Chromosome-specific Aneusomy in Carcinoma of the Breast1 · Vol. 2, 883-888, May 1996 Clinical Cancer Research 883 4 The abbreviations used are: FISH. fluorescence in situ hybridization;

Vol. 2, 883-888, May 1996 Clinical Cancer Research 883

4 The abbreviations used are: FISH. fluorescence in situ hybridization;DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.

Chromosome-specific Aneusomy in Carcinoma of the Breast1

Diane L. Persons,2 Robert A. Robinson,

Ping H. Hsu, Steven A. Seelig, Thomas J. Borell,

Lynn C. Hartmann, and Robert B. Jenkins3

Departments of Laboratory Medicine and Pathology [D. L. P.,T. J. B., R. B. J.l and Medical Oncology [L. C. H.], MayoClinic/Foundation, Rochester, Minnesota 55905; Department ofPathology, University of Iowa Hospital, Iowa City, Iowa 52242[R. A. R.]; and Vysis, Inc., Downers Grove, Illinois 60515[P. H. H., S. A. 5.1

ABSTRACT

Fluorescence in situ hybridization was performed on

touch preparations from 55 primary infiltrating ductal car-

cinomas of the breast to determine numeric chromosome

abnormalities. The frequency of aneusomy, measured by

both nondisomy and chromosomal gain, was determined for

chromosomes X, 4, 6-12, 17, and 18 with the use of chro-

mosome-specific, a-satellite DNA probes. The presence of

chromosome-specific numeric abnormalities was correlated

with established clinicopathological parameters, including

tumor size, lymph node involvement, tumor grade, estrogen

receptor level, and menopause status. In addition, a case-

control study was performed to explore a possible associa-

tion between chromosome-specific aneusomy and recur-

rence in lymph-node-negative patients.

Although chromosomes 8 and 6 were most frequently

aneusomic, numeric abnormalities of chromosomes 4 and 11

were most strongly associated with established prognostic

factors. For chromosomes 4 and 11, strong associations were

found with tumor involvement of lymph nodes and in-

creased tumor size, along with a weaker association with

tumor grade. In addition, numeric abnormalities of the

following chromosomes were associated with the corre-

sponding prognostic factors: chromosomes X, 7, and 12 with

lymph node status; chromosomes 10, 17, and 6 with tumor

size; and chromosomes 7, 12, 17, and X with tumor grade.

No correlations were observed with estrogen receptor level

or menopause status. In the case-control study performed on

isolated nuclei of paraffin-embedded tissue from lymph

node-negative breast cancer patients (19 cases and 19 con-

trols), the gain of chromosome 4 was correlated with disease

progression. These findings suggest that chromosome-

specific aneusomy is associated with certain established

prognostic factors and may be associated with disease pro.

gression.

Received 7/3 1/95; revised 12/28/95: accepted 1/24/96.I This work was supported in part by a grant from Vysis, Inc.2 Present address: Department of Pathology and Laboratory Medicine.

the University of Kansas Medical Center, Kansas City, KS 66160.3 To whom requests for reprints should be addressed, at LaboratoryGenetics, 970 Hilton, Mayo Clinic, 200 1st Street SW., Rochester, MN

55905. Phone: (507) 284-4696; Fax: (507) 284-0043.

INTRODUCTION

Breast cancer is the most common life-threatening malig-

nant neoplasm in women. An estimated 182,000 new cases will

be identified, and 46,000 deaths will occur in U.S. women in

I 994 ( I ). As with other solid tumors, technical difficulties have

resulted in limited cytogenetic information on cultured breast

tumors (2). Efforts to avoid culturing by performing direct

cytogenetic analysis on breast tumors have been hampered by

low mitotic rates and poor chromosome morphology. Chromo-

somes that have thus far been found to be relatively frequently

involved in structural alterations in breast cancer include chro-

mosomes 1, 3, 6, and I 1, whereas numeric abnormalities report-

edly often involve chromosomes 7, 8, 18, and 20 (3-7).

FISH4 using chromosome-specific, a-satellite DNA probes

can be used to evaluate aneusomy in interphase and/or metaphase

cells (8). Large numbers of uncultured interphase cells can be

evaluated rapidly. Several studies have used FISH for analysis of

aneuploidy in solid tumors (9-12). In situ hybridization studies in

breast cancer have been limited to evaluation of aneuploidy in the

breast cancer cell line MCF-7 (13, 14) and the study of seven

primary tumors for abnormalities of chromosomes I and 18 (13).

