Daniela Muhr

BRCA1‐related breast cancer in Austrian breast and ovarian cancer families: Specific BRCA1 mutations and pathological characteristics

We identified 17 BRCA1mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high‐performance liquid chromatography (DHPLC) and the protein<0B> <0R>truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non‐Austrian origin. In addition, 6 rare missense mutations (allele frequency < 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty‐four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty‐eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age‐related, node‐negative control group. The prevalence of grade III tumors was marginally significant . Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age‐related, node‐negative comparison group, showed no statistical difference. Int. J. Cancer 77:354–360, 1998.

Polymerase chain reaction fidelity and denaturing high-performance liquid chromatography.

Incorporation of non-complementary nucleotides during polymerase chain reaction can result in ambiguous denaturing high-performance liquid chromatography profiles that reduce both sensitivity and specificity of mutation analysis. The use of proofreading DNA polymerases increases the fidelity of polymerase chain reaction and, consequently, reduces background noise in the chromatograms. This is demonstrated for several BRCA1 and BRCA2 mutations hat had yielded previously chromatograms of poor quality using non-proofreading enzyme for amplification. Interestingly, despite the reduced level of background heteroduplices, the ability of denaturing high-performance liquid chromatography to detect mutant alleles at a frequency <10% in pools of chromosomes did not improve significantly.

Effect of lifestyle and reproductive factors on the onset of breast cancer in female BRCA 1 and 2 mutation carriers.

The birth year‐dependent onset of breast cancer (BC) in BRCA1/2 mutation carriers suggests a risk‐modifying role for reproductive and life style factors. We therefore examined possible associations between these factors and age at diagnosis.

Central European BRCA2 mutation carriers: birth cohort status correlates with onset of breast cancer.

BACKGROUND Mutations in brca1 and 2 genes lead to a significant increase in the lifetime risk of developing breast (BC) and ovarian cancer (OC). There are indications that birth cohort can influence the cancer risk in brca1 mutation carriers. Therefore, we investigated the risks for BC and OC associated with brca2 mutations in a cohort of female mutation carriers of a genetically heterogeneous Central European population. PATIENTS AND METHODS This study included 246 women in whom a functional mutation in the brca2 gene had been identified at our institution. At the time of analysis, 153 women had developed cancer (142 BC, 9 OC, 2 BC and OC). Risks were estimated using the product limit method. The log rank test was used to compare different strata. RESULTS After correction for risk-reducing surgeries, the cumulative risk of developing cancer to age 70 was found to be 88% for BC (95% CI 81-95%) and 31% for OC (95% CI 17-45%). Female brca2 mutation carriers born in 1958 or later were at a significantly higher risk of developing BC at a younger age (p<0.001), while no such age cohort-dependent correlation was found for OC. CONCLUSION The age cohort-dependent early onset in BC in women born after 1958 strongly suggests the importance of exogenous factors such as lifestyle modification while this does not seem to be the case for OC. Female brca2 mutation carriers should be counseled about their age cohort-dependent breast cancer risk.

Clinical implications of genetic testing for BRCA1 and BRCA2 mutations in Austria

The objective of this study was to describe the experience of genetic testing in Austrian women with a BRCA1 or BRCA2 mutation in terms of preventive measures taken and incident cancers diagnosed. We collected clinical information on 246 Austrian women with a BRCA1 or BRCA2 mutation tested between 1995 and 2012 and followed 182 of them for an average of 6.5 years. Of the 90 women who were cancer‐free at baseline, 21.4% underwent preventive bilateral mastectomy, 46.1% had preventive bilateral salpingo‐oophorectomy, and 1 took tamoxifen; 58.8% of the at‐risk women underwent at least one screening breast magnetic resonance imaging (MRI). Of the 85 women with breast cancer, 69.4% had a unilateral mastectomy or lumpectomy and 30.6% had a contralateral mastectomy. In the follow‐up period, 14 new invasive breast cancers (6 first primary and 8 contralateral), 1 ductal carcinoma in situ case, 2 incident ovarian cancer cases, and 1 peritoneal cancer were diagnosed. In Austria, the majority of healthy women with a BRCA1 or BRCA2 mutation opt for preventive oophorectomy and MRI screening to manage their breast cancer risk; few have preventive mastectomy or take tamoxifen.

HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK

Background HER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood. Methods Using Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively. Results All three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively. Conclusion We have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.

