Ž .Clinica Chimica Acta 308 2001 127–131www.elsevier.comrlocaterclinchim

Chromosomal instability detected by fluorescence in situhybridization in Japanese breast cancer patients

Satoshi Takami a, Chie Kawasome a, Moritoshi Kinoshita a,), Hiroki Koyama b,Shinzaburo Noguchi c

a Gene-Diagnostic Center, Otsuka Assay Laboratories, Otsuka Pharmaceutical Co., Ltd., 224-18 Aza Ebisuno Hiraishi, Kawauchi-cho,Tokushima, Japan

b Department of Surgery, Osaka Medical Center for Cancer and CardioÕascular Diseases, 1-3-3 Nakamichi, Higashinari, Osaka, Japanc Department of Surgical Oncology, Osaka UniÕersity Medical School, 2-2 Yamadaoka, Suita, Osaka, Japan

Received 11 October 2000; received in revised form 8 February 2001; accepted 28 February 2001

Abstract

Ž .The relationship between chromosomal instability CIN and prognostic factors was investigated in 31 breast cancers andŽ . Ž .5 benign breast lesions three fibroadenomas and two papillomas . Using fluorescence in situ hybridization FISH with

chromosome-specific DNA probes of chromosomes 1, 2, 6, 7, 10, 11, 17 and 18, CIN for each case was determined. CINvaried from 8.1% to 59.3% among the breast cancer patients tested, and was significantly higher than that observed in the

Ž . Ž .benign breast lesions p-0.01 . Moreover, CIN showed a significant correlation with lymph node metastases p-0.05Ž .and estrogen receptor negativity p-0.01 . These findings suggest that CIN might be useful in the prediction of the

biological aggressiveness of breast cancers. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Breast cancer; FISH; Chromosomal instability; Genetic instability; Centromere probe

1. Introduction

Recent cytogenetic analyses have revealed a vari-ety of karyotypic changes in a high percentage of

w xbreast cancers 1 . Fluorescence in situ hybridizationŽ .FISH is a very useful technique for cytogeneticanalyses. Indeed, FISH analysis of interphase nucleienables the detection of genetic aberrations that aredifficult to be identified by conventional cytogenetic

) Corresponding author. Tel.: q81-886-65-1743; fax: q81-886-65-1734.

E-mail address: kinoshitamo@assay.otsuka.co.jpŽ .M. Kinoshita .

w x Ž .analyses 2 . Chromosomal instability CIN is aw xterm that has been used by Lengauer et al. 3 in

reference to the presence of chromosomal aneu-ploidy in colorectal cancer cell lines.

In this study, we determined CIN using FISH andattempted to elucidate the clinicopathological charac-teristics of breast cancers in relation to CIN.

2. Materials and methods

2.1. Clinical materials

ŽAll tumor tissue samples 31 breast cancers, 3.fibroadenomal, and 2 papillomas were obtained from

0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0009-8981 01 00473-9

( )S. Takami et al.rClinica Chimica Acta 308 2001 127–131128

female patients treated at Osaka Medical Center forŽ .Cancer and Cardiovascular Diseases Osaka, Japan ,

and were subjected to FISH analyses and a routinehistological examination. The details of 31 breastcarcinomas included 13 scirrhous carcinomas, 4 pa-pillotubular carcinomas, 1 medullary carcinoma, 2mucinous carcinomas, 9 solid-tubular carcinomas, 1metaplastic carcinoma, and 1 ductal carcinoma. Inthe stage classification, 3 carcinomas were at stage 1,27 carcinomas at stage 2, and 1 carcinoma at stage 3,respectively.

2.2. Fluorescence in situ hybridization

The probes used were specific for chromosomesŽ .2, 11, 17 and 18 labeled with biotin Oncor , chro-

mosomes 6, 7 and 10 labeled with digoxigeninŽ .Oncor , and chromosome 1 labeled with Spectrum

Ž .Orange VYSIS . These probes fluoresced with brightto brilliant intensity in interphase nuclei of normallymphocytes, and the percentage of nuclei with twosignals per nucleus was )95%. FISH was per-

w xformed according to standard procedures 4 . Surgi-cally resected samples were gently microsected, in-cubated in 0.5% collagenase buffer for 30 min atroom temperature, filtered through a mesh, and cen-trifuged at 1200 rpm for 5 min. Collected cells werewashed in phosphate-buffered saline and incubatedin 75 mmolrl KCl for 10 min at 378C. The cellswere fixed by the addition of an equal volume of

Ž .methanolracetic acid 3:1 , centrifuged, and resus-Ž .pended in an appropriate volume 20–100 ml of

methanolracetic acid solution. The cell suspensionsŽ .were dropped on slides Matsunami, Osaka, Japan ,

dried overnight at 378C, and incubated for 2–4 h at65–708C.

After denaturation in 70% formamider2=SSC,pH 7.0, for 2 min at 758C, the slides were dehy-drated through an ethanol series. In this study, dual-color FISH was performed. Two differently labeledprobes were hybridized and detected simultaneously.Biotinylated probes were detected with avidin-FITCŽ .Boehringer Mannheim, Germany and digoxigenin-labeled probes were detected with anti-digoxigenin-

Ž .rhodamine Boehringer Mannheim . Cells werecounterstained with DAPI. These slides were viewed

Žunder a fluorescence microscope BX 60, Olympus,.Tokyo .

2.3. Identification of chromosomal instability

Only intact and non-overlapping nuclei were eval-uated, and signals were simultaneously visualizedthrough a double bandpass filter. The results wereevaluated by counting at least 100 nuclei. According

w xto the method described by Lengauer et al. 3 , wedefined the ACINB as the average percentage ofnuclei that had signals except modal chromosomenumber for each chromosome. For example, in acase shown in Table 1, modal chromosome number

Table 1Chromosomal instability data obtained for patient 14

( )S. Takami et al.rClinica Chimica Acta 308 2001 127–131 129

is shown by the shaded number, and the CIN was46.5%.

