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Cytogenetic and molecular analysis of complexchromosome rearrangements in human cancer:
Application of chromosome microdissection.
Item Type text; Dissertation-Reproduction (electronic)
Authors Guan, Xin-Yuan.
Publisher The University of Arizona.
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Download date 02/02/2021 10:50:19
Link to Item http://hdl.handle.net/10150/186228
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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.
Requests for permission for extended quotation from or reprodu9tion of
this maniscript in whole or in part may be granted by the copyright
holder.
," \. ' .. 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
4. G-banded karyotype from the ovarian carcinoma T88-286 .............................. 57
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
7. G-banded karyotype from the ovarian carcinoma T89-321 .............................. 60
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
4. Structural abnormalities of chromosome 7 from 50 cases of ovarian carcinoma ............. 52
5. Structural abnormalities of chromosome 11 from 50 cases of ovarian carcinoma ............. 53
6. Structural abnormalities of chromosome 12 from 50 cases of ovarian carcinoma ............. 54
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.
Biologicals and Reagents:
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
Biologicals and Reagents:
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
I. Deletions I. Deletions
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)
Abnormalities # of Patients Abnormalities # of Patients
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
Abnormalities # of Patients Abnormalities # of Patients
I. Deletions I. Deletions
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
Biologicals and Reagents:
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.).
Biologicals and Reagents:
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.
Biologicals and Reagents:
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).
Biologicals and Reagents:
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.
Biologicals and Reagents:
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.
Biologicals and Reagents:
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.
Biologicals and Reagents:
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
REFERENCES
1. Boveri, T. (1912): Bietrag zum stadius des chromatins in den epithelzellen des carcinoma. Bietr. Path. Anat. AUg. Pathol.,14, 249.
2. Armitage, P. and Doll, R. (1954): The age distribution of cancer and a multi-stage theory of carcinogenesis. Br. J. Cancer, 8., 1-12.
3. Soloman, E., Borrow, J., Goddard, A.D. (1991): Chromosome aberrations and cancer. Science, 254, 1153-1160.
4. Tijo, J.H., Levan, A. (1956): The chromosome number of man. Hereditas, 42, 1-6.
5. Caspersson, T., Zech, L., Johnson, C. (1970): Differential banding of alkylating flourchromes in human chromosomes. Exp. Cell Res., 60, 315-319.
6. Berger, R. (1971): Vne novelle technique dtanalyse dur caryotype . ..h,. R. Acad. Sci., 273, 2620-2622.
7. Heim, S. and Mitelman, F. (1989): Primary chromosome abnormalities in human neoplasia. Adv. Cancer Res., 52. 1-43.
8. Nowell, P.C., Hungerford, D.A. (1960): A minute chromosome in human chronic granulocytic leukemia. Science, 132, 1497.
9. Rowley, J.D. (1973): A new consistent chromosmal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature, 343,290-293.
10. Trent, J., Crickard, K., Gibas, Z., Goodacre, A., Pathak, S., Sandberg, A.A., Thompson, F., Whang-Peng, J., Wolman, S. (1986): Methologic advances in the cytogenetic analysis of human solid tumors. Cancer Genet. Cytogenet., 12, 57 -66.
131
11. Mitelman, F. (1984): Restricted number of chromosomal regions implicated in aetiology of human cancer and leukemia. Nature, 310,325-327.
12. Rowley, J.D. (1984): Biological implications of consistent chromosome rearrangements in leukemia and lymphoma. Cancer Res., 44, 3159-3168.
13. Mitelman, F. (1986): Clustering of breakpoints to specific chromosomal regions in human neoplasia: A survey of 5,345 cases. Hereditas, 104, 113-119.
14. Sandberg, A.A. (1990): The Chromosomes in Human Cancer and Leukemia, Second Edition. Elsevier Science Publishing Co. New York, N.Y.
15. Human Gene Mapping 10 (1989): New Haven Conference, 10th International Workshop on Human Gene Mapping. Cytogenet. Cell Genet., il, 533-562.
16. Groffen, J., Stephenson, J.E., Heisterkamp, N., de Klein, A., Bartram, C.R., Grosveld, G. (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell, 36,93-99.
17. Heisterkamp, N., Stephenson, J.R., Groffen, J., Hansen, P.F., de Klein, A., Bartram, C.R., and Grosveld, G. (1983): Localization of the c-abl oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature, 306, 239-242.
18. Konopka, J. B., Watanabe, S.M., Witte, O.N. (1984): An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosin kinase activity. Cell, 37, 1035-1042.
19. Shtivelman, E., Lifshitz, B., Gale, R. P., Roe, B.A., Canaani, E. (1986): Alternative splicing of RNAs transcribed from the human abl gene and from the ber-abl fused gene. Cell, 47,277-284.
132
20. Zech, L., Haglund, D., Nilsson, K., Klein, G. (1976): Charateristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int. 1. Cancer, 17,47-56.
21. Bernheim, A., Berger, R., Lenoir, G. (1981): Cytogentic studies on African Burkitt's lymphoma cell lines: t(8;14), t(2;8), and t(8;22) translocations. Cancer Genet. Cytogenet., 1, 307-315.