Diagnostic markers for the detection of ovarian cancer in BRCA1 mutation carriers

Background Screening for ovarian cancer (OC) in women at high risk consists of a combination of carbohydrate antigen 125 (CA125) and transvaginal ultrasound, despite their low sensitivity and specificity. This could be improved by the combination of several biomarkers, which has been shown in average risk patients but has not been investigated until now in female BRCA mutation carriers. Methods Using a multiplex, bead-based, immunoassay system, we analyzed the concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, CA125 and human epididymis antigen 4 in 26 healthy wild type women, 26 healthy BRCA1 mutation carriers, 28 wildtype OC patients and 26 OC patients with BRCA1 mutation. Results Using the ROC analysis, we found a high overall sensitivity of 94.3% in differentiating healthy controls from OC patients with comparable results in the wildtype subgroup (sensitivity 92.8%, AUC = 0.988; p = 5.2e-14) as well as in BRCA1 mutation carriers (sensitivity 95.2%, AUC = 0.978; p = 1.7e-15) at an overall specificity of 92.3%. The used algorithm also allowed to identify healthy BRCA1 mutation carriers when compared to healthy wildtype women (sensitivity 88.4%, specificity 80.7%, AUC = 0.895; p = 6e-08), while this was less pronounced in patients with OC (sensitivity 66.7%, specificity 67.8%, AUC = 0.724; p = 0.00065). Conclusion We have developed an algorithm, which can differentiate between healthy women and OC patients and have for the first time shown, that such an algorithm can also be used in BRCA mutation carriers. To clarify a suggested benefit to the existing early detection program, large prospective trials with mainly early stage OC cases are warranted.

Impact of lifestyle factors on preneoplastic changes in prophylactic oophorectomies of BRCA mutation carriers

BRCA mutation carriers are at high risk of developing ovarian cancer. Ovarian malignancies are usually identified at an advanced stage with poor prognosis, attributed to inadequate options of early detection. Because of its risk-reducing effect of nearly 96%, prophylactic salpingo-oophorectomy is still the leading option for risk-reduction in women with a positive BRCA mutation status. The presence of ovarian cancer precursor lesions, such as epithelial inclusion cysts (EICs) or cortical invaginations (CIs), has previously been discussed in several studies with diverse conclusions. We retrospectively investigated a large and consistent population (n=94) of BRCA mutation carriers for the presence of potential preneoplastic and neoplastic changes. We also examined the role of specific lifestyle factors. Ninety-four women with disease-associated germline BRCA1 or BRCA2 mutations were included in this retrospective study. All women had undergone genetic counseling and prophylactic salpingo-oophorectomy, which was performed at a mean age of 43.33 years (range 27–66). Histological slides of both ovaries were reviewed by an independent pathologist. Data concerning lifestyle factors were collected from medical files and questionnaires. Two malignant lesions (2.1%), one bilateral serous papillary adenocarcinoma of the epithelial surface and one adenocarcinoma of the peritoneum with involvement of the left adnexa, and one lesion (1.1%) with obvious malignant potential, one mucinous borderline tumor of the right ovary, have been identified. We registered a high prevalence of CIs (30; 31.9%) and EICs (44; 46.8%) in prophylactically removed ovaries of BRCA mutation carriers. A significant correlation (P=0.002) was found with regard to the presence of EICs in women with increased BMI. Concerning the regular consumption of alcohol as a risk factor for premalignant lesions, in particular CIs, a statistically insignificant trend (P=0.083) was noted. Overweight women seem to be at risk of developing more cortical invaginations than women of normal weight. To improve the final outcome of the disease, women at increased risk of ovarian cancer should be appropriately informed of potential increased risk factors.

Correction: Diagnostic markers for the detection of ovarian cancer in BRCA1 mutation carriers

[This corrects the article DOI: 10.1371/journal.pone.0189641.].

Differential Claudin 3 and EGFR Expression Predicts BRCA1 Mutation in Triple-Negative Breast Cancer

Abstract BRCA-1 mutation-associated triple-negative breast cancer (TNBC) has been hypothesized to exhibit a phenotype that is distinct from non-mutation carriers. We have analyzed immunohistochemically detected cytokeratins 5 and 14, epidermal growth factor receptor (EGFR), claudin (CLDN) 3, 4, and 7, and E-cadherin in 57 TNBC (32 BRCA1 and 8 BRCA2 tumors, 17 WT tumors). Positive staining of CLDN3 and negative EGFR expression in TNBC are associated with a BRCA1 mutation. EGFR and CLDN3 expression was able to predict the presence of BRCA1 mutation (area under curve 0.802, p < 0.001). This could help in guiding the decision for BRCA testing.