Sigridur K. Bodvarsdottir

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

p53 Abnormality and Chromosomal Instability in the Same Breast Tumor Cells

To clarify the important role of the tumor-suppressor gene p53 in maintaining genetic integrity, we estimated chromosome instability and staining of overexpressed p53 protein in the same cells of five primary breast carcinomas. The method included both fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH) on sections from formalin-fixed, paraffin-embedded breast cancer tissue. By using a centromeric FISH probe for chromosome 17 on interphase cells in these sections, we showed that cells with abnormal p53 protein expression had a statistically significant higher number of chromosome 17 than did cells with no p53 protein staining in the same samples as well as cells in four other tumor samples with no p53 protein staining. The samples identified positive for p53 abnormality by immunostaining were shown to have p53 mutation by constant denaturing gel electrophoresis analysis and DNA sequencing. These mutated samples were characterized by high DNA index, high S-phase, abnormal karyotype, and aneuploidy. The results strongly implicate p53 mutation as a cause for chromosomal instability and a crucial step in mammary carcinogenesis.

Genomic and phenotypic analysis of BRCA2 mutated breast cancers reveals co-occurring changes linked to progression.

BackgroundInherited mutations in the BRCA2 gene greatly increase the risk of developing breast cancer. Consistent with an important role for BRCA2 in error-free DNA repair, complex genomic changes are frequently observed in tumors derived from BRCA2 mutation carriers. Here, we explore the impact of DNA copy-number changes in BRCA2 tumors with respect to phenotype and clinical staging of the disease.MethodsBreast tumors (n = 33) derived from BRCA2 999del5 mutation carriers were examined in terms of copy-number changes with high-resolution aCGH (array comparative genomic hybridization) containing 385 thousand probes (about one for each 7 kbp) and expression of phenotypic markers on TMAs (tissue microarrays). The data were examined with respect to clinical parameters including TNM staging, histologic grade, S phase, and ploidy.ResultsTumors from BRCA2 carriers of luminal and basal/triple-negative phenotypes (TNPs) differ with respect to patterns of DNA copy-number changes. The basal/TNP subtype was characterized by lack of pRb (RB1) coupled with high/intense expression of p16 (CDKN2A) gene products. We found increased proportions of Ki-67-positive cells to be significantly associated with loss of the wild-type (wt) BRCA2 allele in luminal types, whereas BRCA2wt loss was less frequent in BRCA2 tumors displaying basal/TNP phenotypes. Furthermore, we show that deletions at 13q13.1, involving the BRCA2wt allele, represents a part of a larger network of co-occurring genetic changes, including deletions at 6q22.32-q22.33, 11q14.2-q24.1, and gains at 17q24.1. Importantly, copy-number changes at these BRCA2-linked networking regions coincide with those associated with advanced progression, involving the capacity to metastasize to the nodes or more-distant sites at diagnosis.ConclusionsThe results presented here demonstrate divergent paths of tumor evolution in BRCA2 carriers and that deletion of the wild-type BRCA2 allele, together with co-occurring changes at 6 q, 11 q, and 17 q, are important events in progression toward advanced disease.

Telomere length is predictive of breast cancer risk in BRCA2 mutation carriers.

Background: Germline BRCA2 mutations increase risk of breast cancer and other malignancies. BRCA2 has been shown to play a role in telomere protection and maintenance. Telomere length (TL) has been studied as a modifying factor for various diseases, including breast cancer. Previous research on TL in BRCA mutation carriers has produced contradicting results. Methods: We measured blood TL, using a high-throughput monochrome multiplex qPCR method, in a well-defined Icelandic cohort of female BRCA2 mutation carriers (n = 169), sporadic breast cancer patients (n = 561), and healthy controls (n = 537). Results: Breast cancer cases had significantly shorter TL than unaffected women (P < 0.0001), both BRCA2 mutation carriers (P = 0.0097) and noncarriers (P = 0.00006). Using exclusively samples acquired before breast cancer diagnosis, we found that shorter telomeres were significantly associated with increased breast cancer risk in BRCA2 mutation carriers [HR, 3.60; 95% confidence interval (CI), 1.17–11.28; P, 0.025] but not in non-carriers (HR,1.40; 95% CI, 0.89–2.22; P, 0.15). We found no association between TL and breast cancer–specific survival. Conclusions: Blood TL is predictive of breast cancer risk in BRCA2 mutation carriers. Breast cancer cases have significantly shorter TL than unaffected women, regardless of BRCA2 status, indicating that samples taken after breast cancer diagnosis should not be included in evaluations of TL and breast cancer risk. Impact: Our study is built on a well-defined cohort, highly accurate methods, and long follow-up and can therefore help to clarify some previously published, contradictory results. Our findings also suggest that BRCA2 has an important role in telomere maintenance, even in normal blood cells. Cancer Epidemiol Biomarkers Prev; 26(8); 1248–54. ©2017 AACR.

