Adjuvant capecitabine-containing chemotherapy benefit and homologous recombination deficiency in early-stage triple-negative breast cancer patients

Background The addition of adjuvant capecitabine to standard chemotherapy of early-stage triple-negative breast cancer (TNBC) patients has improved survival in a few randomised trials and in meta-analyses. However, many patients did not benefit. We evaluated the BRCA1-like DNA copy number signature, indicative of homologous recombination deficiency, as a predictive biomarker for capecitabine benefit in the TNBC subgroup of the FinXX trial. Methods Early-stage TNBC patients were randomised between adjuvant capecitabine-containing (TX + CEX: capecitabine-docetaxel, followed by cyclophosphamide-epirubicin-capecitabine) and conventional chemotherapy (T + CEF: docetaxel, followed by cyclophosphamide-epirubicin-fluorouracil). Tumour BRCA1-like status was determined on low-coverage, whole genome next-generation sequencing data using an established DNA comparative genomic hybridisation algorithm. Results For 129/202 (63.9%) patients the BRCA1-like status could be determined, mostly due to lack of tissue. During a median follow-up of 10.7 years, 35 recurrences and 32 deaths occurred. Addition of capecitabine appears to improve recurrence-free survival more among 61 (47.3%) patients with non-BRCA1-like tumours (HR 0.23, 95% CI 0.08-0.70) compared to 68 (52.7%) patients with BRCA1-like tumours (HR 0.66, 95% CI 0.24-1.81) (P-interaction = 0.17). Conclusion Based on our data, patients with non-BRCA1-like TNBC appear to benefit from the addition of capecitabine to adjuvant chemotherapy. Patients with BRCA1-like TNBC may also benefit. Additional research is needed to define the subgroup within BRCA1-like TNBC patients who may not benefit from adjuvant capecitabine.Peer reviewe

Role of variant allele fraction and rare SNP filtering to improve cellular DNA repair endpoint association

BackgroundLarge cancer genome studies continue to reveal new players in treatment response and tumorigenesis. The discrimination of functional alterations from the abundance of passenger genetic alterations still poses challenges and determines DNA sequence variant selection procedures. Here we evaluate variant selection strategies that select homozygous variants and rare SNPs and assess its value in detecting tumor cells with DNA repair defects.MethodsTo this end we employed a panel of 29 patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines, of which a subset harbors DNA repair defects. Mitomycin C (MMC) sensitivity was used as functional endpoint of DNA crosslink repair deficiency. 556 genes including the Fanconi anemia (FA) and homologous recombination (HR) genes, whose products strongly determine MMC response, were capture-sequenced.ResultsWe show a strong association between MMC sensitivity, thus loss of DNA repair function, and the presence of homozygous and rare SNPs in the relevant FA/HR genes. Excluding such selection criteria impedes the discrimination of crosslink repair status by mutation analysis. Applied to all KEGG pathways, we find that the association with MMC sensitivity is strongest in the KEGG FA pathway, therefore also demonstrating the value of such selection strategies for exploratory analyses. Variant analyses in 56 clinical samples demonstrate that homozygous variants occur more frequently in tumor suppressor genes than oncogenes further supporting the role of a homozygosity criterion to improve gene function association or tumor suppressor gene identification studies.ConclusionTogether our data show that the detection of relevant genes or of repair pathway defected tumor cells can be improved by the consideration of allele zygosity and SNP allele frequencies

Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in theJAK3gene

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene wasTP53(97% mutation frequency) followed byBRCA1(10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, afterBRCA1,JAK3was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-basedin vitrofunctional assay and identified a novel gain-of-function mutation in the kinase domain ofJAK3(p.T1022I). Importantly, p.T1022IJAK3mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entireJAK3coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening ofCDK12in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency ofJAK3andCDK12mutations in a large well characterized cohort. Although p.T1022IJAK3mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation ofCDK12mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.ISSN:1932-620

Long-term outcomes of young, node-negative, chemotherapy-naïve, triple-negative breast cancer patients according to BRCA1 status

