Background High frequencies of loss ofheterozygosity LOH are found - PDF document

Background: High frequencies of loss ofheterozygosity (LOH) are found in familial breastcarcinomas with BRCA mutations. Although LOH of BRCA1does not coincide with somatic BRCA1 mutations, reducedBRCA1 protein expression and hypermethylation indicatethe involvement of BRCA1 in sporadic carcinogenesis. Tofurther investigate the role of BRCA we determined LOH ofBRCA1 and correlated this with LOH in other breastcancer-associated regions. Materials and Methods: A totalof105 sporadic breast carcinomas were analysed for LOHin the regions of BRCA1, BRCA2, TP53, Caveolin1,putative BRCA3Ž, PTEN, ATM and E-cadherin and 3445 Sporadic Breast Carcinomas with Somatic Gene Deletions Share Genotype/Phenotype 0250-7005/2010 $2.00+.40 Materials and MethodsClinical and clinicopathological staging. The study was carried outwith tumor samples from 105 patients who gave written consent forthe study.Tumors were grouped according to the UICC TNM classification(19). The WHO classification was used for histological subtyping(20). Grading was performed according to Bloom and Richardson(1957) (21). ER and progesterone receptor (PR) expression wereassessed by the dextran-coated charcoal (DCC) method or bymonoclonal antibody assay (22). Data are summarized in Table I.Tissue samples. Tissues from primary breast carcinomas were obtainedat surgery, immediately frozen, stored at …80C and processed as statedelsewhere (23). As a source of constitutional DNA, peripheral bloodlymphocytes were collected from each patient. Prior to DNA analysis,each tumor sample was examined on frozen sections for contaminating80% tumor cells were analyzed. DNA was obtained by standardphenol-chloroform extraction. One hundred and five tumors weresubjected to LOH analysis with multiple microsatellite markers. Fordata from 98 tumors were available.PCR amplification and LOH analysis. amplification of microsatellite markers followed by polyacrylamidegel electrophoresis and silver staining was performed as describedelsewhere (23). In order to qualify for allelic loss, a signal reduction ofat least 50% was required. The microsatellite primers were purchasedfrom Research Genetics (Huntsville, USA). The following 32 lociintragenic markers); D7S495, D7S522, D7S523 on 7q31 (caveolin 1,CAV1-region); D8S133, D8S137 on 8q21 (region of putativeregion), D11S488, D11S968, D11S976, D11S1300, D11S1778,ATMregion), D13S260, D13S267, D13S321, D13S765 on 13q12region), D16S540, D16S265, D16S398, D16S516, D16S539region); TP53 on 17p13.1; and D17S5on 17p13.3 (telomeric to TP53). Mapping information is based on theand the other breast carcinoma-associated regions were formally testedtest. A correction for multiple testing was not performed,since all tests were used as descriptive means. Fractional allelic loss(FAL) was calculated for each tumor as the ratio of chromosomal armswith allelic loss divided by the total number of chromosomal armswith informative markers except the region.ResultsLOH frequencies in sporadic breast carcinomas. frequencies of LOH w

ith at least one marker in each regionare summarized in Table II. We analyzed chromosomal regionsknown to be frequent targets of genetic alterations in sporadiccancer. Genes localized in these regions play a key role incarcinogenesis of the breast and are involved in double-strandTP53, BRCA2, ATMCDH1, CAV1). The regionsshowed a mean LOH rate of 33.9%. In the region, 23tumors out of 105 informative cases (21.9%) revealed LOH.The lowest rate of LOH was observed on chromosome 10q22-region, at 10%. The highest rate of LOH(52.6%) was detected on chromosome 16q24 (mean FAL value for the chromosomal loci was 0.35. Tumors with LOH of BRCA1 versus tumors without LOHWe subdivided the tumors