ABSTRACT
BACKGROUND: Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance patterns of BRCA1 epimutations established. METHODS: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 408 patients with 411 primary breast cancers, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Furthermore, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls and 200 newborn boys. Finally, we assessed BRCA1 methylation status among 575 mothers and 531 fathers of girls with (n = 102) and without (n = 473) BRCA1 methylation. RESULTS: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1-30.5). In contrast, we found concordant WBC and tumor methylation in only three out of 220 patients with 221 ER ≥ 10% tumors and zero out of 114 patients with 116 HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals, but no correlation to BRCA1 methylation status either in mothers or fathers. A significantly lower fraction of newborn boys carried BRCA1 methylation (9/200; 4.5%) as compared to girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). CONCLUSIONS: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expression breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance.
Subject(s)
Breast Neoplasms , Triple Negative Breast Neoplasms , Adult , Female , Infant, Newborn , Humans , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Breast Neoplasms/genetics , DNA Methylation , Promoter Regions, Genetic , BRCA1 Protein/geneticsABSTRACT
PURPOSE: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. METHODS: HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. RESULTS: HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). CONCLUSION: The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/genetics , B7-H1 Antigen/genetics , Genes, BRCA2 , Mutation , Homologous Recombination/geneticsABSTRACT
BACKGROUND: While germline CHEK2 mutations have been linked to a moderately elevated cancer risk, to date, a limited number of such mutations have been identified. Recently, we reported a germline nonsense mutation (C283T; R95*), introducing an early stop-codon, in two Norwegian patients diagnosed with locally advanced breast cancer. Both patients were resistant to anthracycline therapy, resembling what has been observed for TP53 mutations. METHODS: In the present study, we screened a large population based sample, including 3748 non-cancer individuals and 7081 incident cancer cases (breast cancer, n = 1717; prostate cancer n = 2501, lung cancer n = 1331 and colorectal cancer n = 1532), for the distribution of CHEK2 R95*. RESULTS: We found that 12 individuals (0.11 %) carried the R95* variant: 4 non-cancer individuals (0.11 %), 4 breast cancer cases (0.23 %), and 4 prostate cancer cases (0.16 %). Although the low number of observations precluded formal statistical assessment, our data may indicate an elevated risk for breast (OR: 2.19, 95 % CI: 0.55-8.75) and prostate cancer (OR: 1.5, 95 % CI: 0.36-6.00) associated with CHEK2 R95*. By mining international databanks, we found no individuals carrying the R95* mutation, indicating it to be restricted to the Norwegian population. CONCLUSION: We provide proof-of-concept that previously unknown CHEK2 germline mutations may be present in certain populations. Notably, germline mutations in tumours are in general missed by contemporary massive parallel sequencing strategies, since tumour mutations are usually filtered against the germline. The fact that the CHEK2 R95* mutation may be associated with resistance to anthracyclines in cancer patients emphasizes its possible clinical importance.
ABSTRACT
Chemoresistance is the main obstacle to cancer cure. Contrasting studies focusing on single gene mutations, we hypothesize chemoresistance to be due to inactivation of key pathways affecting cellular mechanisms such as apoptosis, senescence, or DNA repair. In support of this hypothesis, we have previously shown inactivation of either TP53 or its key activators CHK2 and ATM to predict resistance to DNA damaging drugs in breast cancer better than TP53 mutations alone. Further, we hypothesized that redundant pathway(s) may compensate for loss of p53-pathway signaling and that these are inactivated as well in resistant tumour cells. Here, we assessed genetic alterations of the retinoblastoma gene (RB1) and its key regulators: Cyclin D and E as well as their inhibitors p16 and p27. In an exploratory cohort of 69 patients selected from two prospective studies treated with either doxorubicin monotherapy or 5-FU and mitomycin for locally advanced breast cancers, we found defects in the pRB-pathway to be associated with therapy resistance (p-values ranging from 0.001 to 0.094, depending on the cut-off value applied to p27 expression levels). Although statistically weaker, we observed confirmatory associations in a validation cohort from another prospective study (n = 107 patients treated with neoadjuvant epirubicin monotherapy; p-values ranging from 7.0 × 10(-4) to 0.001 in the combined data sets). Importantly, inactivation of the p53-and the pRB-pathways in concert predicted resistance to therapy more strongly than each of the two pathways assessed individually (exploratory cohort: p-values ranging from 3.9 × 10(-6) to 7.5 × 10(-3) depending on cut-off values applied to ATM and p27 mRNA expression levels). Again, similar findings were confirmed in the validation cohort, with p-values ranging from 6.0 × 10(-7) to 6.5 × 10(-5) in the combined data sets. Our findings strongly indicate that concomitant inactivation of the p53- and pRB- pathways predict resistance towards anthracyclines and mitomycin in breast cancer in vivo.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , DNA Damage , Drug Resistance, Neoplasm/genetics , Retinoblastoma Protein/genetics , Tumor Suppressor Protein p53/genetics , Cohort Studies , DNA Damage/genetics , DNA Mutational Analysis , Doxorubicin/therapeutic use , Female , Fluorouracil/therapeutic use , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Mitomycin/therapeutic use , Prognosis , Retinoblastoma Protein/metabolism , Signal Transduction/genetics , Treatment FailureABSTRACT