Background: Due to the abundant usage of chemotherapy in young triple-negative breast cancer (TNBC) patients, the unbiased prognostic value of BRCA1-related biomarkers in this population remains unclear. In addition, whether BRCA1-related biomarkers modify the well-established prognostic value of stromal tumor-infiltrating lymphocytes (sTILs) is unknown. This study aimed to compare the outcomes of young, node-negative, chemotherapy-naïve TNBC patients according to BRCA1 status, taking sTILs into account. Methods: We included 485 Dutch women diagnosed with node-negative TNBC under age 40 between 1989 and 2000. During this period, these women were considered low-risk and did not receive chemotherapy. BRCA1 status, including pathogenic germline BRCA1 mutation (gBRCA1m), somatic BRCA1 mutation (sBRCA1m), and tumor BRCA1 promoter methylation (BRCA1-PM), was assessed using DNA from formalin-fixed paraffin-embedded tissue. sTILs were assessed according to the international guideline. Patients’ outcomes were compared using Cox regression and competing risk models. Results: Among the 399 patients with BRCA1 status, 26.3% had a gBRCA1m, 5.3% had a sBRCA1m, 36.6% had tumor BRCA1-PM, and 31.8% had BRCA1-non-altered tumors. Compared to BRCA1-non-alteration, gBRCA1m was associated with worse overall survival (OS) from the fourth year after diagnosis (adjusted HR, 2.11; 95% CI, 1.18–3.75), and this association attenuated after adjustment for second primary tumors. Every 10% sTIL increment was associated with 16% higher OS (adjusted HR, 0.84; 95% CI, 0.78–0.90) in gBRCA1m, sBRCA1m, or BRCA1-non-altered patients and 31% higher OS in tumor BRCA1-PM patients. Among the 66 patients with tumor BRCA1-PM and ≥ 50% sTILs, we observed excellent 15-year OS (97.0%; 95% CI, 92.9–100%). Conversely, among the 61 patients with gBRCA1m and < 50% sTILs, we observed poor 15-year OS (50.8%; 95% CI, 39.7–65.0%). Furthermore, gBRCA1m was associated with higher (adjusted subdistribution HR, 4.04; 95% CI, 2.29–7.13) and tumor BRCA1-PM with lower (adjusted subdistribution HR, 0.42; 95% CI, 0.19–0.95) incidence of second primary tumors, compared to BRCA1-non-alteration. Conclusions: Although both gBRCA1m and tumor BRCA1-PM alter BRCA1 gene transcription, they are associated with different outcomes in young, node-negative, chemotherapy-naïve TNBC patients. By combining sTILs and BRCA1 status for risk classification, we were able to identify potential subgroups in this population to intensify and optimize adjuvant treatment

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation

Human induced pluripotent stem cells (hiPSC) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSC as well as the differentiation of hiPSC into target cells that are used for toxicity testing, mainly depend on the presence of extracellular matrix (ECM) coatings. The most widely used ECM coating is Matrigel, an animal product that is derived from mice sarcomas. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, usage of animal rather than human material and ethical concerns for its production. While alternative coatings exist, factors such as higher cost and limited characterizations may hinder a broader uptake by the scientific community. Here, we report an extensive comparison of three commercial human ECM coatings, vitronectin, laminin-511 and laminin-521, to Matrigel in three different hiPSC lines in long term culture (< 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies and karyotype stability by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost alternative, decellularized ECM produced by human neonatal dermal fibroblasts was analyzed. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results showed that all tested coatings were highly comparable to the animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast ECM could be a novel attractive low-cost coating

A regression model for estimating DNA copy number applied to capture sequencing data

International audienceMotivation: Target enrichment, also referred to as DNA capture, provides an effective way to focus sequencing efforts on a genomic region of interest. Capture data are typically used to detect single-nucleotide variants. It can also be used to detect copy number alterations, which is particularly useful in the context of cancer, where such changes occur frequently. In copy number analysis, it is a common practice to determine log-ratios between test and control samples, but this approach results in a loss of information as it disregards the total coverage or intensity at a locus. Results: We modeled the coverage or intensity of the test sample as a linear function of the control sample. This regression approach is able to deal with regions that are completely deleted, which are problematic for methods that use log-ratios. To demonstrate the utility of our approach, we used capture data to determine copy number for a set of 600 genes in a panel of nine breast cancer cell lines. We found high concordance between our results and those generated using a single-nucleotide polymorphsim genotyping platform. When we compared our results with other log-ratio-based methods, including ExomeCNV, we found that our approach produced better overall correlation with SNP data