into two groups, thosewas found for) with a frequency of 80% in group 10.005). In the region8q21 where a putative 0.01). In addition, in the region 10q22-23, a differencein LOH was detected, with 21.1% in group 1 and 5.9% in 3446 TableI. Clinicopathologicalcharacteristicsof thebreastcancercases. Tumor sizepT 134�pT 1 (2, 3, 4)65No data available6Lymph nodespN …53pN +46No data available6G1, G271G331No data available3HistologicaltypeDuctal81Lobular13Medullary, mucinous and phyllodes4, 3, 1No data available3No data available ER/PR7/9mean (range)59 (29-87) 50 years27�50 years71 No data available7 0.043). No differences were demonstrated forallelic losses in other loci. These results are summarized inTable III. The mean FAL of tumors with LOH of (0.27; range, 5.9%-50.8%) revealed no significantdifference (Correlation of LOH of BRCA1 with clinicopathologicalcharacteristics. The two groups of tumors were tested forassociation with ER and PR expression, age at onset,clinicopathological classification, tumor grading and pTNMstatus. A significant association of LOH of negativity was detected (0.003). There was a trend for a0.075). Data are summarized in Table IV.DiscussionOur aim was to elucidate the role of breast cancer. Therefore, we determined the frequency ofLOH in sporadic breast carcinomas from 105 patients andand to LOH in other chromosomal regions as a marker forgenetic instability. We detected LOH of 28). Due to somatic mutations being rarely found (6), it wasinactivation. Several studies investigating the role ofin sporadic breast cancer development revealed(29-33) and almost no somatic mutations (6). The study ofStaff supported evidence that haplo-insufficiency mightMoreover, we detected a highly significant associationand LOHof versus0.005), and significant associations betweenand LOH in the regions 8q21 (72.7%versusversus 3447 TableII. Frequencyof loss of heterozygosityin the BRCA1 geneandother breast cancer-associatedregions. LocusMicrosatellite markersLOH/ % inf. case17q21D17S855, D17S1322, D17S132323/10521.917p13D17S5, D17S13.1 ()24/5642.97q31D7S495, D7S522, D7S52316/7321.98q21D8S133, D8S13723/6137.710q22-23D10S215, D10S564, D10S5739/901011q22-23D11S488, D11S968, D11S976, 47/10544.813q12-13D13S260, D13S267A, 32/10338.816q21-24D16S540, D16S265, D16S398, 40/7652.6 LOH in a chromos

omal locusis defined as LOH of at least onemicrosatellite marker in the respective chromosomal region. inf. case,Informative case; LOH, loss of heterozygosity. TableIII.Frequencyofallelic losses in breast cancer-associatedregionsin tumorswith andwithoutallelic losses in theBRCA1 gene. LocusGeneLOH of With%Without %p-Value23/23100%0/00%-12/1580%17/4934.7%CAV12/1414.3%14/5824.1%0.4268q21Putative 8/1172.7%15/4930.6%4/1921.1%4/685.9%0.043ATM13/2259.1%33/8041.3%0.1369/2142.9%22/7927.8%0.186 8/1457.1%31/6150.8%0.669LOH in a chromosomal region requires at least one microsatellite markerwith LOH in the respective region. LOH, Loss of heterozygosity. TableIV. Relationshipbetweentheloss ofheterozygositystatus intheBRCA1 gene in non-familialbreast cancer and clinical features. Frequency of%-Value 15/7125.3%0.3759/7539.1%12%0.00310/5924.3%16.9%0.377Histological classificationlob, med, phy15/814/2118.5%19%0.956G310/719/3114.1%29%0.075Tumor sizeP-6;द.;瀀pT14/3414/6511.7%21.5%0.231 pN+10/538/4618.9%17.4%0.849Frequency of BRCA1: LOH of informative cases with loss ofheterozygosity in least one marker in the region 17q21; ER/PR, estrogenreceptor/progesterone receptor; lob, lobular; med, medullary; phy, affected genes in breast cancer. In hereditary breast cancerwas detected in about 80% (34) comparedto 32% to 52.4% in sporadic breast cancer, respectivelycarcinomas were significantly more likely to carry 2.8; 95% confidence interval 1.6-4.7; 0.0003) (reviewedin 35). On