Using FISH, touch preparations of 55 primary infiltrating

ductal breast carcinomas were analyzed for numeric abnormal-

ities ofchromosomes X, 4, 6-12, 17. and 18 (for which directly

labeled a-satellite probes were available) at two collaborating

institutions. In addition, a case-control study using isolated

nuclei from paraffin-embedded tissue was performed to exam-

inc possible associations between chromosome-specific aneu-

somy and disease progression in node-negative breast carci-

noma. In this report, we present the chromosome aneusomy

results detected by FISH and correlate the findings with clini-

copathological data.

MATERIALS AND METHODS

Tissue Samples and Slide Preparation. Tumor samples

for FISH analysis of touch preparations were obtained from pa-

tients with primary breast carcinoma at the time of diagnosis. None

of the patients had received prior therapy. Samples were collected

at the two study sites (Mayo Clinic, site 1; and University of Iowa,

site 2) between January 1992 and December 1993. The study was

limited to infiltrating ductal carcinomas. Intaductal carcinoma was

present in a minority of cases; however, the infiltrating component

was the major component in all cases. Fresh tissue from 10 normal

breasts was used as control specimens. Touch preparations were

made from tissue samples obtained adjacent to areas where histo-

logical frozen sections verified the presence of tumor cells. Touch

preparations made from each tissue sample were fixed in cold

methanol for 20 mm, air dried, and stored at -20#{176}Cuntil in situ

hybridization was performed.

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884 Aneuploidy in Breast Carcinoma

In the retrospective case-control study, isolated nuclei were

obtained from paraffin-embedded breast carcinoma from lymph

node-negative cases. Nineteen cases of node-negative breast

carcinoma were matched by tumor size (within 0.5 cm) and

surgical date. Cases were defined by evidence of disease pro-

gression within the first 3 years following surgery, whereas

control patients, who had been followed for a minimum of 6.5

years, showed no evidence of progression.

In Situ Hybridization with Centromere Enumeration

Probes. Detailed FISH methods for touch preparations have

been described previously (15). Briefly, frozen slides of touch

preparations were thawed, fixed for 20 mm in 3: 1 methanol:acetic

acid, dehydrated in an ethanol series, and air dried. For paraffin-

embedded tissue, isolated nuclei were prepared by the method of

Hedley et a!. (16) from two to four 40-p.m sections of tissue. The

sections were deparaffinized in Histo-Clear (National Diagnostics,

Atlanta, GA) for 10 mm (three times), dehydrated for 5 mm in

100% ethanol (twice), and rinsed in distilled water for S minutes

(twice). Two ml of an 8.5-mg/mi pepsin solution [P-70l2; Sigma

Chemical Co., St. Louis, MO; 8.5 mg/ml in 0.9% NaCl (pH 1.5)]

were added, and the sections were incubated at 37#{176}Cfor 2-2.5 h

with vortexing every 30 mm. The digested material was filtered

through 40.jam nylon mesh (Tetko, Briarcliff Manor, NY) and

centrifuged at 2400 rpm for 5 mm. The material was washed twice

in PBS, resuspended in 200-500 �xl PBS, and dropped onto Su-

perfrost/Plus slides (Fischer Scientific, Pittsburgh, PA). The cell

suspension was spread over an approximately 22 X 22-mm area

and oven dried at 60#{176}Cfor 10 mm.

