Hum Genet (1994) 94:231-234 �9 Springer-Verlag 1994

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M o r a i m a Z e l a d a - H e d m a n �9 Mar ta Torroel la Rober to Mesqui ta �9 Magnus Nordenskj61d Lamber t Skoog �9 Ann ika L indb lom

Loss of heterozygosity studies in tumors from families with breast-ovarian cancer syndrome

Received: 28 January 1994 / Revised: 8 March 1994

Abstract A gene ( B R C A 1 ) p r ed i spos ing for fami l ia l breast and ovar ian cancer has been m a p p e d to chromo- some band 17q12-21. Based on the observat ion that ovar- ian tumors f rom famil ies with breas t and ovar ian cancer lose the wi ld - type al lele in the region for the BRCA1 lo- cus, it has been sugges ted that the gene funct ions as a tu- mor suppressor gene. We have s tudied ch romosomal dele- t ions in the BRCA1 region in seven breas t tumors, three ovar ian tumors, one b ladder cancer, and one colon cancer f rom patients in six famil ies with breas t -ovar ian cancer, in order to test the hypothes is o f the tumor suppressor mech- an i sm at this locus. We have found a low f requency of loss o f he te rozygos i ty at this region, and our results do not support the idea that BRCA1 is a tumor suppressor gene. Al ternat ively , the d isease segregat ing in these famil ies is l inked to one or more different loci.

Introduction

Between 10% and 14% of all breas t cancer cases be long to famil ies wi th a genet ic pred ispos i t ion to the d isease (Bcrresen 1992). In some o f these famil ies , other tumors occur in a manner suggest ing that they represent different enti t ies o f inher i ted famil ia l syndromes . Mos t c o m m o n after famil ies with b reas t -on ly cancer are famil ies with an

M. Zelada-Hedman �9 M. Torroella National Institute of Oncology and Radiobiology, Havana City, Cuba

M. Zelada-Hedman �9 M. Nordenskj61d - A. Lindblom (1~) Department of Clinical Genetics, Karolinska Hospital, S-171 76 Stockholm, Sweden

R. Mesquita Immunopathology Laboratory, Karolinska Hospital, S-17176 Stockholm, Sweden

L. Skoog Division of Clinical Cytology, Department of Pathology, Karolinska Hospital, S-17176 Stockholm, Sweden

inheri ted predispos i t ion for breas t and/or ovar ian cancer. A locus on the long arm of c h romosome 17 is l inked to the d isease in breast cancer famil ies , espec ia l ly in cases with ear ly onset (Hal l et al. 1990) and in famil ies with breas t -ovar ian cancer syndrome (Narod et al. 1991). This locus, BRCA1, has been further m a p p e d by the breas t cancer consor t ium to 17q12-21 be tween the markers D17S250 and D17S579 (Easton et al. 1993)). The same study has es t imated that approx imate ly 50% of breas t can- cer famil ies and 90% of the famil ies with breas t -ovar ian cancer are l inked to this locus. Smi th et al. (1992) re- por ted that nine of e leven ovar ian cancers and one of two breas t cancers d i sp layed loss o f he te rozygos i ty (LOH) with markers in the BRCA1 region. In all informat ive cases, the wi ld - type al lele was lost, suggest ing that the gene pred ispos ing for the tumors in their four famil ies was a tumor suppressor gene (Smith et al. 1992). We have s tudied seven breas t tumors, three ovar ian tumors, one colon cancer, and one b ladder cancer f rom famil ies with the breas t -ovar ian cancer syndrome in an a t tempt to con- f i rm the observat ion by Smith et al. (1992).

Materials and methods

Patients

Six families with breast-ovarian cancer syndrome (Fig. 1) were as- certained through patients at the clinic for familial cancer at the Karolinska Hospital. Paraffin-embedded tissues representing nor- mal lymph nodes and corresponding tumors were obtained from 12 patients from these families. The patients whose tumors were in- cluded in the study are marked with numbers from 1-12 in Fig. 1.

