy and Photography

The counterstained slide was scanned using a Zeiss Axiophot

microscope equipped with a dual bandpass fluorescein-rhodamine filter.

The quality of fluorescent signals and metaphase spreads were

determined using an 100X oil immersion objective. Selected

99

metaphases with clear fluorescent signals were photographed with

Kodak. Ektachrome 160T or Kodak Gole Plus 400.

Results

Preparation of Chromosome Region-Specific Libraries

Based principally upon the compiliation of cytogenetic

information (44) and to a lesser degree LOH information (159), the

ovarian carcinoma and melanoma deletion region is assumed to

encompass 6qI6-q21. Accordingly, chromosome microdissection was

carried out in an effort to generate clones within this region. Forty

copies of 6q21 were dissected with representative examples of

metaphase chromosomes following physical microdissection shown in

Figure 10. Figure 10a-d shows sequential photographs illustrating the

process of microdissction of this chromosome band region. Figure 10e

h shows a series of dissected chromosomes to illustrated the

reproducibility of dissection. After proteinase-K treatment, the

dissected DNA fragments were amplified with the UNI primer by PCR.

As the primer includes a random hexamer adjacent to the 3' specific

hexamer, the annealing temperature of first the 8 cycles amplification

was reduced to 30°C to allow sufficient priming to initiate DNA

synthesis at frequent intervals along the template. The defined

sequence at the 3' end of the primer tends to separate initiation sites,

increasing product size. As the PCR product molecules all contain a

100

common specific 5' sequence, the annealing temperature was raised to

56° C after the first 8 cycles. A blank reaction consisting of an

identically processed empty collection drop was performed with each

experiment. The experiment was only continued if DNA synthesis was

not apparent in the blank reaction as determined by ethidium bromide

staining of agarose gels. Figure 11 illustrates a representative size

distribution of PCR-amplified DNA from dissected fragments. The

negative control (no DNA) was blank after PCR amplification, while

both a positive control (5 ng human placenta DNA) and dissected DNA

resulted in a smear ranging from approximately 200-900 bp. Before

microcloning, the direct PCR-amplified dissected DNA (labeled with

biotin-ll-dUTP by second round PCR) was used as a probe to

hybridize to normal human metaphase chromosomes by FISH. Figure

10i illustrates the FISH results which clearly confirm the

sublocalization of the labeled probe to the dissected region (6q21).

After purified, PCR products were then directly inserted into aT-tailed

derivative of pGEM5fZ( +), generating a library of 20,000 clones.

Examples of 12 clones from this library are shown in Figure 12, with

representative insert sizes ranging from 50-800 bp.

Forty-one of the microclones from this library were radiolabeled

and tested as probes against Southern blots composed of our hybrid

mapping panel (151). Of these probes, 21/41 (51 %) generated single

copy hybridization signals, 9/41 (22%) contained repetitive DNA

sequence, and 11/41 (27%) failed to provide a detectable signal on

hybridization to human DNA. The 21 single-copy probes ranged in

101

size from 350-620 bp with an average size of approximately 500 bp.

The chromosomal sublocalization of the 21 single-copy micro cloning

was comfinned by mapping relative to our recently published hybrid

mapping panel (151). Figure 13 illustrates the pattern of hybridization

of microclones to Southern blots of this mapping panel. The

chromosomal content of these hybrids that subdivide the long ann of

chromosome 6 into seven different regions is illustrated in the lower

portion of Figure 13. The targeted region of dissection was 6q21, with

the majority of probes tightly distributed around the dissected region

and no clone originated from a chromosome other than 6. Although

redundancy is expected in a library generated by PCR, each of these 21

single-copy probes tested recognized a distinct restriction fragment in

human DNA.

Preparation of Whole Chromosome Painting Probes

As mentioned before, karyotypes in many human cancers

(particularly ovarian tumors) are so complex that complete analysis by

conventional banding techniques are often impossible. This technique

shortcoming seriously hampers accurate karyotype analysis.