Fluorescent, directly labeled a-satellite DNA probes for

chromosomes X, 4, 6-12, 17, and 18 and the hybridization

solution were provided by Vysis, Inc. Probes were used in either

single-color or in two-color combinations. A total of 10 p.1 probe

and hybridization mix was placed on the slide, which was

coverslipped and sealed with rubber cement. Following simul-

taneous denaturing of the probe and target DNA in a 90#{176}Coven

for 1 mm for touch preparations, the slides were incubated

overnight at 41#{176}Cin a moist chamber. For isolated nuclei from

paraffin-embedded tissue, slides were denatured at 80#{176}Cfor 10

mm and incubated at 50#{176}Cfor approximately I h. Posthybrid-

ization washes (10 mm each) for touch preparations included

three 50% formamide washes in 2X SSC (300 mmol/L sodium

chloride and 30 mmol/L sodium citrate) at 45#{176}Cand single

washes in 2X SSC at 45#{176}C,2X SSC-0.l% NP4O at 45#{176}C,and

2X SSC-0. 1% NP4O at room temperature. Times for posthy-

bridization washes for isolated nuclei were reduced to 1-5 s in

one 50% formamide wash and 5-10 s in 2X SSC at 45#{176}C,2X

SSC-0.l% NP4O at 45#{176}C,and 2X SSC-0.l% NP4O at room

temperature. Nuclei were counterstained with 1 jxg/ml DAPI in

the antifade p-phenylenediamine dihydrochloride.

Analysis of Interphase in Situ Hybridization. Hybrid-

ization signals were enumerated within 500 interphase nuclei per

specimen. Criteria for analysis of nuclei have been described pre-

viously (15). Overlapping nuclei and nuclei lacking any hybridiza-

tion signals were excluded from analysis. All specimens had more

than approximately 85% ofnuclei with hybridization signals. Anal-

ysis and photography were performed on fluorescence microscopes

equipped with a triple-pass filter (DAPI/Green/Orange; Vysis) for

simultaneous detection of SpectrumGreen, SpectrumOrange, and

DAPI, or a dual-pass filter (DAPI/Orange) for detection of Spec-

trumOrange and DAPI.

Two methods of analysis were used to evaluate the signif-

icance of numerical chromosome abnormalities. In method 1,

the presence of significant aneusomy was determined by using

a cutoff value of 40% nondisomy (any signal pattern other than

two signals) for each chromosome. In method 2, the presence of

25% of the nuclei with more than two signals was considered

abnormal for any given chromosome. The associations between

chromosome-specific aneusomy by either method and clinico-

pathological variables were determined by x2 analysis (lymph

node status, estrogen receptor status, and menopause status) and

ANOVA (tumor size and tumor grade). A one-tailed, paired

test was performed for analysis of cases and their corresponding

controls in the case-control study.

RESULTS

Touch Preparation Studies. The use of dual-color FISH

for chromosome centromere enumeration is illustrated in Fig. 1.

The numbers of centromeric signals (one, two, three, four, etc.)

for the 1 1 chromosomes were counted in each of 55 breast

carcinoma touch preparations. It should be emphasized that the

use of centromere probes identifies the copy numbers of mdi-

vidual chromosomes but does not determine whether the chro-

mosomes are structurally normal or abnormal.

Because a standard approach for analysis of chromosome-

specific aneusomy has not been developed, the chromosome

copy number data were analyzed by two methods. In the first

method, the frequency of chromosome-specific aneusomy

within the 55 cases was determined by using a cutoff value

based on normal controls. The mean nondisomy (patterns other

than two signals) among the 1 1 chromosomes in the I 0 normal

breast touch preparations varied between 9 and 14%. The mean

nondisomy values plus 3 SD varied between 26 and 37% (data

not shown). Therefore, to assure identification of only signifi-

cant aneusomic populations, a 40% cutoff value was chosen.

Significant aneusomy (>40% nondisomy) for at least one of the

chromosomes examined was observed in 44 of 55 cases. Fig. 2

is an example of the pattern of the percentage of nondisomic

cells observed in one case for the I I chromosomes. In this case,

chromosomes 6, 7-12, and 18 were classified as aneusomic.

The second method of analysis of numeric chromosome ab-

normalities focused on specific chromosome gains (more than than

two signals). In control specimens, the mean gain for individual

chromosomes varied between 1 .5 and 7.2%. The mean values plus

3 SD varied between 1.9 and 10.4%. Observing more than two

signals for a specific chromosome reflects either endoreduplication

of the specific chromosome or a tetraploidization event resulting in

doubling of the number of all chromosomes. This method was

chosen because cytogenetic evidence has suggested that trisomies

may be relevant as early changes in breast cancer (7). In addition,

it was thought that analyzing the chromosomal gain would be a

sensitive measurement of aneusomy that would avoid certain tech-

nical artifacts, such as decreased hybridization efficiency and ran-

dom chromosomal loss.