22. Klein, G. (1983): Specific chromosomal translocations and the genesis of B-cell derived tumors in mice and men. Cell, 32, 311-315.
23. Magrath, I. (1986): Clinical and pathobiological features of Burkitt's lymphoma and their relevance to treatment. p631-643. In Levine P.H., Ablarhi D.V., Pearson G.R., and Kattarides S.D. Epstein-Barr virus and associated diseases. Martinus Nijhoff Publishing, Boston.
24. Bishop, I.M. (1987): The molecular genetics of cancer. Science, 235, 305-311.
25. Lammie, G.A., Fantl, V., Smith, R., Schuuring, E., Brookes, S., Michalides, R., Deckson, C., Arnold, A., and Peters, G. (1991): D11S287, a putative oncogene on chromosome llql3, is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-I. Oncogene, Q, 439-444.
26. Arnold, A, Kim, H.G., Gaz, R.D., Eddy, R.L., Fukushima, Y., Byers, M.G., Shows, T.B., and Kronenberg, H.M. (1989): Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. 1. Clin. Invest., 83. 2034-2040.
27. Rosenberg, C.L., Kim, H.G., Shows, T.B., Kronenberg, H.M., and Arnold, A. (1991): Rearrangement and overexpression of DI1S287E, a candidate oncogene on chromosome l1qI3 in benign parathyroid tumors. Oncogene, ~ 449-453.
133
28. Mellentin, J.D., Smith, S.D., and Cleary, M.L. (1989): Lyl-l, a novel gene altered by chromosomal translocation in T cell leukemia, codes for a protein with a helix-loop-helix DNA binding motif. Cell, 58, 77-83.
29. Alitalo, K., and Schwab, M. (1986): Oncogene amplification in tumor cells. Adv. Cancer Res., 47,235-281.
30. Cox, D., Yuncken, C., Spriggs, A.I. (1965): Minute chromatin bodies in malignant tumors of childhood. Lancet., ii, 55-58.
31. Biedler, J.L., Spengler, B.A. (1976): Metaphase chromosome anomaly: Association with drug resistance and cell-specific products. Science, 911, 185-187.
32. Biedler, J.L., Spengler, B.A. (1976): A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lines in culture. J. Natl. Cancer Inst., 57, 683-695.
33. Brodeur, G.M., Green, A.A., Hayes, F.A., Williams, KJ., Williams, D.L., Tsiatis, A.A. (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res .. 41, 4678-4686.
34. Schwab, M., Varmus, H.E., Bishop, J.M., Grzeschik, K.H., Sakaguchi, A.Y., Brodeur, G., Trent, J. (1984): Chromosome localization in normal human cells and neuroblastomas of a gene related to c-myc. Nature, 308, 288-291.
35. Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., Ullrich, A., and Press, M.F. (1989): Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science, 244, 707-712.
36. Wilson, M.G., Towner, J.W., and Fujimoto, A. (1973): Retimoblastoma and D-chromosome deletions. Amer. J. Hum. Genet., 25, 57-61.
134
37. Yunis, J.J. and Ramsay, N. (1978): Retinoblastoma and subband deletion of chromosome 13. Am. 1. Dis. Child, 132. 161-163.
38. Cavenee, W.K., Dryja, T.P., Phillips, R.A., Benedict, W.F., Godbout, R., Gallie, B.L., Murphree, A.L., Strong, L.C., and White, R.L. (1983): Expression of recessive allels by chromosomal mechanisms in retinoblastoma. Nature. 305, 779-784.
39. Dryja, T.P., Cavenee, W., White, R., Papaport, J.M., Petersen, R., Albert, D.M., and Bruns, G.A. (1984): Homozygosity of chromosome 13 in retinoblastoma. N. Eng!. 1. Med., 310, 550-553.
40. Bookstein, R., Shew, 1-Y., Chen, P-L., Scully, P., Lee, W-H. (1990): Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutant RB gene. Science, 247, 712-715.
41. Trent, 1.M., Stanbridge, E.l., McBride, H.L., Meese, E.U., Casey, G., Araujo, D.E., Witkowski, C.M., Nagle, R.B. (1990): Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6. Science, 247, 568-571.
42. Trent, 1.M., Rosenfeld, S.B., and Meyskens, F.L. (1983): Chromosome 6q involvement in human malignant melanoma. Cancer Genet. Cytogenet., 2.,. 177-180.
43. Pathak, S., Drwinga, H.L., and Hsu, T.C. (1983): Involvement of chromosome 6 in rearrangements in human malignant melanoma cell lines. Cytogenet. Cell Genet., 36, 573-579.
44. Trent, 1.M., Thompson, F.H., and Meyskens, F.L. (1989): Identification of a recurring translocation site involving chromosome 6 in human malignant melanoma. Cancer Res., 49, 420-423.
45. Call, K.M., Glaser, T., Ito, C.Y., Burkler, A.l., Pelletier ,I." Haber, D.A., Rose, E.A., Karl, A. Yeger, H., Lewis, W.H.,
135
Jones, C., and Housman, D.E. (1990): Isolation and charaterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell, 60, 509-520.