DNA analysis

DNA was extracted from paraffin-embedded tissue as described (Greer et al. 1991). The microsatellite markers used included D17S250/mfd15 (Weber et al. 1990), D17S800 (Weissenbach et al. 1992), D17S579/mfd188 (Hall et al. 1992) found in the BRCA1 region, the proximal marker D17S587/46E2, and the more distal markers D17S588/42D6 and NME/nm23 (kindly provided by M. Skolnick and D. Black through the breast cancer consortium).

232

BrOvl

CML Mes Br28 Br40 19 4Z

BrOv2 BrOv3

kov,

Br39,48 Br34,37

Gi4Z OvSZ 60

BrOv4

Mel2$

BrOv5 B r 4 %

Br38 ]

Mel Co Br 50 43 42

PaSO Br46

BrOv6

Fig. 1 Pedigree showing the families with breast-ovarian cancer syndrome. Black symbols Affected with breast or ovarian cancer, hatched symbols affected with some other form of cancer. The type of cancer and the age of onset are given below the symbols as follows: Br breast cancer, Ov ovarial cancer, CML chronic myeloic leukemia, Mes mesothelioma, Gi gastrointestinal cancer, Bl bladder cancer, Pa papilar cancer, Co colon cancer, Mel melanoma. The individuals from whom the tumors were obtained are indicated with a case number above and to the right of the symbol

Fig.2 Autoradiographs of labeled PCR product of microsatellite markers in the BRCA1 region for three cases showing LOH. T Tu- mor tissue, C constitutional tissue

The standard polymerase chain reaction (PCR) was carried out in a 1 l-[al volume containing 20-40 ng genomic DNA template, 1 pmol each oligodeoxynucleotide primer (one of which was endla- beled with 32p), 100 [aM nucleotides (25 [aM dGTP, 25 [aM dATP, 25 [aM TTP, and 25 [aM CTP), PCR buffer ( 1.5 mM MgC12, 50 mM KCI, 10 mM TR1S, pH 8.3) and 0.2 U Taq polymerase (AmpliTaq, Perkin-Elmer-Cetus). Samples were overlaid with mineral oil, heated for 4 rain to 95~ and processed through 35 temperature cycles at 95~ for 30 s, 55~ for 30 s, and 72~ for 30 s, with a final step for 4 min at 72~ in a Perkin-Elmer-Cetus thermo cy- cler. Aliquots were then denatured with formamide, and elec- trophoresed on standard denaturing 6% polyacrylamide DNA se- quencing gels. Gels were fixed, dried, and subjected to autoradiog- raphy for about 24 h. Lod scores were calculated using the com- puter program LIPED (Ott 1976).

Chrom. 17

233

1Br 2Br 3Br 4BL

D 1 7 S 5 8 7 / 4 6 E 2 + +

D 1 7 S 2 5 0 / m f d 1 5 + + + +

D17S800 + + + +

D17S579/mfd188 + + +

D 1 7 $ $ 8 8 / 4 2 D 6 + + + +

5Or-- 6Ov 7Co 8Br 90v 10Br 11Br 12Br

+ + t- + + + + + +

+

+ + +

+ + + +

Fig.3 Chromosome 17 and the markers tested. + Retention of both alleles, - loss of heterozygosity, space the patient was consti- tutionally homozygous for the marker or no information was avail- able

Resu#s

Linkage analysis was used to estimate the likelihood that the disease was segregating with the BRCA1 locus on chromosome 17 in one family with breast-ovarian cancer, and thus whether the disease was caused by a mutation in the BRCA1 gene. The linkage analysis, assuming various recombination fractions in family BrOv-6, showed a two- point lod score of 0.67 at a recombination fraction 0 = 0 for the marker D17S800, located very close to the BRCA1 region. Allele frequencies were assumed equal. Three cousins shared one haplotype from D17S800- NM23. As shown by the case where the unaffected father was available, this haplotype is maternal. There was one recombination distal to the marker D17S250 in one of the patients studied. This marker is considered to be the prox- imal flanking marker to the BRCA1 gene (Easton et al. 1993). Thus, the data are consistent with the disease being linked to the BRCA1. In the five additional families, the available material did not allow linkage analysis.