The frequency of aneusomy by both methods for each

chromosome in the cohort of 55 cases is illustrated in Fig. 3. The

most commonly aneusomic chromosome was chromosome 8,



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Clinical Cancer Research 885

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Fig. I Photomicrograph ( X630) illustrating(SpectrumOrange) and 1 1 (SpectrumGreen).

100

90

80

70

60

50

40

Chromosome

nuclei with tour signals and nuclei with two signals for the centromeres of chromosomes 6

Fig. 2 Example of chromosome-specific aneusomy (percentage ofnondisomic nuclei ) for I 1 chromosomes in an individual case of infil-

trating breast carcinoma. Horizo,,tal li,,e, 40% cutoff value for signifi-

cant aneusomy.

which had an abnormal copy number (nondisomy) in 53% of

cases and was gained in 58% of the cases. Likewise, chromo-

some 6 was aneusomic in a higher proportion of cases (47C/�

nondisomic and 50% gain) than were the remaining chromo-

somes. Nondisomy and/or gain of the remaining nine chromo-

somes was observed in 24-44% of the cases.

Clinicopathological data, including lymph node status. tu-

mor size, tumor grade, estrogen receptor status, and menopausal

status for the 55 cases, are summarized in Table 1 . Significant

[iri[IrI.rI[1.x 4 6 7 8 9 10 11 12 17 18

Chromosome

Fig. 3 Frequency (percentage of cases) of chromosome-specific aneu-

somy (LI, nondisomy. #{149},gain) in 55 cases of brcast carcinoii�a.

statistical correlations (P < 0.05) between the clinicopatholog-

ical variables and the frequency of aneusomy (40% nondisomy)

and gain (more than two signals in 25% cells) for each chro-

mosome are shown in Table 2. Aneusomy for chromosomes 4

(P = 0.007, nondisomy; P 0.009. gain) I 1 (P 0.05,

nondisomy: P = 0.001, gain). and X (P = 0.006. nondisoniy:

P = 0.01. gain) was found to be significantly associated with

tumor involvement of lymph nodes. An association with lymph

node status was also observed with the gain of chromosomes 7

(P = 0.005) and 12 (P = 0.03).



Table 1 Clinicopathological patient data (n = 55)

Characteristic No. of patients

Table 2 Chromosome-specific aneusomy: associations withclinicopathological variables

See text for description of methods I and 2. All values P < 0.05.

ChromosomeMethod 1

(40% Nondisomy)Method 2

(>2 signals)

Lymph node status4 0.007 0.0097 0.00511 0.05 0.001

12 0.03

X 0.006 0.01

Tumor size4 0.04 0.00056 0.01

10 0.0002 0.00111 0.01 0.0003

17 0.03 0.002

Tumor grade4 0.05

7 0.03 0.004

11 0.0212 0.009 0.02

17 0.03

X 0.02


Age (yr)<4040-49

50-59

60-69

>69Lymph nodes

PositiveNegative

Tumor size (cm)<2

2-5>5

Tumor grade2

3

4

Estrogen receptorPositiveNegative

MenopauseBefore

After

4

5

12

1618

21

34

12

394

8

20

27

45

10

4510

Increasing tumor size was found to be associated with

numeric chromosome abnormalities determined by both meth-

ods involving chromosomes 4 (P = 0.04, nondisomy; P =

0.0005, gain), 10 (P = 0.0002, nondisomy; P = 0.001, gain), 11

(P = 0.01, nondisomy; P = 0.0003, gain), and 17 (P = 0.03,

nondisomy; P = 0.002, gain). In addition, chromosome 6 (P =

0.01) was associated with tumor size.

Tumor grade was associated with abnormal chromosomes

7 (P = 0.03, nondisomy; P - 0.004, gain) and 12 (P = 0.009,

nondisomy; P = 0.02, gain), as determined by both methods.

Nondisomy of chromosome X (P = 0.02) was also associated

with tumor grade, as was the gain of chromosomes 4 (P = 0.05),I 1 (P = 0.02), and 17 (P = 0.03).

No associations were observed between aneusomy by ci-

ther method for any of the 1 1 chromosomes and estrogen

receptor or menopause status.