Chromosome microdissection combined with FISH provide an

inportant mean to overcome this shortcoming. As mentioned

previously, whole chromosome composite "painting" probes (WCPs)

have been constructed from chromosome-specific DNA libraries for

several human chromosomes (147-150). However, the generation of

whole chromosome painting probes prepared by chromosome

102

microdissection are easier to generate, and appear to provide a more

complex pattern of hybridization than WCP's generated from somatic

cell hybrids. Only 20 copies of each chromosome needs to be dissected

in order to prepare a WCP. The present procedure for preparing a WCP

probe is rapid; synthesis of useful fluorescence probes can be

accomplished in less than 24 hours. After chromosome

microdissection the dissected DNA is amplifiied with the UNI primer

by PCR. Second-round PCR with 2 J..lI first round PCR products and

Biotin-l1-dUTP is performed to generate the WCP probe. Figure 14

illustrates the FISH results using WCP probes derived from

chromosomes 6 and X.

Preparation of Chromosome Region-specific Painting Probes

One of the many possible applications of chromosome

microdissection is the generation of chromosome band- or region

specific painting probes. As mentioned before, at least 149 nonrandom

chromosome rearrangements involving chromosome specific-regions

have been identified in 43 different malignancies (15). Identification of

a consistent chromosomal region alteration associated with a type of

malignancy by cytogenetic analysis often provide the initial basis in

determining the underlying molecular change at the locus.

Chromosome microdissection combining with FISH (Micro-FISH)

provide a easy way to generate chromosome band- or region-specific

painting probes (Micro-FISH probes) which can be used to identify the

visible chromosome rearrangement involving the target band or region.

103

~ o

~ a~ b ry-

~'" ~ ~J .~.J • ,.. :& V ~." .~. , ~i ~ ~:i ~~.!~ . ~~

f' e ,-~

,I! Ii ~\,

/71

f

~

j /'U

" .. t;

"" ""f"': ,It::" ...

J.

, £

.?IU

Figure I O. Chromosome microdissection. a-d, Sequential photographs illustrating the microdissection of chromosome band 6q21-23. e-h, Series of dissected chromosome 6's to illustrate the reproducibility of dissection. i, A Micro-FISH probe for 6q21 was hybridized to a normal peripheral blood lymphocyte metaphase demonstrating the band-specific fluorescence. Inset shows hybridization to the domains of interphase nuclei that encompass chromosome 6q2I.

bp

2176-

1230-

653-517-394-

~~8= 154-

1 2 3

Figure 11. peR products from microdissection of chromosome

band 6q21 resulting in a smear ranging from 200 to

900 bp (Lane 3). Lane 1, peR reaction with no DNA;

Lane 2, peR products of 5 ng total human DNA; peR

products (10 lll) were size fractionated on 1 % agarose

gel and stained with ethidium bromide.

105

Clone :"Julllher 21 ~2 3.1 X ~l) J3 56 50 .1X 5X 5~ 3~

VCl:lOr -

bp I 76()

123() I(m

653 517

3l)~

2l)X

15~

Figure 12. Inserts recovered by PCR from 12 individual microclones

size fractionated on 1 % agarose gel and stained with ethidium bromide. These peR products include 200 bp of vector sequence as indicated in left-hand lane. The average size of the microclones (-500 bp) reflects the size distribution of the primary PCR products.

106

Clone Number

67

7

16

:'.linol'lonc rcgional mapping

'l. -;, 7, X. II, Ill. 1'1.21. 2:'.

2(1, '" .. \ I. .'2. 'fl. lX .. ll).

,"'! ( - ~ ~: .,--,I' I ;.,.-;'::i'~~ ,"I,

;';'--

t'" '., r' - ., ---_.--- ... !'JII8a

, ---••• ~::t ~.:;.~:.'" ~:t ••• •• -. --

- l).--Ikh

- 3.6kl1

- X.Xkh

Figure 13. Regional assignment of microclones. (Top) Southern blots using clones 67, 7, and 16 against EcoRI-digested DNA from somatic cell mapping panel comprised of eight cell lines. (Bottom) The chromosome 6 content of this panel is shown by representative idiograms of panel members. The region of 6 dissected is illustrated on the right of the idiograms for this hybrid mapping panel, with the regional assignment of microclones illustrated adjacent to the left of the idiograms.

107

Figure 14. Whole chromosome paints generated by microdissection. Whole chromosome painting probes for X and 6 were hybridized to nonnallymphocyte metaphase demonstrating chromosome-specific fluorescence. a, Spectrum orangelabeled whole X chromosome painting probe (red). b, Biotinlabeled whole chromosome 6 painting probe (yellow).