Abstract A33: Expression of BRCA2 and Mir-21 in sporadic and BRCA2 mutated breast cancer in Iceland

The BRCA2 protein plays an important role in the repair of double-stranded DNA breaks (DSBs) by homologous recombination. An inherited founder mutation in the BRCA2 gene accounts for 6-7 % of all breast cancers in Iceland. Breast cancer in BRCA2 mutation carriers is associated with poor long-term prognosis. It has been reported that > 20% of sporadic breast cancers display a BRCA-like phenotype (BRCA‘ness), suggesting a defect in the DSB repair pathway. Little is known about the role of miRNAs in BRCA2 related breast cancer. Analysis of array CGH data from BRCA2 breast tumors shows that the mir-21 locus on chromosome 17 is amplified in > 70% of BRCA2 breast tumors. Mir-21 is a known oncomir with known target genes involved in tumor suppression, amongst others. High expression of mir-21 has been associated with poor-prognosis in other breast cancer populations. The aim of the study is to investigate the level and variation of BRCA2 and mir-21 expression in breast tumors from BRCA2 mutation carriers and breast tumors from non-carriers. Furthermore we want to see if there is association between BRCA2 and mir-21 expression. Expression analysis was performed by qPCR on RNA extracted from fresh frozen tumor tissue from BRCA2 mutation carriers and sporadic breast tumors. Expression of mir-21 was analyzed using specific mir-21 Taqman probes, with RNU48 as an endogenous control. Expression of BRCA2 was analyzed using gene specific primers detected by SYBR® Green, with HPRT1 as an endogenous control. To examine clinical relevance, we examine clinicopathological parameters including grade, nodal status, time to relapse and overall survival. Results from the BRCA2 expression analysis reveal significantly lower expression in the BRCA2 group compared to the sporadic group (as expected). However, BRCA2 expression shows a large degree of variation within both groups, with a greater than 20 fold difference between the highest and the lowest expression values. Results from the mir-21 expression analysis reveal a tendency towards a higher mir-21 expression in the BRCA2 group, although not statistically significant. When analyzing the BRCA2 and mir-21 expression data together we see a definite trend towards a negative correlation, where lower BRCA2 expression tends to associate with higher mir-21 expression. Our preliminary data thus show variability in BRCA2 expression, both among BRCA2 mutation carriers and non-carriers, as well as strong indications of negative association between BRCA2 and mir-21 expression. This information could be useful with respect to therapeutic strategies, e.g. making use of PARP inhibitors. Inhibitors targeting mir-21 have been shown to reduce tumor growth in animal models of multiple myelomas. The BRCA2 group shows high expression of mir-21 raising the possibility of using mir-21 inhibitors in patients with BRCA2 deficiency. Our results also indicate that mir-21 has an important role in BRCA2 carrier tumors as well as in sporadic cases. Note: This abstract was not presented at the conference. Citation Format: Sigridur Thora Reyisdottir, Sigridur Thora Reynisdottir, Olafur Andri Stefansson, Sigridur Klara Bodvarsdottir, Arnar Palsson, Jorunn Erla Eyfjord. Expression of BRCA2 and Mir-21 in sporadic and BRCA2 mutated breast cancer in Iceland. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A33.

Selection for EGFR gene amplification in a breast epithelial cell line with basal-like phenotype and hereditary background

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.

AURKA and Breast Cancer in BRCA1/2 Mutation Carriers

To the Editors: A recent article reported, “the F31I polymorphism in AURKA is not associated with a modified risk of breast cancer found in 3,884 breast cancer BRCA1 and BRCA2 carrier cases compared to unaffected BRCA1/2 mutation carriers” ([1][1]). This agrees with our previously published