46. Gessler, M., Poustka, A., Cavenee, W., Neve, R.L., Orkin, S.H., and Bruns, G.A. (1990): Homozygous deletion in Wilms' tumors of a zinc-finger gene identified by chromosome jumping. Nature, 343, 774-778.
47. Riccardi, K.M., Sujansky, E., Smith, A.C., Francke, D. (1978): Chromosomal imbalance in the aniridia-Wilms' tumor association: IIp interstitial deletion. Pediatrics, .61, 604-610.
48. Francke, D., Holmes, L.B., Atkins, L., Riccarki, V.M. (1979): Aniridia-Wilms' tumor association: Evidence for specific deletion of llpl3. Cytogenet. Cell Genet., 24, 185-192.
49. Koufos, A., Hansen, M.F., Lampkin, B.C., Workman, M.L., Jankins, N.A., and Cavenee, W.K. (1984): Loss of alleles at loci on human chromosome 11 during genesis of Wilms' tumor. Nature, 309, 170-172.
50. Baker, S.J., Fearon, E.R., Nigro, J.M., Hamilton, S.R., Preisinger, A.C., Jessup, M., van Tuinen, P., Ledbetter, D.H., Barker, D.F., Nakamura, Y., White, R., Vogel stein, B. (1989): Chromosome 17 deletion and p53 gene mutations in colorectal carcinomas. Science, 244, 217-221.
51. Vogelstein, B., Fearon, E.R., Hamilton, S.R., Kern, S., Preisinger, A.C., Leppert, M., Nakamura, Y., White, R., Smith, A.M.M., and Bos, J.L. (1988): Genetic alterations during colorectal-tumor development. N. Eng!. J. Med., 319, 525-532.
52. Cawthon, R.M., Weiss, R., Xu, G., Viskochil, D., Culver, M., Stevens, J., Robertson, M., Dunn, D., Gesteland, R., O'Connell, P., And White, R. (1990): A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic struture and point mutation. Cell, 62, 193-201.
136
53. Viskochil, D., Burchberg, A.M., Xu, G., Cauthon, R.M., Steven,s J., Wolff, R.K., Culver, M., Carey, J.C., Copeland, N.G., Jenkins, N.A., White, R., and O'Connell, P. (1990): Deletion and translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell, 62, 187-192.
54. Wallace, M.R., Marchuk, D.A., Audersen, L.B., Letcher, R., Odeh, H.M., Saulino, A.M., Fountain, J.W., Brereton, A., Nicholson, J., Mitchell, A.L., Brownstein, B.H., and Collins, F.S. (1990): Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NFl patients. Science, 249, 181-186.
55. Ledbetter, D.H., Rich, D.C., O'Connell, P., Leppert, M., and Carey, J.C. (1989): Precise localization of NFl to 17ql1.2 by balanced translocation. Am. J. Hum. Genet., 44,20-24.
56. Schmidt, M.A., Michels, V.V., and Deward, W. (1987): Cases of neurofibromatosis with rearrangements of chromosome 17 involving band 17ql1.2. Am. J. Med. Genet., 28, 771-777.
57. Young, R.C., Knapp, R.C., Zvi, F., Disaia, P.J. (1985): Cancer of the ovary. In De Vita, V.T., Hellman, S., Rosenberg, S.A. (eds): Cancer-Principles and Practice of Oncology, 2nd Ed., Philadelphia: JB Lippincott Company, p 1083-1116.
58. Mitelman, F. (1991): Catalog of Chromosome Aberrations in Cancer. 4th Ed., New Youk: Wiley Liss, Inc., p2056.
59. Atkin, N.B., Baker, M.C., Robinson, R., and Gaze, S.E. (1974): Chromosome studies on 14 near-diploid carcinomas of the ovary. Europ. J. Cancer, 10, 143-146.
60. Atkin, N.B. and Pickthall, V.J. (1977): Chromosomes 1 in 14 ovarian cancers: Heterochromatin variants and structural changes. Hum. Genet., 38, 25-33.
61. Wake, N., Hreshchyshyn, M.M., Piver, S.M., Matsui, S-I., and Sandberg, A.A. (1980): Specific cytogenetic changes in ovarian
137
cancer involving chromosomes 6 and 14. Cancer Res. 40. 4512-4518.
62. Wake, N., Hreshchyshyn, M.M., Piver, S.M., Matsui, S-I., and Sandberg, A.A. (1980): Specific chromosome change in ovarian cancer. Cancer Genet. and Cytogenet.,.b 87-88.
63. Atkin, N.B. and Baker M.C. (1981): Specific chromosome changes in ovarian cancer. Cancer Genet. and Cytogenet., 1, 275-276.
64. Trentn I.M. and Salmonn S.E. (1981): Karyotypic analysis of human ovarian carcinoma cells cloned in short term agar culture. Cancer Genet. and Cytogenet., 1,279-291.
65. Trent, I.M., Salmon, S.E. (1979): Karyotypic analysis of human ovarian carcinoma cells cloned in agar. Am. I. Hum. Genet., n,112A.
66. Trent, I.M. (1980): The clinical significance of non-random chromosome changes in human hematopoietic and solid tumors. Advences in Cancer Treatment and Research, Baltimore, Maryland, March 3-6.