108

As described previously, chromosome 6 is frequently altered in ovarian

carcinoma, and is a candidate chromosome on which a tumor

suppressor gene is localized. In order to obtain a series of Micro-FISH

probes for chromosome 6, 11 different dissections were made, allowing

the generation of painting probes for subbands of the entire

chromosome. The painting probes were generated by directly labeling

probes with Biotin-ll-dUTP by PCR. Figure 15 shows examples of

FISH results using several band-specific painting probes on

chromosome 6 generated by chromosome microdissection.

Two applications of the region-specific painting probes in molecular

cytogenetic analysis are illustrated in Figure 16. First, a painting probe

was generated for the specific chromosomal band 6q21-22 (Fig. 16a).

This probe was hybridized to metaphase cells carrying a reciprocal

translocation involving the 6q22 band region [t(6;7)(q22;q21)]. In this

experiment, the 6q21-22 Micro-FISH probe was applied simultaneously

with a chromosome 6-WCP (Imagenetics, Naperville, IL) to localize

unequivocally the dissected material in this cell (Fig. 16a). The results

illustrate the application of this approach to span a translocation

breakpoint. By selecting different colored fluorochromes for the

Micro-FISH probe (green), and the chromosome 6-WCP (red/orange),

areas binding both probes appeared yellow, allowing the normal

chromosome 6 and both of its derivatives to be identified definitively.

The second application of Micro-FISH probes illustrated in

Figure 16 is the identification of interstitial deletions (Fig. 16b, c).

Here chromosomes were obtained directly from a malignant melanoma

109

tumor biopsy and analyzed by Micro-FISH for the possible loss of

chromosome 6q21-22 sequences. Loss of sequences from the long arm

of chromosome 6 have been implicated in the genesis or progression of

ovarian carcinoma and malignant melanoma. Simultaneous

hybridization with a chromosome 6-WCP and 6q21-22 Micro-FISH

probe demonstrates an interstitial deletion in one chromosome 6. The

deletion chromosome was also recognizable in interphase nuclei

(Figure 16c). These results demonstrate the potential value of Micro

FISH probes for assessing chromosomal deletion, providing a general

approach for identifying deletion of specific chromosome regions in

biopsies of human cancers.

Detection of Nonreciprocal Translocations and Breakpoints

Another application of chromosome microdissection combining

FISH is to dissect the unknown part of a nonreciprocal translocation

and generate a specific Micro-FISH probe. This probe is then used to

determine the origin of the unidentified chromosomal fragment by

hybridizing back to normal metaphase chromosomes. The

microdissection can also be used to span a translocation breakpoint, in

order to determine the specific band involved in the breakpoint on both

chromosomes.

Figure 17 illustrates the use of a Micro-FISH probe to identify

chromosomal rearrangements which were impossible identified by

standard cytogenetic analysis. A malignant melanoma cell line

(UACC-383) was determined by G-banding analysis to contain an

110

Figure 15. Examples of FISH results using band- or region-specific painting probes on chromosome 6 preparing from microdissection. a.6q13-14. b.6q22-23. c.6qter(q27). d. Whole 6 (red) combining three region-specific probes (yellow); 6pter(p25), 6cen(p 12-q 1 1), and 6qter( q27).

11 1

Figure 16. Mapping of a translocation and deletion chromosomes with Micro-FISH probes. a, (Top) G-banded normal chromosome 6 (center) and derivative (der) chromosome 6 (left) and 7 (right) present in NIGMS Human Genetic Mutant Cell Repository line No. GM05184. (Bottom) Simultaneous hybridization with a chromosome 6-WCP (orange) and a Micro-FISH probe for band 6q21-22 (green). Regions hybridizing to both probes appear yellow. Left, der(6) chromosome illustrating the short arm and proximal long arm of chromosome 6 (the yellow band represents the microdissected material spanning the translocation breakpoint).Center, normal chromosome 6 showing band location of the Micro-FISH probe. Right, der(7) showing the distal long arm of chromosome 6 (painted by the 6-WCP and 6q21-22 Micro-FISH probe). b, Illustration of the use of Micro-FISH probe to assess an interstitial deletion of chromosome 6 from a biopsy sample of a melanoma tumor (UM91-023). The two center chromosomes represent a normal chromosome 6 and a chromosome 6 with an interstitial deletion of 6q [del(6)(q15q23)]. Adjacent to each chromosome is an example of the simultaneous of the chromossome 6-WCP (red/orange) and the 6q21-22 Micro-FISH probe (green) which document the loss of sequences recognized by this probe. c, Interphase nucleus from the tumor used in b, demonstrating the two red/orange domains recognized by the 6-WCP. Only one chromosome 6 domain (arrow) demonstrates a yellow area from hybridization to both the 6-WCP and the 6q21-22 Micro-FISH probe.