3. Results

ŽA total of 36 breast tumors 31 malignant and 5.benign were examined. Photomicrographs of two

representative cases in which FISH has been used toassay chromosome number and by inference CIN areshown in Fig. 1. Panel AaB illustrates the appearanceof nuclei in which CIN for the assayed chromosomesis not present. All five of the nuclei representedcontain two red chromosome 6 signals and two green

Ž . Ž .Fig. 1. FISH analysis of breast tumors. a and b were hybridizedwith labelled centromeric DNA probes specific for chromosomesŽ . Ž . Ž . Ž .6 red and 11 green . a is a stable pattern while b is unstable.

Table 2Pathological and CIN data of the breast tumors

Ž .No. Histology CIN %

Benign tumors1 Fibroadenoma 10.02 Fibroadenoma 10.33 Fibroadenoma 14.64 Papilloma 12.35 Papilloma 11.8

No. Histology Stage Lymph ER CINŽ .node %

Malignant tumors1 Sci 2 y q 28.02 PT 1 y q 34.03 Med 2 y y 40.14 Muc 2 y q 8.15 PT 2 q q 29.56 Sci 2 y q 10.57 Muc 2 y q 18.88 ST 2 q N.D. 16.99 Sci 1 y y 25.9

10 ST 2 y y 48.611 Sci 2 y q 21.812 Sci 2 y q 17.913 Sci 1 y y 22.014 ST 3 q y 46.515 ST 2 q y 59.316 PT 2 y y 56.817 DCIS 2 y N.D. 11.818 ST 2 q q 12.419 ST 2 y q 31.220 PT 2 y y 47.021 Sci 2 q q 39.422 Sci 2 y y 38.123 PT 2 y q 20.124 ST 2 y y 23.325 Met 2 y y 27.826 Sci 2 q y 27.527 Sci 2 q y 54.028 Sci 2 q q 52.629 Sci 2 y q 20.630 ST 2 y y 22.631 Sci 2 y q 22.6

Sci: scirrhous carcinoma; PT: papillotubular carcinoma; med:medullary carcinoma; Muc: mucinous carcinoma; ST: solid-tubu-lar carcinoma; Met: metaplastic carcinoma; DCIS: ductal carci-noma in situ; ER: estrogen receptor.

chromosome 11 signals. In contrast, two or threechromosome 6 signals, and two to four chromosome11 signals are seen in the nuclei shown in panel AbB.

( )S. Takami et al.rClinica Chimica Acta 308 2001 127–131130

Fig. 2. CIN in breast cancers and benign breast lesions. CIN ofbreast cancers was about 2.5 times higher than that of benignbreast lesions.

Table 2 shows histologic type, stage, lymph nodeŽ .metastasis, estrogen receptor ER status and CIN.

CIN was not correlated with histologic type, buttended to increase with stage. In addition, the meanof CINs in benign lesions was 11.8%, while that inbreast cancers was 30.2%. CIN in breast cancers wasabout 2.5 times higher than that in benign lesionsŽ . Ž .p-0.01 Fig. 2 . Furthermore, CIN in carcinomawas related to the other several pathological factors.CIN in lymph node positive- and ER-negative tu-mors was significantly higher than that in lymphnode negative- and ER-positive tumors, respectivelyŽ .Fig. 3 .

4. Discussion

Conventional cytogenetic studies have revealednumerical and structural changes of chromosomes in

w xbreast cancers 5–7 . In the present study, we havefound that CIN is significantly higher in breast can-cer than benign breast lesions. These results seem tosuggest an important role for numerical chromoso-mal aberrations in carcinogenesis and developmentof breast cancers.

We have also found a significant associationbetween CIN and lymph node metastasis and ER-negativity. These data suggest that chromosomalaberrations may increase according to tumor aggres-

siveness, and that CIN may be useful in the predic-tion of breast cancer aggressiveness.

Since it is cumbersome and time-consuming toassay CIN using eight chromosomes, as was done inthe present study, we also tried using various combi-nations of two chromosomes to determine CIN. Fig.4 shows that CIN calculated from eight chromo-

Ž .somes 1, 2, 6, 7, 10, 11, 17 and 18 correlated veryŽ .strongly with that calculated from two 7 and 10

Fig. 3. CIN according to lymph node status and ER status. CIN inlymph node-positive and ER-negative tumors was significantlyhigher than that in lymph node-negative and ER-positive breastcancers, respectively.

( )S. Takami et al.rClinica Chimica Acta 308 2001 127–131 131

Ž .Fig. 4. Correlation between CIN calculated from eight chromosomes 1, 2, 6, 7, 10, 11, 17 and 18 and that calculated from twoŽ . Ž 2 .chromosomes 7 and 10 . Two CINs showed a strong correlation r s0.927 .

Ž 2 .r s0.963 . These results demonstrate that twoŽ .chromosomes 7 and 10 rather than eight can be

used to assess CIN without loss of accuracy.In conclusion, we have shown that CIN is signifi-

cantly higher in breast cancers than in benign breastlesions, and CIN is also associated with lymph nodemetastases and ER-negativity. These results suggestthat CIN might be useful in diagnosing aggressivebreast cancers and necessary in addition to conven-tional cytogenetic procedures. If the abnormality ofthe chromosomal structure is further examined by

w xusing multicolor FISH 8,9 or comparative genomicw xhybridization 10 , and its relationship with pro-

tooncogene is studied, the developmental mechanismof breast carcinoma, advancement of the carcinoma,and its application to the diagnosis of the malignancymight be known.

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