67. Trent, I.M.(1980): Cytogenetic analysis of human tumor cells cloned in agar. In: Cloning of Human Tumor Stem Cells, S SImon. ed. A Liss, Inc., New Youk, p 165-178.
68. Whang-Peng, I., Knutsen, T., Douglass, E.C., Chu, E., Ozols, R.F., Hogan, W.M., and Young, R.C. (1984): Cytogenetic studies in ovarian cancer. Cancer Genet. and Cytogenet., 11, 91-106.
69. Trent, I.M., Floyd, H.T., and Buick, R.N. (1985): Generation of clonal variants in a human ovarian carcinoma studied by chromosome banding analysis. Cancer Genet. and Cytogenet., l4..,. 153-161.
138
70. Mackillop, W.I., Trent, I.M., Stewart, S.S., Buick, R.N. (1983): Tumor progression studied by analysis of cellular features of serial ovarian carcinoma ascites. Cancer Res., 43, 874-878.
71. Panani, A. and Ferti-Passantonopoulou, A. (1985): Common marker chromosomes in ovarian cancer. Cancer Genet. and Cytogenet.. 16,65-71.
72. Crickard, K., Marinello, M.I., Crickard, D., Satchidanand, S.K., Yoonessi, M., and Caglar, H. (1986): Borderline malignant serous tumors of the ovary maintained on extracellular matrix: Evidence for clonal evolution and invasive potential. Cancer Genet. and Cytogenet., 23. 135-143.
73. Augustus, M., Bruderlein, S., and Gerhart, E. (1986): Cytogenetic and cell cycle studies in metastatic cells from ovarian carcinomas. Anticancer Res., ~ 283-290.
74. Smith, A., Roberts, C., van Haaften-Day, C., den Dulk, G., Russell, P., and Tattersall, H.N. (1987): Cytogenetic findings in cell lines derived from four ovarian carcinomas. Cancer Genet. Cytogenet., 24. 231-242.
75. Iendyn, DJ. and McCartney, AJ. (1987): A chromosome study of three ovarian tumors. Cancer Genet. Cytogenet., 26. 327-337.
76. Atkin, N. and Baker, M.C. (1987): Abnormal chromosomes including small metacentrics in 14 ovarian cancers. Cancer Genet. Cytogenet., 26, 355-361.
77. Sheer, D., Sheppard, D.M., Gorman, P.A., Ward, B., Whelan, R.D.H., and Hill, B.T. (1987): Cytogenetic analysis of four human ovarian carcinoma cell lines. Cancer Genet. Cytogenet .. ~ 339-349.
78. Zhou, D.I., Gonzalez-Cadavid, N., Ahuja, H., Battifora, H., Moore, G.E., and Cline, M.l (1988): A unique pattern of protooncogene abnormalities in ovarian adenocarcinomas. Cancer, Qb 1573-1576.
139
79. Samuelson, J., Katz, S., Schwartz, P.E., Yang-Feng, T.L. (1988): Chromosomal evolution through tumor progression in ovarian cancer. Am. J. Hum. Genet., 43. Suppl A16.
80. Tanaka K., Boice C.R., and Testa J.R. (1989): Chromosome aberrations in nine patients with ovarian cancer. Cancer Genet. Cytogenet., 43. 1-14.
81. Guan, X-Y., Alberts, D., Burgess, A.C., Thompson, F.H., and Trent, J.M. (1989): Chromosomal analysis of 90 cases of ovarian carcinomas. Cancer Genet. Cytogenet., 41, 291.
82. Pejovic, T., Heim, S., Mandahl, N., Elmfors, B., Floderus, UM., Furgyik, S., Helm, G., Willen, H., and Mitelman, F. (1989): Consistent occurrence of a 19p+ marker chromosome and loss of IIp material in ovarian seropapillary cystadenocarcinomas. Genes. Chromosomes & Cancer. L 167-171.
83. Pejovic, T., Heim, S., Mandahl, N., Elmfors, B., Floderus, UM., Furgyik, S., Helm, G., Willen, H., and Mitelman, F. (1990): Trisomy 12 is a consistent chromosomal aberration in benign ovarian tumors. Genes, Chromosomes & Cancer,,2, 48-52.
84. Samuelson, J., Leung, W.Y., Schwartz, P.E., Ng, H-t., and Yang-Feng, T.L. (1990): Trisomy 12 in various ovarian tumors of benign to low malignancy. Am. l. Hum. Genet., 47. Suppl A16.
85. Bello, M.J. and Rey, l.A. (1990): Chromosome aberrations in metastatic ovarian cancer: Relationship with abnormalties in primary tumors. Int. l. Cancer, 45,50-54.
86. Bello, M.J., Moreno, S., and Rey, J.A. (1990): Involvement of 9p in metastatic ovarian adenocarcinomas. Cancer Genet. Cytogenet., 45,223-229.
87. Sasano, H., Garrett, C., Wilkinson, D., Silverberg, S., Comerford, l., and Hyde, l. (1990): Proto oncogene amplification and tumor ploidy in human ovarian neoplasms. Hum. Pathology, 21, 382-391.
140
88. Roberts, C.G. and Tattersall, M.H.N. (1990): Cytogenetic study of solid ovarian tumors. Cancer Genet. Cytogenet., 48. 243-253.