112

unidentifiable de novo translocation [t(6;?)(q14;?)]. A Micro-FISH

probe for this unknown chromosomal material involving the

translocation was generated by microdissecting 40 copies of the

unknown fragment. The Micro-FISH probe was then hybridized to

UACC-383 chromosomes (Figure 17d) and to normal lymphocyte

chromosomes (Figure 17e, 0. The results indicate that the dissected

material was derived from the terminal long arm of chromosome 21

(q21-qter). This experiment demonstrates the potential of Micro-FISH

probes to reveal information previously hidden in the banded

karyotype.

One additional application of the Micro-FISH procedure in

molecular cytogenetic analysis of a de novo translocation is illustrated

in Figure 18. A malignant melanoma cell line (UACC-837) was

determined by G-banding analysis to contain a terminal deletion of

6q(q21-qter). In order to determine the breakpoint, a Micro-FISH

probe was generated by microdissecting the terminal material of this

deletion marker. The results of hybridization of this Micro-FISH probe

to UACC-837 chromosomes and normal lymphocyte chromosomes

indicate that this terminal deletion was in fact a nonreciprocal

translocation t(6;8) (Figure 18).

Detection of Homogeneously Stained Region (HSRs)

As decribed in the Introduction, HSRs are cytogenetic

manifestation of gene amplification. Microdissection and Micro-FISH

can also be applied on HSRs in order to determine the composition of

113

the HSR. For example, G-banding analysis of a neuroblastoma cell line

(NGP-127) demonstrated that there is an HSR on the long ann

chromosome 12. A Micro-FISH probe was generated by dissecting 20

copies of about 1/6 of the 12q HSR. Figure 19 illustrates the Micro

FISH results using PCR products from the microdissected hsr DNA as a

probe. Figure 19a-c shows the microdissection of the 12q HSR, and

19d shows an example of the Micro-FISH back to a NGP-127

metaphase cell.

Discussion

As dis us sed in the previous chapter, chromosome micro

dissection is a very powerful technique and can be applied in many

areas. Chromosome microdissection combined with microcloning can

be used to generate chromosome band- or region-specific libraries for

physical mapping. Chromosome microdissection combined with

fluorescence in situ hybridization generates Micro-FISH probe which

can be applied to identify verturally any kind of chromosomal

rearrangements unidentifiable by conventional chromosome banding

analysis. Prior to our advance in this area, chromosome micro

dissection and micro cloning procedures were so complex and labor

intensive that they had not readily been applied to cytogenetic

charaterization. The majority of investigators using this technique have

used highly demanding microchemical techniques requiring specialized

114

a

" -~

.(' ;f-e

Figure 17. Microdissection of an unidentifiable translocation. a, Partial metaphase spread denoting a transli)Cation unidentifiable by routine banding analysis interpreted by G-banding as t(6;?)(qI4;?) (arrow).b-c, Microdissection of the distal to 6q14 for the generation of a Micro-FISH probe. d, The dissected Micro-FISH probe hybridized back to tumor metaphase chromosome. Upper, (left) G-banded normal chromosome; (right) normal 6 painted with a FITC-Iabeled 6-WCP. Lower, (left) G-banded unidentifiable translocation chromosome; (center) FITC painting by 6-WCP (red) illustrating the presence of the short arm and proximal long arm of chromosome 6; (right) simultaneous painting with the Spectrum Orange 6-WCP resulting in the redlorange signal on the short arm, the FITC-Iabeled Micro-FISH probe, results in the identification of the translocation chromosome. e, Hybridization of the Micro-FISH probe from b and c to normal lymphocyte metaphase chromosomes. (left) G-banded partial metaphase with arrow to chromosome 21; (right) identical cell following Micro-FISH which indicates that the dissected material is from chromosome 21 (q21-qter). f, Illustration of the Micro-FISH probe generated in b and c hybridized to a normal lymphocyte metaphase documenting probe specificity for human chromosome 21. -

