Microsatellite instability in in situ and invasive sporadic breast cancers of Japanese women

ELSEVIER

CancerLetters 108(1996) 205-209

CANCER LETTERS

Microsatellite instability in in situ and invasive sporadic breast cancers of Japanese women Tatsuya ToyamaaT*, Hirotaka Iwasea, Hiroji Iwataa, Yasuo Haraa, Yoko Omotoa, Mariko Suchib, Taiji Katoc, Takaaki Nakamurad, Shunzo Kobayashi” “Department of Surgery II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan bDepartment of Pediatrics, Nagoya City University Medical School, I Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan “Department of Bioregulation, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, M&ho-ku, Nagoya 467, Japan ‘Division of Clinical Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan

Received23 May 1996;revision received30 July 1996;accepted30 July 1996

Abstract We studied the timing of microsatellite instability (referred to as replication error; RER) presentation during human breast carcinogenesis using tissue microdissected from both in situ and invasive breast cancers of Japanesewomen. We analyzed 100 breast cancer specimens for RER at nine genomic loci on seven chromosomes. Eight of the 100 cases (8%) were RER-positive at one or more chromosomal loci. Additionally; we obtained genomic DNA from two of four RER-positive patients with an intraductal component, both of which showed microsatellite instability in in situ foci. This finding indicates that microsatellite instability may be an early event during human breast carcinogenesis. Keywords:

Breast cancer; Microsatellite instability; Microdissection; Ductal carcinoma in situ

1. Introduction Human tumors appear to accumulate genetic abnormalities as they develop from a single transformed cell to metastatic carcinoma [ 11. Current models of the development of breast cancer propose that there is progression from atypical hyperplasia to in situ carcinoma, and finally to invasive and metastatic carcinoma [2]. Although many investigators have studied the role of chromosomal abnormalities and genetic mutations in sporadic breast cancer, no clear models of the critical events nor delineation of the -* Corresponding author. Tel.: +81 52 8538231; fax: +81 52 8536440.

0304-38X/96/$12.00 0 1996Elsevier ScienceIreland Ltd. All PII

SO304-3835(96)04414-X

primary abnormalities leading to breast carcinoma have emerged. Microsatellites are short repeated nucleotide sequences that are interspersed throughout the human genome and are useful as markers for genetic mapping in humans, rodents and other vertebrates, because they are stably inherited, vary from individual to individual, and have a relatively low inherent mutation rate. Instability within these sequences has been described in numerous human tumors [3-51, including breast cancer [5--71. In this study, we analyzed tissue from 100 Japanese patients with breast cancer for microsatellite instability at nine genomic loci on seven chromosomes. We used microdissection of tissue sections to further investigate microsatellite instability in in situ and invasive breast cancer.

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2. Materials

and methods

2.1. Tumor samples Resected tumor specimens and corresponding peripheral blood lymphocytes were obtained from 100 Japanese female patients with primary sporadic breast cancer who had been treated at the Nagoya City University Hospital between 1983 and 1995. Two samples frozen in ‘Tissue-Tee, OCT compound’ (Miles, Inc., Elkhart, IN, USA) also available for study. There were 98 invasive ductal and two lobular carcinomas. 2 2. DNA extruction Genomic DNA was extracted using a phenolchloroform procedure employing standard techniques IX]. Normal control DNA for each patient was obtained from peripheral blood lymphocytes. Cells procured by microdissection were resuspended immediately in 20 ~1 of a solution containing 10 mM TrisHCl, 100 mM EDTA, 1% (v/v) Tween-20. and 0. I mg/ml proteinase K (pH 8.0) and incubated overnight at 37°C. The mixture was boiled for 5 min to inactivate proteinase K, and 10% of this solution was used for polymerase chain reaction (PCR) analysis. 2 3. Mic.rodisser,lion Microdissection of in situ and invasive tumor cells from two OCT compound-embedded tissue specimens was performed under direct light microscopic