We studied seven breast tumors, three ovarian tumors, one bladder tumor, and one colon tumor from six breast- ovarian families (Fig. 1) for LOH using markers in the BRCA1 region. The results from the LOH study are sum- marized in Fig. 2. Three tumors, viz., two breast carcino- mas from cases 2 and 11, and the colon cancer from case 7, showed LOH for at least some markers in the region (Fig. 3).

The tumor from case 2 displayed clear LOH for the marker D17S587 outside the BRCA1 region (Figs.2, 3). The same tumor showed retention of both alleles at the two markers D17S250 and D17S800, and again, showed LOH at the marker D17S579 within the BRCA1 region. The loss observed at D17S587 (outside the BRCA1 re- gion) was a complete loss of this allele, suggesting there is little normal cell contamination in this tumor, whereas the loss with the marker D17S579 within the region was a partial loss, with a remaining weak band representing nor- mal DNA amplification. This assay was repeated three times with identical results.

The tumor from case 7 (Figs. 2, 3) was a colon cancer; it showed LOH with two markers in the region D17S587

and D17S579, was uninformative for the other two mark- ers, and showed an allelic imbalance with the marker out- side the region, suggestive of LOH even at this locus. The tumor from case 11 was a breast cancer and showed re- tention of both alleles for the most proximal marker D17S587. The same tumor showed LOH for two other markers, D17S250 and D17S588 (in the BRCA1 region), but was uninformative for the rest (Figs. 2, 3). Thus, we found LOH in the BRCA1 region in two breast carcino- mas and one colon cancer of 11 informative tumors from families with breast-ovarian cancer.

Discussion

In 90% of all families with breast-ovarian cancer, a muta- tion segregates in the BRCA1 gene on 17q. By using polymorphic markers, it is possible to make carrier diag- nosis in families where the disease locus is linked to the BRCA1 gene. Furthermore, if the BRCA1 gene acts as a tumor suppressor gene, all conceivable mechanisms, in- cluding chromosomal rearrangements in the tumor tissue, could cause the elimination or functional inactivation of the wild-type allele. Hence, LOH studies in familial tu- mors using the same microsatellite markers could further support the idea of linkage in a particular family.

We studied LOH in seven breast tumors, three ovarian tumors, one bladder cancer, and one colon cancer from six families with breast-ovarian cancer to confirm the previ- ous finding of LOH in the BRCA1 region. We observed LOH in the BRCA1 region in only three cases, viz., two breast carcinomas and one colon cancer. The losses in the breast carcinoma from case 11 and the colon carcinoma from case 7 were within the region in which the BRCA1 has been mapped. The breast carcinoma from case 2 showed two separate regions with LOH. One was outside the region (D17S587) and seemed to reflect a primary event, since it was a complete loss. The other loss was within the region (D17S579) and seemed to be a sec- ondary event, since this loss was only partial, suggesting a subfraction of the tumor cells carrying this rearrangement. However, the lost allele at D17S579 was not that shared by the affected women; no information in this respect was available for the other loss.

We thus found LOH in fewer tumors than expected and our results are in contrast to the finding in a previous study (Smith et al. 1992). One explanation could be that

234

there has been a functional inactivation of the wild-type allele by small mutational events not detectable by LOH studies. Alternatively, the disease in these families is not l inked to the BRCA1 locus. This could not be tested with l inkage analysis in five of these families, and the answer must wait until the BRCA1 gene has been cloned. On the other hand, the BRCAI gene may not be a tumor suppres- sor gene, or the mechanisms by which the gene in the BRCA1 locus causes the tumors may be different in ovar- ian and breast carcinomas. LOH in the BRCAI region has been reported at a high frequency for ovarian carcinomas from breast-ovarian families (Smith et al. 1992) and spo- radic breast carcinomas (Futreal et al. 1992). However, sporadic ovarian carcinomas show LOH mainly in a re- gion distal to BRCA1 (Jacobs et al. 1993), and familial breast tumors show a low frequency of LOH in the BRCAI region (Lindblom et al. 1993).