VI

Figure 18. Microdissection of an unidentifiable translocation. (Inset) Microdissection of the material distal to 6q 13 of a translocation from a malignant melanoma cell line (UACC-837) for the generation of a Micro-FISH probe. (Top) G-banded normal lymphocyte metaphase chromosomes with arrow to chromosome 8. (Bottom) Identical metaphase chromosomes following hybridization with the Micro-FISH probe which indicates that the dissected material is from chromosome 6 (q13-15) and chromosome 8 (q24.3).

116

Figure 19. Microdissection of an homogeneously stained region (hsr) . a, Partial G-banded metaphase spread from a neuroblastoma cell line (NGP-127) denoting an hsr on chromosome 12(qI4) (arrow). b-c, Microdissection of about 1/6 hsr material for the generation of a Micro-FISH probe. d, Illustration of a metaphase of NGP-127 cell line demonstrating that the probe hybridized to two region on chromosome 12 (q14 and q24.4) and whole homogeneously stained region on the hsr marker.

117

equipments to carry out the usual recombinant DNA manipulations

(phenol extraction, restriction endonuclease digestion, ligation, etc.) in

a nanoliter microdrop under microscope observation. The procedure of

chromosome microdissection and microcloning used in this dissertation

is simplified tremendously compared to the previous methods and

unquestionably is more rapidly performed. At least three rate limiting

steps affecting all previous microdissection procedures have been

greatly improved in this study. First, in previous procedures the

dissected DNA fragments were pooled into a Inl collection microdrop

which was located on a seperate coverslip in a small moist chamber

requiring the use of a micromanipulator under microscope observation.

Keeping a Inl microdrop from evaporation for the several hours

required for microdissection is very difficult. In the present study, the

microdissected DNA fragments are directly transferred into a 5 , . .t1

collection drop in a 0.5 ml microcentrifuge tube. The advantages of

this improvement include: 1) the dissected DNA fragment is transferred

more easily, 2) it eliminates the problem of collection drop evaporation,

and 3) it decreases the possibility of contamination.

Second, before PCR all previous procedures used the normal

recombinant DNA manipulations (such as phenol extraction, restriction

endonuclease digestion, and ligation), which, because of the

exceedingly small amount of microdissected DNA, needed to be

performed using microchemical techniques. In our present procedure,

the microdissected DNA is directly amplified by PCR using

oligonucleotide primers. The advantages of this change include: 1)

118

elimination of the necessity for microchemical manipulations, 2)

decrease in the chance of contamination, and 3) a very significant

increase in the DNA amplification efficiency.

Third, in our present procedure the peR amplified DNA

fragments are much more efficiently cloned by direct ligation with a T

tailed vector derivative of pGEM5tz( +) without any restriction

endonuclease digestion. This change makes micro cloning easier and

the library prepared by this method contains a proportion of useful

clones far in excess of that obtained by the more biochemically rigorous

procedures.

Chromosome microdissection as described this dissertation is an

extremely useful approach to generate band- or region-specific libraries

for physical mapping. This procedure provides a novel method for

direct amplification and cloning of the microdissected template DNA,

which eliminates the necessity for microchemical manipulation. This

study documents the feasibility of this significantly simplified approach

for generating a large number of region-specific probes in a short

period of time. A further increase in rapidity is gained by testing the

primary microdissection peR pro dust in FISH experiments. The

localization and intensity of the FISH signal confirm the specificity of

the library and give a prelimimary indication of its quality. Therefore,

the time-consuming process of testing individual microclones by

Southern blot analysis is reserved for microdissection peR products,

which are already known to contain significant sequence representation

from the targeted chromosomal region. The 6q21 DNA microclone

119

library described in this study should be useful for the long-range

physical mapping of the ovarian carcinoma and melanoma deletion

region of chromosome 6. Further, the size of these clones is convenient

for sequencing without subcloning and their density should be

sufficient to assist significantly in the construction of a saturation STS

map of 6q21 with corresponding Y AC clones. Further studies will

continued the characterization of micro clones from this 6q21 library in

an effort to confirm the presence of clones within the melanoma

deletion region.