Table

visualization. Briefly, IO-pm histologic sections were prepared on glass slides, stained with hematoxylin. and allowed to air dry. The adjacent section was stained with hematoxylin and eosin. Specific cells of interest were selected from the hematoxylin-stained slides and microdissected using a disposable 27. gauge needle. DNA was extracted by incubating each sample in 100 ~1 of a solution containing SC) mM Tris-HCl, 1 mM EDTA. 0.5% (v/v) Tween-30. and 0.5 mg/ml proteinase K (pH 8.0) in a shaking incubator at 37°C overnight. Samplea were then boiled for 10 min and vortexed. Two to 10 yl of the samples were used for PCR analysis depending on the number of cells present in the sample. 2.4. Mic7wsatellite

anatysi.r

Tumor and control DNA were amplified by PCR al the following nine microsatellite markers (Table 1!: chromosome 1, Alu Vpa ( 1~32); chromosome 4, FGA (4q28); chromosome 6, ESR (6q25) 191. HTF and F 13A I (6~24-25 1;chromosome 8, D8S320 (8q); chromosome 11, Dl 1SS54; chromosome 12, PI-AZ; chromosome 17. D17S846. PCR was performed in 15 ~1 volumes of a mixture containing 1 x PCR buffer (50 mM KCI, 10 mM Tris buffer, pH 8.3) and 150 ng of genomic DNA, 3 pmol each of unlabeled primer and S-end-labeled primer, 0.9 LJ Taq DNA Polymerase (TaKaRa. Kyoto, Japan). 200 PM of each deoxynucleotide triphosphate and 1.5-6.7 mM MgC12. The S’-end-labeled primers were labeled using I’4 polynucleotide kinasr

I

Microsatellite

markers

-I__--

I--

Name

Chromosomal localization

Repeat unil six

Annealing temp. (“C)

MgC& (mM)

PCR cycles

Alu Vpa FGA

I 3

AAAG TCTT

56 60

ESR HTF F13Al D8S320 DllS554 PLA2 D17S846

6 6 6 8 II 12 17

TA CAG AAAG AAAG ,4AAG TTA GGAA

58 sx 58

1 1._ 6.7 2 ‘7

78 2x 24 ‘4 24

60

2

24

60 60 58

6.7

20 ‘.I 30

Annealing

temp., annealing

temperature

l_-_-

h.‘i I .5

--.Case number of RER i Y-5 Y-5. Y-80. 933, 945 Y-5 Y-5, 933. 935 Y-3. Y-S. Y-28. ) .i -----_____

Y-44

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(TaKaRa) and [T-~*P]ATP (-3000 Ci/mmol) (Amersham, Tokyo, Japan). PCR was initiated by denaturation at 94°C for 5 min, followed by 20-30 cycles consisting of 1 min of denaturation at 94”C, 1 min of annealing at 56-60°C (Table 1). and 90 s of standard elongation at 72°C. Final elongation was performed for 5 min at 72°C using a PCR apparatus (DNA thermal cycler 480, Perkin Elmer, Foster City, CA, USA). A 4 ~1 aliquot of each PCR reaction mixture was diluted 1: 1 with loading buffer (80% (v/ v) formamide, 50 mM Tris-borate, 1 mM EDTA (pH 8.0), 0.1% (w/v) bromophenol blue, and 0.1% (w/v) xylene cyanol), heat denatured, and analyzed by electrophoresis on 5% polyacrylamide gel under denaturing conditions at 40 W for 2-3 h. The gel was dried and exposed to autoradiograph film for 12-24 h.

loci. All RER-positive cases were invasive ductal carcinomas. In situ breast carcinomas were found in four of eight RER-positive cases. Genomic DNA was obtained from two (case Y-44 and 93-5) of four RER-positive cases with in situ tumor. Both case Y44 and 93-5 showed RER at a single locus, Dl lS554 and ESR, respectively. For both patients, in situ tumor and invasive tumor were obtained from the same histologic tissue section. The corresponding hematoxylin and eosin stained slides prepared for each case were reviewed and cell populations were identified for microdissection. Both RER-positive cases showed microsatellite instability in both the in situ and invasive tumor component. The pattern of microsatellite instability of the in situ lesion was the same as that of the invasive lesion in each case.