Questions remain about the nature of the BRCA1 lo- cus, and in what way it may be related to the development of breast, ovarian, and other tumors in families with breast-ovarian cancer, breast cancer families, and sporadic breast and ovarian cancer. Other genes close 1o the BRCA1 region could serve as tumor suppressor genes in- volved in tumorigenesis; this also could explain the find- ing of LOH at 17q. When there is no significant lod score to indicate whether BRCAI is l inked to the disease in a particular family, no additional information in this respect can be obtained from LOH studies in the tumors. To be able to help these families with gene carrier diagnosis, we must wait for the gene to be cloned, and then perform mu- tation analysis.

Acknowledgements This work was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), the King Gustav V Jubilee Fund, the Swedish Cancer Society, the Swedish Medical Research Council, and the Axel and Margaret Ax:son Johnson's Foundation. R.M. is a visiting scientist from the Department of Virology, F1OCRUC, Brazil.

References

B0rresen AL (1992) Role of genetic factors in breast cancer sus- ceptibility. Acta Oncol 31:151-155

Easton DF, Bishop DT, Ford D, Crockford GP (1993) Genetic linkage analysis in familial breast and ovarian cancer - results from 214 families. A J Hum Genet 52:678-701

Futreal PA, S6derkvist P, Marks JR, Iglehart JD, Cochran C, Bar- rett JC, Wiseman RW (1992) Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma us- ing microsatellite length polymorphisms. Cancer Res 52: 2624-2627

Greer CE, Peterson SL, Kiviat NB, Manos MM (1991) PCR am- plification from paraffin-embedded tissues. Am J Clin Pathol 95:117

Hall JM, Lee MK, Newman B, Morrow JE, Andersson LA, Huey B, King MC (1990) Linkage of early-onset familial breast can- cer to chromosome 17q21. Science 250 : 1684-1689

Hall JM, Friedman L, Guenther C, Lee MK, Weber JL, Black DM, King MC (1992) Closing in on the breast cancer gene on chro- mosome 17q. Am J Hum Genet 50:1235-1242

Jacobs HJ, Smith SA, Wiseman RW, Futreal PA, Harrington T, Osborne RJ, Leech V, Molyneux, Berchuck A, Ponder BAJ, Bast RC Jr (1993) A deletion unit on chromosome 17q in ep- ithelial ovarian tumors distal to the familial breast/ovarian can- cer locus. Cancer Res 53:1218-1221

Lindblom A, Skoog L, Ikdahl-Andersen T, Rotstein S, Norden- skj61d M, Larsson C (1993) Four separate regions on chromo- some 17 show loss of heterozygosity in familial breast carcino- mas. Hum Genet 91:6-12

Narod SA, Feunteun J, Lynch HT, Watson P, Conway T, Lynch J, Lenoir GM (1991) Familial breast-ovarian cancer locus on chromosome 17q 12~123. Lancet 338 : 82-83

Ott J (1976) A computer program for linkage analysis of general human pedigrees: letter. Am J Hum Genet 28 : 528-529

Smith SA, Easton DF, Evans DGR, Ponder BAJ (1992) Allele losses in the region 17q12-21 in familial breast and ovarian cancer involve the wild-type chromosome. Nature Genet 2: 128 131

Weber JL, Kwitek AE, May PE, Wallace MR, Collins FS, Ledbetter DH (1990) Dinucleotide repeat polymorphisms at the D17S250 and D17S261 loci. Nucleic Acids Res 18:4640

Weissenbach J, Gyapay G, Dib C, Vignal A, Morissette J, Mil- lasseau P, Vaysseix G, Lathrop M (1992) A second-generation linkage map of the human genome. Nature 359:794-801