89. Lee, J.H., Kavanagh, J., Wildrick, D.M., Wharton, J.T., and Blick, M. (1990): Frequent loss of heterozygosity on chromosomes 6q, 11, and 17 in human ovarian carcinomas. Cancer Res., 50, 2724-2728.
90. Ehlen, T. and Dubeau, L. (1990): Loss of heterozygosity on chromosomal segments 3p, 6q and 11 p in human ovarian carcinomas. Oncogene, 5.,219-223.
91. Pejovic, T., Heim, S., Mandahl, N., Baldetorp, B., Elmfors, B., Floderus, U-M., Furgyik, S., Helm, G., Himmelmann, A., Willen, H., and Mitelman, F. (1992): Chromosome aberrations in 35 primary ovarian carcinomas. Genes. Chromosomes & Cancer,~, 58-68.
92. Trent, J.M. and Thompson, F.H. (1987): Methods for chromosome banding of human wxperimental tumors in vitro. Methods Enzymol., 151,267-279.
93. Yunis, J.J., Sawyer, J.R., and Ball, D.W. (1978): Characterization and banding patterns of metaphasprophase chromosomes and their use in gene mapping. Cytogenet. Cell Genet., 22, 679-683.
94. ISCN (1985): International system for cytogenetic nomenclature. Cancer Genet. Cytogenet., 21, 1-117.
95. Awa, A.A. and Neel, J. (1986): Cytogenetic "rogue" cells, What is their frequency, origin, and evolutionary significance? Proc. Nat!. Acad. Sci. USA, 83, 1021-1025.
96. Cruciger, O.V.J., Pathak, S., and Cailleau, R. (1976): Human breast carcinomas: Marker chromosomes involving lq in seven cases .. Cytogenet. Cell Genet., 17, 231-235.
141
97. Kovacs, G. (1978): Abnormalities of chromosome No.1 in human solid malignant tumors. Int. J. Cancer, 21,688-694.
98. Gardner, H.A., Gallie, B.L., Knight, L.A., and Phillips, R.A. (1982): Multiple karyotypic changes in retinoblastoma tumor cells: Presence of normal chromosome no. 13 in most tumors. Cancer Genet. Cytogenet., Q, 201-211.
99. Atkin, N.B., Baker, M.C. (1979): Chromosome 1 in 26 carcinomas of the cervix uteri: Structural and numerical changes. Cancer, 44, 604-613.
100. Wang, N., Trend, B., Bronson, D.L., and Fraley, E.E. (1980): Nonrandom abnormalities in chromosome 1 in human testicular cancers. Cancer Res., 40, 796-802.
101. Atkin, N.B. (1986): Chromosome changes in preneoplastic and neoplastic fenitallesions. Banbury Report 21: Viral Etiology of Cervix Cancer, pp 303-310. Cold Spring Harbor Laboratory.
102. Vanni, R., Peretti, D., Scarpa, R.M., Usai, E. (1985): Derivative 11 marker cheomosome in bladder carcinoma. Cancer Genet. Cytogenet., 16,289-295.
103. Mckusick, V. (1988): Mendelian inheritance in man, 8th edn. Johns Hopkins University Press, Baltimore London.
104. Botstein, D., White, R.L., Skolnick, M., Davis, R.W. (1980): Construction of a genetic linkage map in man using restriction fragment lenght polymorphisms. Am. J. Hum. Genet., 32, 314-331.
105. Carle, G.F., Frank, M., and Olson, M.V. (1986): Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science, 232, 65-68.
106. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Hom, G.T., Erlich, H.A., and Arnheim, N. (1985): Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230, 1350-1354.
142
107. Burke, D.T., Carle, G.F., and Olson, M.V. (1987): Cloning of large segments of DNA into yeast by means of artificial chromosome vector. Science, 236, 806-812.
108. Sternberg, r\. (1990): Bacteriophage PI cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proc. Nat!. Acad. Sci. USA, 87, 103-107.
109. Minoshima, S., Kawasaki, K., Fukuyama, R., Maekawa, M., Kudoh, H., and Shimizu, N. (1990): Isolation of giant DNA fragments from flow-sorted human chromosomes. Cytometry, 11, 539-546.
110. Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Albert, D.M., and Dryia, T.P. (1986): A human DNA segment with properites of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 323, 643-646.
111. Koenig, M., Hoffman, E.P., Bertelxon, CJ., Monaco, A.P., Feener, C., and Kunkel, L.P. (1987): Complete cloning of the Duchenne muscular dystrophy DMD) cDNA and affected individuals. Cell, 50, 509-517.
112. Rommens, J.M., Iannuzzi, M.C., Kerem, B-S, Drumm, M.L., Melmer, G., Dean, M., Rozmahel, R., Cole, J.L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J.R., Trui, L-C, and Collins, F.S. (1989): Identification of the cystic fibrosis gene: chromosome walking and jumping. Science, 245, 1059-1065.
113. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., and Robertson. (1991): Identification and characterization of the familial adenomatous polyposis coli gene. Cell, 66, 589-600.
114. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., Hedge, P., Markham, A., Krush, AJ., Petersen, G., Hamilton, S.R.,
143
Nilbert, M.C., Levy, D.B., Bryan, T.M., Preisinger, A.C., Smith K.B., Su, L-K, Kinzler, K.W., and Vogel stein, B. (1991): Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science, 253, 665-669.
115. McCormick, M.K., Shero, J.H., Kan, Y.U., Hieter, P.A., and Anronarakis, S.E. (1989): Construction of human chromosome 21-specific yeast artificial chromosomes. Proc. Natl. Acad. Sci. DSA, 86, 9991-9995.
116. Scalenghe, F., Turco, E., Edstrom, J-E, Pirotta, V., and Melli, M.L. (1981): Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes. Chromosoma, 82, 205-216.
117. Rohme, D., Fox, H., Herrmann, B., Frischauf, A-M, Edstrom, J-E, Mains, P., Silver, L.M., and Lehrach, H. (1984): Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes. Cell, 36, 783-788.
118. Fisher, E.M.C., Cavanna, J.S., and Brown, S.D.M. (1985): Microdissection and microcloning of the mouse X chromosome. Proc. Natl. Acad. Sci. USA, 82, 5846-5849.
119. Bates, G.P., Wainwright, BJ., Williamson, R., and Brown, S.D.M. (1986): Microdissection of and micro cloning from the short arm of human chromosome 2. Molecular and Cellular Biology, ,6, 3826-3830.
120. Kauser, R., Weber, J., Grzeschik, K-H, Edstrom, J.E., Driesel, A., Zengerling, S., Buchwald, M., Tsui, L.C., and Olek, K. (1987): Microdisection and micro cloning of the long ann of human chromosome 7. Molecular Biology Reports,.12., 3-6.
121. Liidecke, H-J, Senger, G., Claussen, D., and Horsthemke, B. (1989): Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature, 338, 348-350.
144
122. Senger, G., Liidecke, H-J, Horsthemke, B., and Claussen, U. (1990): Microdissection of banded human chromosomes. Hum. Genet., 84,507-511.
123. Davis, L.M., Senger, G., Liidecke, H-J, Claussen, U., Horsthemke, B., Zhang, S.S., Metzroth, B., Hohenfellner, K., Zabel, B., and Shows, T.B. (1990): Somatic cell hybrid and long-range physical mapping of 11p13 microdissection genomic clones. Proc. Natl. Acad. Sci. USA, 87, 7005-7009.
124. Cotter, F.E., Lillington, D., Hampton, G., Riddle, P., Nasipuri, S., Gibbons, B., and Young, B.D. (1991): Gene mapping by microdissection and enzymatic amplification: Hetergeneity in leukaemia associated breakpoints on chromosome 11. Genes, Chromosomes & Cancer, 1,8-15.
125. Newsham, I., Claussen, U., Ltidecke, H-J, Mason, M., Senger, G., Horsthemke, B., and Cavenee, W. (1991): Microdissection of chromosome band llpI5.5: Charaterization of probes mapping distal to the HBBC locus. Genes, Chromosomes & Cancer,,3., 108-116.
126. Han, J., Lu, C-M, Brown, G.B., and Rado, T.A. (1991): Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: A human brain sodium channel gene is on chromosome 2q22-q23. Proc. Natl. Acad. Sci. USA, 88,335-339.
127. MacKinnon, R.N., Hirst, M.C., Bell, M.V., Watson, J.E.V., Claussen, U., Ltidecke, H-J, Senger, G., Horsthemke, B., and Davies, K.E. (1990): Microdissection of the fragile X region. Am. J. Hum. Genet., 47,181-187.
128. Djabali, M., Nguyen, C., Biunno, I., Oostra, B.A., Mattei, M-G, Ikeda, J-E, and Jordan, B.R. (1991): Laser microdissection of fragile X region: Identification of cosmid clones and of conserved sequences in this region. Genomics, ill, 1053-1060.
129. Trautmann, U., Leuteritz, G., Senger, G., Claussen, U., and Ballhausen, W.G. (1991): Detection of APC region-specific
145
signals by nonisotopic chromosomal in situ suppression (CISS)-hybridization using a microdissection library as a probe. Hum. Genet., 87, 495-497.
130. Lengauer, C., Eckelt, A., Weith, A., Endlich, N., Ponelies, N., Lichter, P., Greulich, K.O., and Cremer, T. (1991): Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes. Cytogenet. Cell Genet., 56, 27-30.
131. Deng, H-X, Yoshiura, K-I, Dirks, R.W., Harada, N., Hirota, T., Tsudamoto, K., Jinno, Y., and Niikawa, N. (1992): Chromosome-band-specific painting: chromosome in situ suppression hybridization using PCR products from a microdissected chromosome band as a probe pool. Hum. Genet., 89, 13-17.
132. Nelson, D.L., Ledbetter, S.A., Corbo, L., Victoria, M.P., Ramirez-Solis, R., Webster, T.D., Ledbetter, D. H., and Caskey, C.T. (1989): Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA, 86, 6686-6690.
133. Brooks-Wilson, A.R., Goodfellow, P.N., Povey, S., Nevanlinna, H.A., de Jong, P.l, and Goodfellow, PJ. (1990): Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR. Genomics, 1, 614-620.