Detection of numerical and structural aberrations with WCPs

may facilitate many areas of cytogenetics. For example, WCPs can be

used to visualize any specific chromosome and its derivatives. Previous

WCPs were constructed using fluorescence-activated chromosome

sorting and human/rodent cell hybrids. The WCPs prepared from these

flow-sorted libraries have several disadvantages. First, these libraries

are often contaminated with fragments from other chromosomes during

the process of chromosome-sorting, resulting in increases in

nonspecific hybridization. Second, the WCPs prepared from flow

sorted chromosomes often hybridized unevenly to the target

chromosome and frequently leave gaps along the chromosome. In

contrast, WCPs prepared by using chromosome microdissection are

more rapid (generated in less than 24 hours) and simple (only 20 copies

of target chromosome are dissected). The probes hybridize to the target

chromosome evenly without gaps and far less nonspecific background

120

is obsetved. (Figure 14). Therefore, chromosome microdissection is an

easier, more rapid and effective method for preparing WCPs.

Because of the labor intensive and time consuming nature of

previously published microdissection techniques, they were restricted to

normal metaphases (prepared from peripheral blood lymphocytes) and

had not been applied to abnormal human chromosomes. In the present

study, microdissection was successfully applied to several abnormal

human chromosomes. This application significantly extends the limits

of conventional cytogenetic analysis by providing Micro-FISH probes

from any unknown chromosome markers. Theoretically, the

chromosomal derivation of previous unidentifiable de novo

translocations and unknown marker chromosome can be determined by

Micro-FISH. The application of Micro-FISH may also detect the

breakpoints of a translocation more accurately. For example,

conventional G-banding analysis on a translocation involving

chromosome 6 in a malignant melonoma cell line (UACC-383) could

not determine the origin of the non-6 material in the translocation. The

breakpoint on chromosome 6 was localized to 6q 15-16 by G-banding

analysis. The Micro-FISH results showed that the unknown portion of

the translocation was derived from chromosome 21, and the breakpoint

on chromosome 6 could be more accurately localized to 6q 14 (Fig. 15).

Application of Micro-FISH also revealed some previously

unidentifiable chromosome rearrangements. Conventional banding

analysis on a malignant melanoma cell line (UACC-837) found a

terminal deletion of chromosome 6 [del (6)(q21-qter)]. However, the

121

Micro-FISH results reveal this deletion 6 marker was a nonreciprocal

translocation t(6;8). This approach to mapping tumor-associated

breakpoints has significant advantages over conventional methods.

Further, it can be directly applied to tumor material without the

establishment of cell lines or somatic cell hybrids.

Application of Micro-FISH to HSRs has also been performed as

part of this study. Cytogenetic study of a neuroblastoma cell line

(NGP-127) demonstrated an HSR on the long arm of chromosome 12.

The amplification of a gene(s) within this region is considered to playa

role in the development of neuroblastoma. A Micro-FISH probe was

used to examine the chromosomal composition of this HSR and is the

first time microdissection has been used for this purpose.

Chromosome microdissection clearly is a very powerful and

useful technique. However, there are still several steps which can be

improved including decreasing the dissected DNA fragment number,

increasing the amplified products from dissected DNA, and decreasing

the contanimation during the PCR amplification. We have attempted to

address these problems by the following approaches.

1) As discussed before, the anealing temperature of UNI primer is

30°C, and Taq DNA polymerase works with low efficiency at this

temperature. The initiation of PCR amplification is also very difficult

because the amount of microdissected DNA is exceedingly small.

Initially, a minimum of 40 dissected DNA fragments were required to

initiate the PCR amplification. Recently, we have decreased the DNA

fragment number to 17 using Sequenase to replace Taq DNA

122

polymerase in first 5-10 peR cycles (for more information, see

reference 172).

2) Recently, Zhang et al. (173) developed an in vitro method for

amplifying a large fraction of the DNA sequences present in a single

haploid cell by repeated primer-extension pre amplification (PEP) using

a mixture of IS-base random oligonucleotides. Although the working

condition needs to be optimized, PEP is hoped to be a useful means to

decrease the dissected DNA copy number. It will be a significant

advance if a single copy of dissected DNA can be effectively amplified.