the one hand, loss of BRCA1/2DNA repairfunction leads to an overall increase of geneticalinstability, selecting somatic mutations which contribute tocritical event in that in the presence of a defective BRCA pathway, TP53alterations may confer tumor growth advantage. Ourfindings give evidence for a subset of sporadic breastMoreover, we detected a significant correlation of LOHwith the absence of ER expression (39% versus0.003), whereas no association was evidentfactors analyzed. Our finding of a significant relationshipbetween ER negativity and LOH showed asignificant correlation between decreased levels of mRNA and LOH 0.006) and ER-negative status0.0004), respectively (9). Staff negativity, but did with amplification and aneuploidyas other markers of aggressive breast cancer behaviour (28).shorter relapse-free interval (0.03) and poorer overallsurvival ((36, 38). Therefore, the association of LOH of and ER negativity indicate an unfavourable prognosis.In conclusion, we report a consistent association of LOHwith an overall increased rate of genomicregion. In addition, aand ER negativity indicativeof a poor prognosis was detected. Our results indicate thatregion mimicpathway. Further investigations are needed to define if thesetumors may require specific therapeutic approaches asseems to be true for familial breast carcinomas.AcknowledgementsWe thank Gabi Krebsbach and Brigitte Kau for their excellentReferences1Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P,Bishop DT, Weber B, Lenoir G, Chang-Claude J, Sobo

l H, TeareMD, Struewing J, Arason A, Scherneck S, Peto J, Rebbeck TR,Tonin P, Neuhausen S, Barkardottir R, Eyfjord J, Lynch H,Ponder BA, Gayther SA, Zelada-Hedman M genes in breast cancer families. The Breast Cancer LinkageConsortium. Am J Hum Genet 2Szabo CI and King MC: Population genetics of . Am J Hum Genet 3Cropp C, Nevanlinna H, Pyrhonen S, Ulf-Hakan S,Salminkangan P, Albertsen H, White R and Callahan R:Evidence for involvement of BRCA1 in sporadic breast4Kerangueven F, Eisinger F, Noguchi T, Allione F, Wargniez V,Eng C, Padberg G, Theillet C, Jacquemier J, Longy M, Sobol H,Birnbaum D: Loss of heterozygosity in human breast carcinomain the ataxia telangiectasia, Cowden disease and regions. Oncogene 5Johnson SM, Shaw JA and Walker RA: Sporadic breast cancer inyoung women: prevalence of loss of heterozygosity at 6Futreal PA, Liu Y, Shatuck-Eidens D, Cochran C, HarshmanK, Tavtigian S, Bennett LM, Haugen-Strano A, Swensen J,Miki Y, Edditon K, McClure M, Frye C, Weaver-Feldhaus J,Ding W, Gholami Z, Soderkvist P, Terry L, Jhanwar S,Berchuck A, Iglehart JD, Marks J, Ballinger DG, Barrett JC,Skolnick MH, Kamb A and Wiseman R: primary breast and ovarian carinomas. Science 7Khoo US, Ozcelik H, Cheung AN, Chow LW, Ngan HY, DoneSJ, Liang AC, Chan VW, Au GK, Ng WF, Poon CS, Leung YF,Loong F, Ip P, Chan GS, Andrulis IL, Lu J and Ho FC: Somaticovarian cancer. Oncogene 8Thompson ME, Jensen RA, Obermiller PS, Page DL and HoltTJ: Decreased expression of accelerates growth and is9Sourvinos G and Spandidos DA: Decreased expressionlevels may arrest the cell cycle through activation of 10Welcsh PL and King MC: of breast and ovarian cancer. Hum Mol Gen 11Staff S, Isola J and Tanner M: Haplo-insufficiency of sporadic breast cancer. Cancer Res 12Dobovric A and Simpfendörfer D: Methylation of the gene in sporadic breast cancer. Cancer Res 13Esteller M, Silva JM, Dominguez G, Bonilla F, Matias-Guiu X,Lerma E, Bussaglia E, Prat J, Herkes IC, Repasky EA,inactivation in sporadic breast andovarian tumors. J Natl Cancer Inst 14Foulkes WD, Stefansson IM, Chappuis PO, Begin LR, GoffinJR, Wong N, Trudel M and Akslen LA: Germline mutations and a basal epithelial phenotype in breast cancer. J 3448 15Armes JE, Trute L, White D, Southey MC, Hammet