Lymph Node-negative Case-Control Study. Table 3

summarizes the results of aneusomy of specific chromosomes in

breast carcinomas from node-negative patients whose disease

progressed in the first 3 years following diagnosis (cases) com-

pared with matched control patients (controls), in whom no

evidence of disease progression was observed within at least 6.5

years following diagnosis. The difference in the mean percent-

age of aneusomic (other than two signals) cells and the differ-

ence in the mean percentage of gain (more than two signals)

between matched cases and controls are illustrated for each

chromosome. The most significant difference in the mean values

for both aneusomy and gain was observed with chromosome 4

(P = 0.09 and 0.05, respectively).

DISCUSSION

In this study, we have described chromosome-specific,

numeric abnormalities in a group of 55 primary infiltrating

ductal carcinomas of the breast. Most previous cytogenetic

studies of breast cancer have suggested that most breast tumors

have clonal chromosomal abnormalities (17-22). In contrast,

other authors have reported that a substantial proportion of

primary tumors has a normal diploid karyotype (23, 24). The

difference in these results could be due to a number of factors,

including case selection and various culture artifacts. The use of

FISH on uncultured tumor nuclei provides the advantage of

accurately reflecting the in vivo chromosome complement. In

addition, culture artifacts, such as overgrowth of normal diploid

cells or selection of subclones possessing in vitro growth ad-

vantages, are avoided. The observation of significant aneusomy

for at least one of the chromosomes examined in 44 of 55 of the

tumors in this study supports the previous reports of the pres-

ence of clonal abnormalities in a large fraction of primary breast

tumors. Because our method detected only numeric abnormal-

ities and examined only one-half of the chromosomes, it is likely

that an even higher proportion of primary tumors contains either

structural and/or numeric clonal abnormalities.

In this study, chromosomes 8 and 6 were found to be the most

freq uently aneusomic chromosomes in the 55 breast carcinomas.

Both the gain and loss of chromosome 8 have been described as

frequent aneusomy in breast cancer cytogenetic studies (25-27).

Rohen et aL (7) recently described trisomy 8 as a clonal aberration

in 10 of 39 clonally abnormal breast carcinomas. In addition, they

reported trisomy 8 as the most frequent nonclonal aberration. The

investigators performed FISH in two cases in which nonclonal

trisomy 8 was observed. Three signals for the centromere of chro-

mosome 8 were present in 1 1 and 14% of the nuclei of the two

cases. The authors suggested that their findings indicated that

trisomy 8 was present in microclones. The present study confirms

previous reports and shows that an abnormal copy number of

chromosome 8 was present in more than 50% of the cases.

Chromosome 6 is one of the most frequently structurally

abnormal chromosomes present in breast cancer (2 1 ). However,

the gain or loss of the entire chromosome has not been reported

as a frequent finding by conventional cytogenetic studies. In

addition, 6q is a frequent site of loss of heterozygosity in breast

tumors (28). Numeric abnormalities of chromosome 6, includ-



Clinical Cancer Research 887

Table 3 Node-negativ e breast cancer case- control study

Nondisomy

(%; mean ± SD) DifferenceI test

one-tailedCases Controls

Chromosome (ii 19) (n = 19) (mean ± SE) P

4 21.3 ± 15.1 15.9 ± 8.6 5.4 ± 3.7 0.09

6 27.1 ± 19.9 20.4 ± 16.8 6.7 ± 6.3 0.157 22.4 ± 16.1 21.2 ± 20.9 1.1 ± 5.8 0.43

8 26.7 ± 18.7 22.3 ± 17.1 4.4 ± 7.0 0.27

9 18.3 ± 15.3 22.1 ± 21.6 -3.8 ± 6.4 0.72

10 19.0 ± 15.2 18.8 ± I 1.4 0.1 ± 4.6 0.491 1 21.9 ± 16.5 19.3 ± 16.2 2.6 ± 6.0 0.34

12 18.0 ± 13.8 19.3 ± 18.0 -1.3 ± 5.7 0.59

17 21.5 ± 16.3 20.7 ± 13.5 0.8 ± 5.2 0.45

18 21.8 ± 15.7 17.1 ± 6.1 4.7 ± 4.2 0.14X 24.0 ± 16.2 23.3 ± 16.8 0.7 ± 6.2 0.46

ing those that were possibly structurally abnormal, were de-

tected in approximately 50% of the cases in this study.