134. Aslanidis, C. and de Jong, PJ. (1991): Coincidence cloning of Alu PCR produsts. Proc. NatI. Acad. Sci. USA, 88,6765-6769.
135. Telenius, H., Carter, N.P., Bebb, C.E., NordenskjOld, M., Ponder, B.A.J., and Tunnacliffe, A. (1992): Degenerate oligonucleotide primer PCR: General amplification of target DNA by a single degenerate primer. Genomics, U, 718-725.
136. Telenius, H., Pelmear, A.H., Tunnacliffe, A., Carter, N.P., Behmel, A.,Perguson-Smith, M.A., NordenskjOld, M., Pfragner, R., and Ponder, B.AJ. (1992): Cytogenetic analysis by
146
chromosome painting using degenerate oligonucleotide-primedpolymerase chain reaction amplified flow-sorted chromosomes. Genes, Chromo & Cancer,~, 257-263.
137. Kovalic, D., Kwak, J.H., and Weisblum, B. (1991): General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acids Res., 19,4560.
138. Trent, J., Meyskens, F.L., Salmon, S.E., Ryschon, K., Leong", S.P.L., Davis, J.R., and McGee, D.L. (1990): Relation of cytogenetic abnormalities and clinical outcome in metastatic melanoma. New Engl. J. Med., 322, 1508-1511.
139. Weinberg, R.J. (1991): A short guide to oncogene and tumorsuppressoe genes. NIH Res ... ,3., 45-49.
140. Nowell, P. and Croce, C. (1990): Chromosome translocations and oncogenes in human lymphoid tumors. Am. J. Clin. Pathol., 94, 229-237.
141. Rowley, J. (1990):Molecular cytogenetics: Rosetta stone for understanding cancer-twenty-ninth G.H.A. clowes menorial award lecture. Cancer Res., 50, 3816-3825.
142. Fearon, E. and Vogelstein, B. (1990): A genetic model for colorectal tumorigenesis. Cell, 61, 759-767.
143. Fountain, J.W., Wallace, M.R., Bruce, M.A., Seizinger, B.R., Menon, A.G., Guselia, J.F., Michels, V.V., Schmidt, M.A., Dewald, G.W., and Collins, F.S. (1989): Physical mapping of a translocation breakpoint in neurofibromatosis. Science, 244, 1085-1087.
144. Warburton, D. (1991): De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: Clinical significance and distribution of breakpoints. Am. J. Hum. Genet., 49, 995-1013.
145. Callen, D., Eype, H., Ringenbergs, M.L., Freemantle, C.J., Woodroffe, P., and Haan, E.A. (1991): Chromosomal origin of
147
small ring marker chromosomes in man : Charaterization by molecular genetics. Am. J. Hum. Genet., 48. 769-782.
146. Mitelman, F., Kaneko, Y. and Trent, J. (199]): Report of the committee on chromosome changes in neoplasia. Cytogenet. Cell Genet., 58, 1053-1079.
147. Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. (1988): Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA, 85, 9138-9142.
148. Lichter, P., Cremer, T., Borden, J., Mannelidis, L., and Ward, D.C. (1988): Deletion of individural human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet., 80, 224-234.
149. Lichter, P., Chang-Tang, C-J, Call, K., Hennanson, G., Evans, G.A., Housman, D., Ward, D.C. (1990): High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science, 247, 64-69.
150. Kou, W-L, Tenjin, H., Segraves, R., Pinkel, D., Golbus, M.S., and Gray, J. (1991): Detection of aneuploidy involving chromosomes 13, 18, or 21 by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes. Am. J. Hum. Genet., 49, 112-119.
151. Meese, E., Witkowski, C.M., Zoghbi, H.Y., Stanbridge, E.J., Meltzer, P.S., and Trent, J.M. (1992): Development and utilization of a somatic cell hybrid mapping panel to assign Not I linkage probes to the long ann of human chromsome 6. Genomics, 12, 542-548.
152. Tkachuk, D.C., Westbrook, C.A., Andreeff, M., Donlon, T.A., Cleary, M.L., Suryanarayan, K., Homge, M., Redner, A., Gray, J., Pinkel, D. (1990): Detection of bcr-abl fusion in chronic
148
myelogeneous leukemia by in situ hybridization. Science, 250, 559-562.
153. Kuwano, A., Ledbetter, S.A., Dobyus, W.B., Emamuel, B.S., and Ledbetter, D.H. (1991): Detection of deletions and cryptic translocations in Miller-Dieker Syndrome by in situ hybridization. Am. J. Hum. Genet., 49, 707-714.
154. Knudson, A.G. (1971): Mutations and cancer: Statistical study of retinoblastoma. Proc. Natl. Acad. Sci. USA, 68, 820-823.
155. Stanbridge, E.J. (1990): Human tumor suppressor genes. Annu. Rev. Genet., 24,615-657.
156. Marshall, C.J. (1991): Tumor suppressor genes. Cell, 64, 313-326.
157. Kinzler, K.W., Nilbert, M.C., Vogelstein, B., Bryan, T.M., Levy, D.B., Smith, K.J., Preisinger, A.C., Hamilton, S.R., Hedge, P., Markham, A., Carlson, M., Joslyn, G., Groden, J., White, R., Miki, Y., Miyoshi, Y., Nishisho, I., and Nakamura, Y. (1991): Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers. Science, 251, 1366-1370.
