6 research outputs found
An Experimental Model of Breast Cancer Cells: Informative Protocol for In Vitro Culture
Background: Cryopreservation of ovarian tissue for patients with cancer comes with the concern for the existence of cancer cells in the grafts. Data about cancer cell viability after cryopreservation are currently lacking. For the experimental study of cryo-stability of cancer cells, different protocols of in vitro culture of various cell lines are used. The existing protocols are not able to mimic the effectiveness of in vivo/in situ development of cancer cells. This study aimed to test the new protocol for in vitro culture of breast cancer cells. Materials and Methods: ZR-75-1 and MDA-MB-231 cells were in vitro-cultured in standard [RPMI 1640 and Dulbecco's modified Eagle's medium (DMEM), respectively] and experimental (AIM V) medium. Cell phenotypes were tested by CCK-8 cell proliferation measurement, wound-healing assay, transmembrane cell migration and invasion assay and immunofluorescent staining. A 10-day in vitro culture without cell passaging was conducted on both cell lines in culture medium to observe the generation of a cell layer as a model of solid tumor tissue. The density of the cell layers was revealed by immunofluorescent staining. Results: AIM V significantly promoted continued proliferation of both cell lines. Cell motility of ZR-75-1 cells was increased considerably more in AIM V than in RPMI-1640. For MDA-MB-231 cells cultured in AIM V and DMEM, no significant differences in mobility and invasion were observed. Both cell lines maintained in AIM V medium generated a cell layer on day 7 and formed a compact structure on day 10 of in vitro culture. Conclusion: In vitro culture of ZR-75-1 and MDA-MB-231 cells in AIM V medium is more informative than culture in standard medium. The described protocol provides a means for the formation of compact structures from in vitro-cultured cancer cells as a model of solid tumor tissue
Association Between Loss-of-Function Mutations Within the FANCM Gene and Early-Onset Familial Breast Cancer
IMPORTANCE Germline mutations in established moderately or highly penetrant risk genes for breast cancer (BC) and/or ovarian cancer (OC), including BRCA1 and BRCA2, explain fewer than half of all familial BC and/or OC cases. Based on the genotyping of 2 loss-of-function (LoF) variants c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), the FANCM gene has been suggested as a novel BC predisposition gene, while the analysis of the entire coding region of the FANCM gene in familial index cases and geographically matched controls is pending. OBJECTIVES To assess the mutational spectrum within the FANCM gene, and to determine a potential association of LoF germline mutations within the FANCM gene with BC and/or OC risk. DESIGN, SETTING, AND PARTICIPANTS For the purpose of identification and characterization of novel BC and/or OC predisposition genes, a total of 2047 well-characterized familial BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for LoF mutations within the FANCM gene by next-generation sequencing. All patients previously tested negative for pathogenic BRCA1 and BRCA2 mutations. All data collection occurred between June 1, 2013, and April 30, 2016. Data analysis was performed from May 1, 2016, to July 1, 2016. MAIN OUTCOMES AND MEASURES FANCM LoF mutation frequencies in patients with BC and/or OC were compared with the FANCM LoF mutation frequencies in geographically matched controls by univariate logistic regression. Positive associations were stratified by age at onset and cancer family history. RESULTS In this case-control study, 2047 well-characterized familial female BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for truncating FANCM alterations. Heterozygous LoF mutations within the FANCM gene were significantly associated with familial BC risk, with an overall odds ratio (OR) of 2.05 (95% CI, 0.94-4.54; P = .049) and a mutation frequency of 1.03% in index cases. In familial patients whose BC onset was before age 51 years, an elevated OR of 2.44 (95% CI, 1.08-5.59; P = .02) was observed. A more pronounced association was identified for patients with a triple-negative BC tumor phenotype (OR, 3.75; 95% CI, 1.00-12.85; P = .02). No significant association was detected for unselected OC cases (OR, 1.74; 95% CI, 0.57-5.08; P = .27). CONCLUSIONS AND RELEVANCE Based on the significant associations of heterozygous LoF mutations with early-onset or triple-negative BC, FANCM should be included in diagnostic gene panel testing for individual risk assessment. Larger studies are required to determine age-dependent disease risks for BC and to assess a potential role of FANCM mutations in OC pathogenesis
Germline loss-of-function variants in the BARD1 gene are associated with early-onset familial breast cancer but not ovarian cancer
BackgroundThe role of the BARD1 gene in breast cancer (BC) and ovarian cancer (OC) predisposition remains elusive, as published case-control investigations have revealed controversial results. We aimed to assess the role of deleterious BARD1 germline variants in BC/OC predisposition in a sample of 4920 BRCA1/2-negative female BC/OC index patients of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC).MethodsA total of 4469 female index patients with BC, 451 index patients with OC, and 2767 geographically matched female control individuals were screened for loss-of-function (LoF) mutations and potentially damaging rare missense variants in BARD1. All patients met the inclusion criteria of the GC-HBOC for germline testing and reported at least one relative with BC or OC. Additional control datasets (Exome Aggregation Consortium, ExAC; Fabulous Ladies Over Seventy, FLOSSIES) were included for the calculation of odds ratios (ORs).ResultsWe identified LoF variants in 23 of 4469BC index patients (0.51%) and in 36 of 37,265 control individuals (0.10%), resulting in an OR of 5.35 (95% confidence interval [CI]=3.17-9.04; P<0.00001). BARD1-mutated BC index patients showed a significantly younger mean age at first diagnosis (AAD; 42.3years, range 24-60years) compared with the overall study sample (48.6years, range 17-92years; P=0.00347). In the subgroup of BC index patients with an AAD <40years, an OR of 12.04 (95% CI=5.78-25.08; P<0.00001) was observed. An OR of 7.43 (95% CI=4.26-12.98; P<0.00001) was observed when stratified for an AAD <50years. LoF variants in BARD1 were not significantly associated with BC in the subgroup of index patients with an AAD 50years (OR=2.29; 95% CI=0.82-6.45; P=0.11217). Overall, rare and predicted damaging BARD1 missense variants were significantly more prevalent in BC index patients compared with control individuals (OR=2.15; 95% CI=1.26-3.67; P=0.00723). Neither LoF variants nor predicted damaging rare missense variants in BARD1 were identified in 451 familial index patients with OC.ConclusionsDue to the significant association of germline LoF variants in BARD1 with early-onset BC, we suggest that intensified BC surveillance programs should be offered to women carrying pathogenic BARD1 gene variants
BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer
Background: Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. Methods: To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. Results: BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. Conclusions: To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded
BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer
Additional file 1: Table S1. of BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer
Inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) for BRCA1 and BRCA2 germline testing. Table S2. Heterozygous protein-truncating mutations identified in the BRIP1 gene. Figure S1. Characterization of the c.507G > A variant within the BRIP1 gene (rs876660937) on transcript level. Table S3. Genotypes and phenotypes of heterozygous BRIP1 mutation carriers identified within the BC/OC index patient cohorts. Table S4. Potentially damaging missense variants identified in the BRIP1 gene. (PDF 215 kb