3. Results

4. Discussion

Of the 100 sporadic breast cancers of Japanese women studied, eight (8%) showed RER at one or more microsatellite chromosomal loci. Six showed RER at single locus, one at two loci, and one at six

Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasia to in situ carcinoma, and finally to invasive and metastatic carcinoma [2]. Nascent in situ carcinomas fre-

Fig. 1, Microdissection

of an in situ intraductal

carcinoma.

(A) A hematoxylin-stained

section ( x 100). (B) A microdissected

section (

x

100).

DllS554

ESR

IV IS N

IV IS N

Fig. 2. Microsatellite (IV) in breast cancer pound-embedded (XI. Arro\~s. iumors cponding

instability obtained

ol iio m SIILI (6) from microdissected

and

an inwsivc OC‘T com-

tissue sections and a paired normal blood control the positions of all&~ from the in GILI and inv;tsiw.~

with different electrophoretic normal DNA of the paticnr.

mobility

than

that

of corre

quently arise in association with a spectrum of epitheha1 hyperplasias, and larger invasive tumors are often associated with regions of carcinoma in situ at the tumor periphery. The currenl model of the development of breast cancers proposes that invasive breast cancers originate as atypical hyperplasias of epithelium. progress to in situ lesions, and eventually develop into invasive cancers. We are interested in examining the timing fur presentation of microsatellite instability in human breast carcinogenesis. Because carcinoma in situ and foci of invasive cancers are typically small microscopic lesions, it was necessary to use tissue microdissection to obtain specific populations of cells for study. Zhuang et al. [lo] and Koreth et al. [ 111 have investigated the loss of heteroLygosity (LOH) of chromosome 1lq in human breast cancer using tissue microdissection for PCR-LOH analysis at polymorphic microsatellite loci. We used a similar tissue microdissection technique to detect microsatellite instability in both in situ and invasive lesions of the breast cancers. As a result, both two RER-positive cases showed microsatellite instability in the in situ components ot invasive tumors. This finding indicates that microxtellite instability is an early event in human breast carcinogenesis. Aldaz et al. [12] reported microsatellite instability in ductal carcinoma in situ (DCIS) bj means of microdissection. Their study was. however.

performed only on pure DCXS tumors with no invasive component. Microsatellite instability is a phenomenon consisting of abnormalities in the electrophoretic mobility of alleles from tumor tissue compared with those from matched normal control tissue 131. This phenomenon has been described as a characteristic of tumors obtained from patients carrying the autosomal dominant predisposition to tumors of the colon and endometrium known as hereditary non-polyposix colorectal cancer IHNPCC) 131. These studies identified :I group of human DNA mismatch repair genes responsible for this generalized genomic instabilit! phenomenon. The four mismatch repair genes known to be mutated in the germline of HNPCC patient!, are izMSH2. hMLH1, hPMS1, and hPMS2 j l.‘i- 171.These genesare the human homologucsol Ihr prokaryotic ~rutS and nrutl, genesthat are essential 10F,‘~lle/Yc$lirxc,~,iifor the initiation of the repair ot mismatchedDNA [ IX J.Therefore. it is suggestedthat abnormalitiesof the genesresponsiblefor DNA mismatch repair occur early in carcinogenesis.Microsatellite instability appears to be a novel molecular marker of carcinogenesisand is thought to reflect KER induced by the dysfunction of the previouslq discussedmismatch repair genes.Mutations of DNA mismatch repair genes are expected to increase the frequency of other gene mutations, resulting in the activation of protooncogenes or the inactivation ot rumor suppressorgenes. We observed the RER -t phenotype in eight of 100 Japanesebreast cancer patients (8%). This figure is similar to that reported previously in Caucasianbreast cancer patients 15-Y 1, Aldaz et al. 1121have reported that microsatellite instability occurred at a high frequency in invasive lobular breast carcinomas, suggesting that those tumors ariseby mechanismsof carcinogenesisdiffercnt from those leading to ductal breast carcinoma. However, mutations in the DNA tnismatch repair genehave not beenreported in sporadicbreastcancer. and therefore, the mechanismsresponsiblefor RER in sporadic breastcancer remain unknown.

We gratefully acknowledge the technical assistance of Mrs. Mariko Nishio, Department of Surgery II,

T. Toyama

Nagoya City University Japan.

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