INTRODUCTION: Mutations affecting p53 or its upstream activator Chk2 are associated with resistance to DNA-damaging chemotherapy in breast cancer. ATM (Ataxia Telangiectasia Mutated protein) is the key activator of p53 and Chk2 in response to genotoxic stress. Here, we sought to evaluate ATM's potential role in resistance to chemotherapy. METHODS: We sequenced ATM and assessed gene expression levels in pre-treatment biopsies from 71 locally advanced breast cancers treated in the neoadjuvant setting with doxorubicin monotherapy or mitomycin combined with 5-fluorouracil. Findings were confirmed in a separate patient cohort treated with epirubicin monotherapy. Each tumor was previously analyzed for CHEK2 and TP53 mutation status. RESULTS: While ATM mutations were not associated with chemo-resistance, low ATM expression levels predicted chemo-resistance among patients with tumors wild-type for TP53 and CHEK2 (P = 0.028). Analyzing the ATM-chk2-p53 cascade, low ATM levels (defined as the lower 5 to 50% percentiles) or mutations inactivating TP53 or CHEK2 robustly predicted anthracycline resistance (P-values varying between 0.001 and 0.027 depending on the percentile used to define "low" ATM levels). These results were confirmed in an independent cohort of 109 patients treated with epirubicin monotherapy. In contrast, ATM-levels were not suppressed in resistant tumors harboring TP53 or CHEK2 mutations (P > 0.5). CONCLUSIONS: Our data indicate loss of function of the ATM-Chk2-p53 cascade to be strongly associated with resistance to anthracycline/mitomycin-containing chemotherapy in breast cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm/genetics , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Anthracyclines/administration & dosage , Anthracyclines/therapeutic use , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/therapeutic use , Ataxia Telangiectasia Mutated Proteins , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Checkpoint Kinase 2 , Cohort Studies , Doxorubicin/administration & dosage , Doxorubicin/therapeutic use , Epirubicin/administration & dosage , Epirubicin/therapeutic use , Female , Fluorouracil/administration & dosage , Fluorouracil/therapeutic use , Gene Expression Regulation, Neoplastic , Humans , Mitomycin/administration & dosage , Mitomycin/therapeutic use , Mutation , Paclitaxel/administration & dosage , Paclitaxel/therapeutic use , Predictive Value of Tests , Promoter Regions, Genetic , Prospective Studies , Tumor Suppressor Protein p53/therapeutic useABSTRACT
PURPOSE: The cyclin-dependent kinase inhibitor p21 acts as a main executor of p53-induced growth arrest. Recently, a second transcript, p21B, was found to code for a protein expressing proapoptotic activity. We investigated p21 and p21B for mutations and epigenetic silencing in locally advanced breast cancers treated with doxorubicin or 5-fluorouracil/mitomycin and correlated our findings with treatment response and TP53 status. EXPERIMENTAL DESIGN: We used reverse transcription-PCR to analyze p21/p21B mutation status in 73 breast cancer samples. The p21 promoter region was sequenced and analyzed for hypermethylations by methylation-specific PCR. In addition, a selection of patients were analyzed for mutations in the p21B promoter. RESULTS: The p21 gene was neither mutated nor silenced by promoter hypermethylation in any of the tumors examined. One patient harbored a novel p21 splice variant in addition to the wild-type transcript. We observed two base substitutions in the p21 transcript, C93A and G251A, each affecting six patients (8.2%). The G251A variant had not been reported previously. In 12 patients (16.4%), we observed a novel base substitution, T35C, in p21B. All three base substitutions were observed in lymphocyte DNA and therefore considered polymorphisms. The polymorphisms did not correlate with p21 staining index, treatment response to doxorubicin or 5-fluorouracil/mitomycin, or TP53 status. CONCLUSIONS: Our findings do not suggest that genetic or epigenetic disturbances in p21 or p21B cause resistance to doxorubicin or mitomycin/5-fluorouracil in breast cancer. Future studies should assess potential associations between these novel polymorphisms and breast cancer risk.
Subject(s)
Breast Neoplasms/drug therapy , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Fluorouracil/pharmacology , Mitomycin/pharmacology , Proto-Oncogene Proteins p21(ras)/biosynthesis , Proto-Oncogene Proteins p21(ras)/genetics , rap GTP-Binding Proteins/biosynthesis , Antibiotics, Antineoplastic/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Breast Neoplasms/metabolism , Cell Line, Tumor , Cloning, Molecular , DNA Methylation , DNA Primers/chemistry , Gene Silencing , Humans , Immunohistochemistry , Lymphocytes/metabolism , Models, Genetic , Polymorphism, Genetic , Promoter Regions, Genetic , RNA/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Tumor Suppressor Protein p53/metabolismABSTRACT
Objective--Evaluation of the centrifugal pump vs roller pump concerning effects upon platelet function, hemolysis and clinical outcome in elective coronary artery bypass surgery. Design--Thirty-four patients were randomized to centrifugal or roller pump. Platelet activation was studied by flow cytometry before, during and up to 3 days after bypass. Results--Duration of bypass, ischemic period, peripheral anastomoses, hospital stay and mortality did not differ. In roller pump patients, platelet aggregates increased by 250% between end of bypass and 3 h postoperatively (p < 0.001). A secondary, fivefold increase in number of platelet aggregates was found on the 3rd postoperative day (p < 0.001). In the centrifugal pump group, these changes were not significant. Hemolysis increased (20%) at end of bypass and 3 h postoperatively (p < 0.005), and decreased to preoperative levels the next day without group difference. Conclusion--Platelet aggregation was significantly increased in roller compared with centrifugal pump patients, indicating higher susceptibility to postoperative thrombotic complications with the roller pump. Otherwise, there was no clinical evidence for superiority of the centrifugal pump.