158. Vogelstein, B., Fearon, E.R., Kern, S.E., Hamliton, S.R., Preisinger, A.C., Nakamura, Y., and Whit,e R. (1989): Allelotype of colorectal carcinomas. Science, 244, 207-211.
159. Millidin, D., Meese, E., Vogelstein, B., Witkowski, C., and Trent, J. (1991): Loss of heterozygosity for loci on the long arm of chromosome 6 in human malignant melanoma. Cancer Res., n, 5449-5453.
160. Mitelman, F. (1991): "Catalog of Chromosome Aberrations in Cancer." 4th ed., pp 393-474, Wiley-Liss, New York.
161. Meltzer, P.S., Guan, X-Y, Burgess, A., and Trent, J.M. (1992): Rapid generation of region specific probes by chromosome microdissection and their application. Nature Genetics, 1, 24-28.
149
162. Guan, X-Y, Meltzer, P.S., Cso, J., and Trent, J.M. (1992): Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma. Genomics, 14,680-684.
163. Dower, W.J., Miller, J.F., and Ragsdale, C.W. (1988): High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res., 16,6127.
164. Li, S.J. and Landers, T.A. (1990): Electroporation of plasm ids into plasmid-containing E. coli. Focus, 12, 72-74.
165. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989): Molecular cloning, 2nd ed., AI. Cold Spring Harbor Laboratory Press.
166. Runnebaum, LB., Syka, P., and Sukumar, S.(1991): Vector PCR. BioTechniques, il, 446-449. .
167. Feinberg, A.P. and Vogelstein, B. (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132,6-13.
168. Feinberg, A. and Vogelstein, B. (1984): A technique for rediolabeling DNA restriction fragments to high specific activity. Anal. Biochem .• 137,66-67.
169. Graham, D.E., Mekina, D., and Smith, G.H. (1984): Increased concentration of an indigenous proviral mouse mammary tumor virus long terminal repeat-containing transcript is associated with neoplastic transformation of mammary epithelium in C3H/SM mice. J. Virol., 49, 819-827.
170. Meese, E., Meltzer, P.S., Witkowski, C.M., and Trent, J.M. (1989): Molecular mapping of the oncogene MYB and rearrangements in malignent melanoma. Genes Chromo Cancer, L 88-94.
150
171. Langer, P.R., Waldrop, A.A., and Ward, D.C. (1981): Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA, 78, 6633-6637.
172. Zhang, J., Meltzer, P., Jenkins, R., Guan, X-Y., and Trent, J. (1992): Application of chromosome microdissection probes for elucidation of bcr-abl fusion and variant Philadelphia chromsome trans locations in chronic myelogenous leukemia.
173. Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidi, W., and Arnheim, N. (1992): Whole geneome amplification from a single cell: Implications for genetic analysis. Proc. Natl. Acad. Sci. lJSA,89,5847-5851.
174. Zhu, Y.S., Isaacs, S.T., Cimino, G.D., and Hearst, J.E. (1991): The use of exonuclease TIl for polymerase chain reaction sterilization. Nucl. Acids Res., 19,2511.
175. (1990): Elimination ofPCR contamination with UV light. Foto Dynamics, 2, 1-2.
176. Sarker, G. and Sommer, S., (1990): Shedding light on PCR contamination. Nature, 343, 27.
177. Deragan, J-M., Sinnett, D., Mitchell, G., Potier, M., and Labuda, D. (1990): Use of Gamma irradiation to eliminate DNA contamination in PCR. Nucl. Acids Res., lfi, 6149.
178. Jinno, Y., Yoshiura, K., and Niidawa, N. (1990): Use of psoralin as wxtinguisher of contaminated DNA in PCR. Nucl. Acids Res., lfi, 6739.
179. Schwarz, K., Hansen-Hagge, T., and bartram, C. (1990): Improved yields of long PCR products using gene 32 protein. Nucl. Acids Res., lfi, 1079.
180. Ruano, G., Pagliaro, E.M., Schwartz, T.R., Gaensslen, R.E., and Lee, H.C. (1992): Heat-soaked PCR: An efficient method for DNA amplification with applications to forensic analysis.
151
BioTechniques, 11,266-274.
181. Shuldiner, A.R., Scott, L.A., and Roth, J. (1990): PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products. NucI. Acids Res 18, 1920.
182. Stoker A.W. (1990): Cloning of PCR products after defined cohesive tennini are created with T4 DNA polymerase.NucI. Acids Res., la, 4290.
183. Buchman G.N., Schuster D.M., and Rashtchian A. (1992): Rapid and dfficient cloning of PCR products using the CloneAmpTM system. Focus, 14,41-45.
184. Nisson P.E., Rashtchian A., and Watkins P.C. (1991): Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Meth. and AppI., 1, 120-123.
185. Chumakov I.M., Le Gall I., Billault A., Ougen P., Soularue P., Guillou S., Rigault P., Bui H., De Tand M.-F., Barillot E., Abderrahim H., Cherif D., Berger R., Le PasHer D., and Cohen D. (1992): Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library. Nature Genetics, 1, 222-225.
152