This should decrease the contamination opportunity during

microdissection and collection, and also increase the specificity of the

amplified library.

3) Treatment of dissected DNA with proteinase-K was considered

an important step for peR amplification because proteinase-K was

assumed to remove chromosome binding proteins and thus enhance the

efficiency of peR amplification. Recently, I have amplified dissected

DNA with and without proteinase-K, and the results showed no

significant difference (data is not shown). Elimination of proteinase-K

may also decrease the opportunity for contamination.

4) DNA contamination remains a big problem because the initial

amount of dissected DNA is exceedingly small (-10-15 gm). Any

amount of contaminated DNA could potentially be amplified during the

peR and influence the quality of the amplified library. Recently,

several investigators used exonuclease III (174), ultraviolet light (175,

176), Gamma irradiation (177), and psoralin treatment (178) to

123

eliminate PCR contamination. I have recently used UV -treated PCR

reagents (PCR buffer, dNTP, H20, MgCh, and primers) to amplify

microdissected DNA. The PCR results suggested that the

contamination is significantly inhibited, and the amplification of the

specific dissected DNA is equal or even better than that amplified

without UV -untreated reagents.

5) Enhancing PCR amplification is another important approach to

increasing microdissection efficiency. Schwarz et al. (179)

demonstrated that using the gene 32 protein of the phage T4, (which

enhances T4 DNA polymerase activity and accuracy), improved the

yield of long PCR products (3-6.5 kb) at least tenfold. Alternatively,

Ruano et al. (180) developed heat-soaked PCR, a method for enhancing

PCR amplification, which is performed by heating the DNA sample at

940

C for 30 minutes before addition of Taq DNA polymerase and

primers. Both methods can be applied to enhance the specifity and

sensitivity of PCR amplification from microdissected DNA templates.

Despite its broad applicability, the direct cloning of PCR products

remains inefficient and problematic (181, 182). Recently, Buchman et

al. (183, 184) developed a novel method which uses uracil DNA

glycosylase (UDO) to enable cloning of PCR products. This technique,

which relies on the incorporation of dUTP-containing primers into PCR

products, allows rapid and efficient cloning of PCR amplified DNA

with negligible vector background. Chromosome microdissection

combined with UDO cloning offers a powerful tool for preparing

124

chromosome band-specific libraries of 200,000 or more

recombinations.

Chromosome microdissection has been applied to several areas of

cytogenetic analysis and molecular investigation. The application

described in this Dissertation show that this novel technique will open

intriguing new avenues in basic and applied molecular cytogenetics.

As more and more genetic and cancer-associated traits are being

mapped to specific chromosome regions, narrowly spaced DNA

markers for these regions are needed to assist in finding the disease

genes. Recently, Chumakov et al. (185) developed a new approach for

isolating chromosome-specific subsets from a human genomic yeast

artificial chromosome (YAC) library. They screened a megabase

insert-size Y AC library with Alu PCR products amplified from hybrid

cell lines containing human chromosome 21, and identified a subset of

63 clones representative of chromosome 21. Using Alu PCR products

amplified from microdissected band-specific DNA to screen the YAC

library may rapidly and accurately subdivide the library into

chromosome band-specific sublibraries which will dramatically

increase the speed for mapping genes.

Chromosome microdissection may also be applied in several other

areas. Microdissection can be used to generate subband-specific DNA

libraries by dissecting high-resolution chromosomes at a level of 800

bands. The library may contains clones from DNA fragments of only

few megabase length.

125

Another potential application of chromosome microdissection is

to clone translocation breakpoints in cancer cells and map the genes

involved in the development of the cancer. Mapping of the Neurofibro

matosis gene (NFl) (53, 54) is a successful example of mapping the

location of an unknown gene based on the molecular analysis of a

translocation breakpoint. Molecular analysis of two balanced

chromosomal translocations involving l7ql1.2 (55, 56) in two different

patients with neurofibromatosis resulted in the cloning of the NFl gene.

With NFl and other successful cases of positional cloning however, the

progression from identification of a cytogenetically visible aberration to

the cloning of the breakpoint or deleted region was the most time

consuming and labor intensive step of the process. This again is most

frequently due to the problem of lack of a large number of clones at the

breakpoint region. With the help of chromosome microdissection, the

progression from identification of a translocation to positional cloning

of the breakpoint should be great facilitated.