F, TesorieroA, Hutchins AM, Dite GS, McCredie MR, Giles GG, Hopper JLand Venter DJ: Distinct molecular pathogeneses of early-onsetbased study. Cancer Res 16Lakhani SR, van de Vijver MJ, Jacquemier J, Anderson TJ, OsinPP, McGuffog L and Easton DF: The pathology of familialbreast cancer: predictive value of immunostochemical markersestrogen receptor, progesterone receptor, HER-2 and p53 in17Hanby AM, Kelsell DP, Potts HW, Gillett CE, Bishop DT, SpurrNK and Barnes DM: Association between loss of heterozygosityand morphological attributes of sporadicbreast cancer. Int J Cancer 18Wilson CA, Ramos L, Villasenor MR, Anders KH, Press MF,Clarke K, Karlan B, Chen JJ, Scully R, Livinston D, Zuch RH,19Spiessl B, Beahrs OB and Hermanek P: UICC TNM Atlas.Illustrated Guide to the

TNM/pTNM Classification of MalignantTumors. Berlin, Springer, 1990.20Tavassoli FA and Devilee P: Pathology and Genetics, Tumoursof the Breast and Female Genital Organs, IARC WHOClassification of Tumours, No 4, 2003.21Bloom HJ, Richardson WW. Histological grading and prognosisin breast cancer. A study of 1049 cases of which 359 have beenfollowed for 15 years. Br J Cancer 22Remmele W and Stegner HE: Vorschlag zur einheitlichenDefinition eines Immunreaktives Scores (IRS) für denMammakarzinomgewebe. Pathologe 23Schmutzler RK, Bierhoff E, Werkhausen T, Fimmers R, SpeiserR, Kubista E, Krebs D, Zeillinger R, Wiestler OD and vonDeimling A: Genomic deletions in the regions associate with low expression of the estrogen receptor in24Sengupta S and Harris CC: P53: traffic cop at the crossroads ofDNA repair and recombination. Nat Rev Mol Cell Biol 25Gudmundsdottir K and Ashworth A: The roles of stability. Oncogene 26Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP andYin Y: Essential role for nuclear chromosomal integrity. Cell 27Osorio A, de la Hoya M, Rodriguez-Lopez R, Martines-RamiresA, Cazorla A, Granizo JJ, Esteller M, Rivas C, Caldés T andtumor samples from patients with familial breast cancer. Int J28Staff S, Isola JJ, Johannsson O, Borg A and Tanner MM:vice versa29Niederacher D, Picard F, van Roeyen C, An HX, Bender HG andBeckmann MW: Patterns of allelic loss on chromosome 17 in30Rio PG, Pernin D, Bay JO, Albuisson E, Kwiatkowski F, DeLatour M, Bernard-Gallon DJ and Bignon YJ: Loss ofATMinvasive ductal breast carcinoma. Int J Oncol 31Sorlie T, Andersen TI, Bukholm I and Borresen-Dale AL:17q21 in breast carcinomas from familial and non-familial cases.Breast Cancer Res Treat 32Sato T, Tanigami A, Yamakawa K, Akiyama F, Kasumi F,Sakamoto G and Nakamura Y: Allelotype of breast cancer:cumulative allele losses promote tumor progression in primarybreast cancer. Cancer Res 33Phelan CM, Borg A, Cuny M, Crichton DN, Baldersson T,Andersen TI, Caligo Ma, Lidereau R, Lindblom A, Seitz S,Kelsell D, Hamann U, Rio P, Thorlacius S, Papp J, Olah E,Ponder B, Bignon YJ, Scherneck S, Barkardottir R, Borresen-Dale AL, Eyfjörd J, Theillet C, Thompson AM, Larsson C chromosome 17 targets subregions associated with family history34Luo L, Chen J, Du Q, Dumanski J, Blennow E, Kockum I,Luthman H and Lindblom A: A region close to showsLOH in familial breast cancer. Int J Mol Med 35Greenblatt MS, Chappuis PO, Bond JP, Hamel N and FoulkesWD: TP53mutations in breast cancer associated with germ-line mutations: distinctive spectrum and structuraldistribution. Cancer Res 36Querzoli P, Albonico G, di Iasio MG, Ferretti S, Rinaldi R, Carielloand survival of women. Breast Cancer Res Treat 37Nagahata T, Hirano A, Utada Y, Tsuchiya S, Takahashi K, Tada T,Makita M, Kasumi F, Akiyama F, Sakamoto G, Nakamura Y andEmi M: Correlation of allelic losses and clinicopathological factors38Ansquer Y, Gautier C, Fourquet A, Asselain B and Stoppa-Lyonnet D: Survival in early-onset Received October 6, 2010Revised May 11, 2010 344