Summary In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS without adjacent invasive carcinoma also lacks E-cadherin expression and whether the gene mutations present in ILC are already present in LCIS is not known. We report here that E-cadherin expression is absent in six cases of LCIS and present in 150 cases of ductal carcinoma in situ (DCIS), both without an adjacent invasive component. Furthermore, using mutation analysis, we could demonstrate the presence of the same truncating mutations and loss of heterozygosity (LOH) of the wild-type E-cadherin in the LCIS component and in the adjacent ILC. Our results indicate that E-cadherin is a very early target gene in lobular breast carcinogenesis and plays a tumour-suppressive role, additional to the previously suggested invasion-suppressive role.Keywords: ductal carcinoma in situ; lobular carcinoma in situ; mutations; breast cancer; loss of heterozygosity The E-cadherin gene is located on chromosome band 16q22.1 and encodes a calcium-dependent cellular adhesion molecule crucial for epithelial organization and adhesion (Takeichi, 1991).In breast carcinoma, reduced expression of E-cadherin has been found in 50% of invasive ductal carcinoma (IDC). In most cases of invasive lobular carcinoma (ILC), however, a complete loss of expression has been observed (Gamallo et al, 1993;Moll et al, 1993;Rasbridge et al, 1993;Berx et al, 1995). Mutations in the E-cadherin gene have been found previously in invasive lobular breast carcinomas (Kanai et al, 1994;Berx et al, 1995). We have shown that E-cadherin gene mutations and loss of the wild-type allele by loss of heterozygosity (LOH) is the predominant mechanism by which E-cadherin protein expression is lost (Berx et al, 1995(Berx et al, , 1996. These results indicate that E-cadherin acts as a classical tumour-suppressor gene. Recently, we and others found loss of immunohistochemical E-cadherin expression in lobular carcinoma in situ (LCIS), adjacent to ILC (Moll et al, 1993). This is surprising as, on the basis of the in vitro experiments (Frixen et al, 1991;Vleminckx et al, 1991), E-cadherin inactivation would be expected to play a role in the transition of in situ carcinoma to invasive carcinoma or even metastatic cancer.In this study we have investigated the expression of E-cadherin in six cases of LCIS and for comparison also in 150 cases of ductal carcinoma in situ (DCIS), both without an invasive component. In addition, we have performed mutation analysis on the microdissected LCIS component adjacent to invasive lobular carcinoma of two cases with known E-cadherin mutations. For these two tumours and for six cases of LCIS without invasion, LOH was studied on 16q22. 1, where the E-cadherin gene is located. MATERIALS AND METHODS TumoursWe collected 156 paraffin-embedded cases of ...
SummaryWe analysed the involvement of known and putative tumour suppressor-and oncogene loci in ductal carcinoma in situ (DCIS) by microsatellite analysis (LOH), Southern blotting and comparative genomic hybridization (CGH). A total of 78 pure DCIS cases, classified histologically as well, intermediately and poorly differentiated, were examined for LOH with 76 markers dispersed along all chromosome arms. LOH on chromosome 17 was more frequent in poorly differentiated DCIS (70%) compared to well-differentiated DCIS (17%), whereas loss on chromosome 16 was associated with well-and intermediately differentiated DCIS (66%). For a subset we have done Southern blotand CGH analysis. C-erbB2/neu was amplified in 30% of poorly differentiated DCIS. No amplification was found of c-myc, mdm2, bek, flg and the epidermal growth factor (EGF)-receptor. By CGH, most frequent alterations in poorly differentiated DCIS were gains on 8q and 17q22-24 and deletion on 17p, whereas in well-differentiated DCIS amplification on chromosome 1q and deletion on 16q were found. In conclusion, our data indicates that inactivation of a yet unknown tumour suppressor gene on chromosome 16q is implicated in the development of most well and intermediately differentiated DCIS whereas amplification and inactivation of various genes on chromosome 17 are implicated in the development of poorly differentiated DCIS. Furthermore these data show that there is a genetic basis for the classification of DCIS in a well and poorly differentiated type and support the evidence of different genetic routes to develop a specific type of carcinoma in situ of the breast.
PARP inhibitor (PARPi) sensitivity is related to tumor-specific defects in homologous recombination (HR). Therefore, there is great clinical interest in tests that can rapidly and reliably identify HR deficiency (HRD). Functional HRD tests determine the actual HR status by using the (dis)ability to accumulate RAD51 protein at sites of DNA damage as read-out. In this study, we further improved and calibrated a previously described RAD51-based functional HRD test on 74 diagnostic formalin-fixed paraffin-embedded (FFPE) specimens (RAD51-FFPE test) from endometrial cancer (EC n = 25) and epithelial ovarian cancer (OC n = 49) patients. We established optimal parameters with regard to RAD51 foci cut-off (≥ 2) and HRD threshold (15%) using matched endometrial and ovarian carcinoma specimens for which HR status had been established using a RAD51-based test that required ex vivo irradiation of fresh tissue (RECAP test). The RAD51-FFPE test detected BRCA deficient tumors with 90% sensitivity and RECAP-HRD tumors with 87% sensitivity, indicating that it is an attractive alternative to DNA-based tests with the potential to be applied in routine diagnostic pathology.
BackgroundCancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression.MethodsWe combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) in cervical cancer-associated stromal cell fractions (n = 57) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to confirm LOH findings.ResultsSmooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. However, we found no evidence for DNA aneuploidy, somatic genetic alterations in the vimentin-positive stromal cell fractions of any samples, while high frequencies of DNA content abnormalities (43/57) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions. LOH hot-spots on chromosomes 3p, 4p and 6p found were confirmed by microsatellite analysis.ConclusionFrom our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Using flow cytometry and SNP-arrays, stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0061-5) contains supplementary material, which is available to authorized users.
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