As the final major application of this technology I would like to

present a novel strategy to clone a translocation breakpoint.

1). Microdissect the region containing breakpoint on the

translocation marker and prepare a Micro-FISH probe. Use the Micro

FISH probe to determine the chromosomes involved in translocation,

and to accuratly determine the breakpoint on each chromosome. 2).

Generate two breakpoint region libraries from normal peripheral blood

lymphocyte chromosomes by microdissection of both bands involved.

The dissected region at the breakpoint should be as narrow as possible

126

(ideally less than 10 Mb). The breakpoint-region libraries are then

generated by micro cloning techniques. 3). Digestion of DNA from the

tumor cell or cell line containing the translocation chromosome is

accomplished with a rare-cutting restriction endonuclease (such as NotI,

Mlu I, and Sal I) followed by pulse field gel electrophoresis. 4).

Detection of a NotI-DNA fragment containing the breakpoint would be

examined by using 50 clones from each breakpoint-region libraries.

Because the average NotI-restriction fragment size is -1 Mb, if the

breakpoint-region dissected from normal lymphocyte chromosome is

<10 Mb, about 10 DNA fragments will be required to be hybridized

from each breakpoint-region library. Only fragments containing the

breakpoint will be hybridized by micro clones from both libraries.

Accordingly, the breakpoint region can be located by comparing the

hybridization pattern between probes from the two libraries. The

microclones hybridized to the breakpoint region can then be isolated by

several Southern blotting analysis. 5). The DNA fragment containing

the breakpoint may then be isolated and further analyzed.

Alternatively, all clones that hybridize to the fragment containing the

breakpoint can be used for screening a yeast artificial chromosome

(YAC) library. YAC clones crossing the breakpoint will be chosen and

subcloned into phage. Fragments from the phage clone crossing the

breakpoint will be used to isolate transcripts mapping in this region.

Further study of candidate sequences would then lead to find the target

gene.

127

In summary, chromosome microdissection is an increasinly

important bridge connecting cytogenetic analysis and molecular study.

The application of microdissection has closed the gap between

cytogenetics and molecular biology resulting in significant progress in

marker analysis and generation of band-specific genomic library.

Further application of chromosome microdissection should greatly

facilitate both cytogenetic analysis and molecular analysis of complex

chromosome rearrangements.

128

CHAPTER 4

SUMMARY

The central questions that I focused on in this dissertation are:

1) the cytogenetic analysis of 50 cases of human ovarian carcinomas in

order to determine recurring sites of chromosomal changes, and 2) the

development and application of a novel chromosome microdissection

technique.

There is no single chromosome change that was charateristic of aU

ovarian carcinomas in this study but, on the other hand, several

chromosomal regions show a nonrandom involvement. Chromosome 1

is the most frequently involved in structural changes as it is equally in

other types of tumors. The most commonly involved regions on

chromosome 1 are Ip31-p36 and Ip13-qll. Chromosome 3 structural

abnormalities appear to be particularly common in ovarian carcinoma.

This study find deletion of chromosome 3 in 5 patients involving the

long arm (particularly band 3q21) rather than involving the short arm

which was more commonly described by other reports. The other

chromosomal bands and regions which are commonly involved in

structural abnormalites in this study are 6q22-q25, 7pI5-p22, 7q31-q34,

llp15, and 12pll-p13. Rearrangements of above regions may playa

role in the development of human ovarian carcinoma. Loss of X

chromosome and gain of chromo-some 7 may also playa role in the

development of ovarian carcinoma.

Chromosome microdissection is a bridge connecting cytogenetic

analysis and molecular study. The application of microdissection has

129

closed the gap between cytogenetics and molecular biology. In this

study, microdissection was used to generate chromosome band-specific

library for physical mapping, whole chromosome painting probes,

band- or region-specific painting probes (Micro-FISH probes) and their

applications, Micro-FISH probes generated from dissection of

unidentifiable translocations and homogeneously stianed region for

detennining the unknown part of the translocation and hsr. Further

improvement and application of chromosome microdissection will

facilitate both cytogenetic and molecular studies.

130

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