Aneusomy, as evaluated by either specific chromosome

nondisomy or chromosomal gain, was found to be associated

with select prognostic factors. Evaluation of chromosome-spe-

cific gain seemed to be a more sensitive measurement than

nondisomy for correlating aneusomy with prognostic variables.

It is of interest that the chromosomes that most commonly had

numeric abnormalities, chromosomes 8 and 6, were not found to

be associated with prognostic factors, with the exception of a

weak association of chromosome 6 with tumor size. In contrast,

some of the other less commonly aneusomic chromosomes

showed strong associations with recognized prognostic factors

in this disease, namely, lymph node status (chromosomes 4, 7,

11, 12, and X), tumor size (chromosomes 4, 10, 11, and 17), and

tumor grade (chromosomes 4, 7, 1 1, 12, 17, and X).

Numeric abnormalities of chromosome 4 were strongly

associated with lymph node status. A statistically significant

association with tumor size and a weak association with tumor

grade were also found. Lu et a!. (5) reported the loss of chro-

mosome 4 to be a relatively common finding (5 of 22 cases) by

direct cytogenetic analysis of primary breast tumors. In this

study, however, a significant association with the prognostic

factors was seen with the gain of the chromosome, along with

nondisomy. In the retrospective case-control study comparing

disease progression and chromosome-specific aneusomy in

lymph node-negative cases, disease progression was found to be

most closely associated with abnormalities of chromosome 4.

Although (due to the small sample size) the finding was not

statistically significant with nondisomy (P = 0.09), marginally

significant associations were observed with the gain of chromo-

some 4 and disease progression (P = 0.05).

Chromosome 1 1 had a similar pattern of association with the

same prognostic factors as chromosome 4. Structural abnormalities

of chromosome 1 1, including loss of chromosomal material and

amplification of sequences, have been reported in breast cancer.

Mackay et a!. (29) correlated the loss of sequences at 1 lpl3-lS to

the lack of estrogen receptors and increased tumor size. Another

study (30) reported associations between the loss of sequences on

1 lp and grade III tumors, distal metastases, and the lack of estrogen

and progesterone receptors. Amplification of sequences harboring

the genes int-2, bcl-l, and hst, located at 1 1q13, has been reported

in 4-17% of breast tumors (31, 32).

Strong associations between tumor size and abnormal copy

numbers of chromosomes 10 and 17 were observed. Although not

a frequent finding, clonal numeric abnormalities of chromosome 17

have occasionally been reported in conventional cytogenetic stud-

ies of breast tumors (21, 33). Abnormalities of chromosome 17 are

of great interest for several reasons. A region on the short arm of

chromosome 17, including the TP53 gene, has the highest fre-

quency of allele loss (deleted in >50% of informative cases) in

primary breast carcinomas (28, 34-36). Furthermore, approxi-

mately 40% of breast carcinomas have loss of heterozygosity on

the long arm of chromosome 17 (34, 36). Several candidate tumor

suppressor genes reside on l7q, including BRCA-1 (37), the pro-

hibitin gene (38), nm23 (39), and Wnt-3 (40).

Numeric abnormalities, especially gains, of chromosome 7

were found to be associated with lymph node status and tumor

grade. Trisomy 7 has been described as a frequent clonal and

nonclonal abnormality in primary breast carcinomas (7). It has

also been noted as the sole cytogenetic abnormality in some

breast tumors (21, 41).

This study demonstrated a high frequency of chromosome

copy number aberrations in breast carcinoma. Although chro-

mosomes 8 and 6 were the most frequently abnormal chromo-

somes, chromosomes 4 and I I showed the strongest associa-

tions with established prognostic factors. For both chromosomes

4 and I I , strong associations were noted with lymph node status

and tumor size. In addition, aneusomy of chromosome 4 was

most closely associated with disease progression in node-nega-

tive breast cancer patients.

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1996;2:883-888. Clin Cancer Res D L Persons, R A Robinson, P H Hsu, et al. Chromosome-specific aneusomy in carcinoma of the breast.

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Chromosome-specific Aneusomy in Carcinoma of the Breast1 · Vol. 2, 883-888, May 1996 Clinical Cancer Research 883 4 The abbreviations used are: FISH. fluorescence in situ hybridization;