Ovarian Cancer-Specific BRCA-like Copy-Number Aberration Classifiers Detect Mutations Associated with Homologous Recombination Deficiency in the AGO-TR1 Trial

Purpose: Previously, we developed breast cancer BRCA1-like and BRCA2-like copy-number profile shrunken centroid classifiers predictive for mutation status and response to therapy, targeting homologous recombination deficiency (HRD). Therefore, we investigated BRCA1- and BRCA2-like classification in ovarian cancer, aiming to acquire classifiers with similar properties as those in breast cancer. Experimental Design: We analyzed DNA copy-number profiles of germline BRCA1- and BRCA2-mutant ovarian cancers and control tumors and observed that existing breast cancer classifiers did not sufficiently predict mutation status. Hence, we trained new shrunken centroid classifiers on this set and validated them in the independent The Cancer Genome Atlas dataset. Subsequently, we assessed BRCA1/2-like classification and obtained germline and tumor mutation and methylation status of cancer predisposition genes, among them several involved in HR repair, of 300 ovarian cancer samples derived from the consecutive cohort trial AGO-TR1 (NCT02222883). Results: The detection rate of the BRCA1-like classifier for BRCA1 mutations and promoter hypermethylation was 95.6%. The BRCA2-like classifier performed less accurately, likely due to a smaller training set. Furthermore, three quarters of the BRCA1/ 2-like tumors could be explained by (epi)genetic alterations in BRCA1/2, germline RAD51C mutations and alterations in other genes involved in HR. Around half of the non-BRCA-mutated ovarian cancer cases displayed a BRCA-like phenotype. Conclusions: The newly trained classifiers detected most BRCAmutated and methylated cancers and all tumors harboring a RAD51C germline mutations. Beyond that, we found an additional substantial proportion of ovarian cancers to be BRCA-like. _2021 The Authors; Published by the American Association for Cancer Research.</p

Ovarian Cancer-Specific BRCA-like Copy-Number Aberration Classifiers Detect Mutations Associated with Homologous Recombination Deficiency in the AGO-TR1 Trial

Purpose: Previously, we developed breast cancer BRCA1-like and BRCA2-like copy-number profile shrunken centroid classifiers predictive for mutation status and response to therapy, targeting homologous recombination deficiency (HRD). Therefore, we investigated BRCA1- and BRCA2-like classification in ovarian cancer, aiming to acquire classifiers with similar properties as those in breast cancer. Experimental Design: We analyzed DNA copy-number profiles of germline BRCA1- and BRCA2-mutant ovarian cancers and control tumors and observed that existing breast cancer classifiers did not sufficiently predict mutation status. Hence, we trained new shrunken centroid classifiers on this set and validated them in the independent The Cancer Genome Atlas dataset. Subsequently, we assessed BRCA1/2-like classification and obtained germline and tumor mutation and methylation status of cancer predisposition genes, among them several involved in HR repair, of 300 ovarian cancer samples derived from the consecutive cohort trial AGO-TR1 (NCT02222883). Results: The detection rate of the BRCA1-like classifier for BRCA1 mutations and promoter hypermethylation was 95.6%. The BRCA2-like classifier performed less accurately, likely due to a smaller training set. Furthermore, three quarters of the BRCA1/ 2-like tumors could be explained by (epi)genetic alterations in BRCA1/2, germline RAD51C mutations and alterations in other genes involved in HR. Around half of the non-BRCA-mutated ovarian cancer cases displayed a BRCA-like phenotype. Conclusions: The newly trained classifiers detected most BRCAmutated and methylated cancers and all tumors harboring a RAD51C germline mutations. Beyond that, we found an additional substantial proportion of ovarian cancers to be BRCA-like. _2021 The Authors; Published by the American Association for Cancer Research.Pattern Recognition and Bioinformatic