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Kaplan-Meier curves of the non-BRCA1-like cohort split by XIST-53BP1 and chemotherapy regimen.

Analysis of genomic loss of the XIST locus by array comparative genomic hybridization

Supplemental Table 1 - Description of the study according to REMARK criteria. Supplemental Table 2 - Survival analysis of XIST and 53BP1 in non-BRCA1-like patients. Supplemental Table 3 - Clinical characteristics of the full trial population for which 53BP1 status was available. Supplemental Table 4 - Cox Proportional Hazards Model for Recurrence Free Survival including prognostic effect of 53BP1 and chemotherapy effect by 53BP1 in the full trial population for which 53BP1 status was available. Supplemental Table 5 - Clinical characteristics of the triple negative trial population for which 53BP1 status was available. Supplemental Table 6 - Cox Proportional Hazards Model for Recurrence Free Survival including prognostic effect of 53BP1 and chemotherapy effect by 53BP1 in triple negative trial population for which 53BP1 status was available. Supplemental Table 7 - Cox Proportional Hazards Model for Recurrence Free Survival including prognostic effect of 53BP1 and chemotherapy effect by 53BP1 in the full trial population for which 53BP1 status was available. Supplemental Table 8 - Cox Proportional Hazards Model for Recurrence Free Survival including prognostic effect of 53BP1 and chemotherapy effect by 53BP1 in triple negative trial population

Supplemental Figure Legends

Analysis of genomic loss of the 53BP1 locus by array comparative genomic hybridization

Supplementary Material and Methods

Purpose:BRCA1-deficient breast cancers carry a specific DNA copy-number signature (“BRCA1-like”) and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors.Experimental Design:EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1–like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model.Results:The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1–like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents.Conclusions:Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.

Purpose:The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics.Experimental Design:Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort.Results:Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001).Conclusions:HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.

Immunohistological stainings of breast tumors for EZH2 (representative examples of used antibodies)

Supplementary Table 1. Description of the study according to REMARK.

Figure S1 shows that the Oncoscan and CGH agilent platform yield similar results.

Table S1 shows the clinicopathological characteristics of the cases included in final analysis.

Purpose: Breast cancers in carriers of inactivating mutations of the BRCA1 gene carry a specific DNA copy-number signature (“BRCA1-like”). This signature is shared with cancers that inactivate BRCA1 through other mechanisms. Because BRCA1 is important in repair of DNA double-strand breaks through error-free homologous recombination, patients with a BRCA1-like tumor may benefit from high-dose alkylating (HD) chemotherapy, which induces DNA double-strand breaks.Experimental Design: We investigated a single institution cohort of high-risk patients that received tandem HD chemotherapy schedule comprising ifosfamide, epirubicin, and carboplatin or conventional chemotherapy. We classified copy-number profiles to be BRCA1-like or non–BRCA1-like and analyzed clinical associations and performed survival analysis with a treatment by biomarker interaction design.Results: BRCA1-like status associated with high-grade and triple-negative breast cancers. BRCA1-like cases benefitted from the HD compared with a conventional regimen on disease-free survival (DFS): [hazard ratio (HR), 0.05; 95% confidence interval (CI), 0.01–0.38; P = 0.003]; distant DFS (DDFS): (HR, 0.06; 95% CI, 0.01–0.43; P = 0.01); and overall survival (OS; HR, 0.15; 95% CI, 0.03–0.83; P = 0.03) after correction for prognostic factors. No such benefit was observed in the non–BRCA1-like cases on DFS (HR, 0.74; 95% CI, 0.38–1.46; P = 0.39), DDFS (HR, 0.79; 95% CI, 0.41–1.52; P = 0.47), and OS (HR, 0.93; 95% CI, 0.52–1.64; P = 0.79). The P values for interaction were 0.01 (DFS), 0.01 (DDFS), and 0.045 (OS).Conclusions: BRCA1-like tumors recurred significantly less often after HD than conventional chemotherapy. BRCA1-like copy-number profile classification may be a predictive marker for HD alkylating chemotherapy. Clin Cancer Res; 21(4); 763–70. ©2014 AACR.

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The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy. Cancer Res; 72(9); 2350–61. ©2012 AACR.

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How the tumor’s genetic make up (i.e. molecular profiles) is associated with changes in the tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC) is incompletely understood, while these factors influence survival. To investigate this we analyzed the TME and molecular profiles (homologous recombination deficiency (HRD) and Cyclin E1 (CCNE1) amplification) of HGSOC and assessed their associations with each other and overall survival (OS). Patients with advanced-stage HGSOC treated in three Dutch hospitals between 2008-2015 were included. Patient, treatment, and outcome data were collected from medical records. BRCA1/2 mutation, BRCA1 promotor methylation analyses, and copy number variations were used to define five molecular profiles. Immunecell densites were assessed with immunohistochemical staining for T- (CD8 and CD103), B- cells (CD20) and macrophages (CD68). This resulted in the assessability of 348 patients who were categorized as; BRCA mutation or promotor methylation (BRCAm) (30%), non-BRCA mutated HRD (19%), CCNE1-amplification (13%), non-BRCAmut HRD and CCNE1-amplification (double classifier) (20%), and no specific molecular profile (NSMP) (18%). BRCAm tumors showed the highest immune cell densities and CCNE1-amplification the lowest. BRCAm showed the most favorable OS (52.5months), compared to non-BRCAmut HRD (41.0months), CCNE1-amplification (28.0months), double classifier (27.8months), and NSMP (35.4months). CCNE1-amplification and double classifier remained to have a significantly worse OS in multivariable analysis. Higher immune cell densities showed a favorable OS compared to lower densities, also within the molecular profiles. CD8+, CD20+, and CD103+ cells remained associated with OS in multivariable analysis. These results depict that both molecular profiles and TME are associated with OS. Immune composition for lymphocytes and macrophages differ per molecular profile, with higher immune cell densities showing a favorable OS, even within the profiles. HGSOC does not reflect one entity but comprises different variants based on molecular profiles and TME, which ideally is translated into tailored treatment approaches.

BackgroundThe 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 theBRCA1-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.MethodsEarly-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). TumourBRCA1-like status was determined on low-coverage, whole genome next-generation sequencing data using an established DNA comparative genomic hybridisation algorithm.ResultsFor 129/202 (63.9%) patients theBRCA1-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 withBRCA1-like tumours (HR 0.66, 95% CI 0.24–1.81) (P-interaction = 0.17).ConclusionBased on our data, patients with non-BRCA1-like TNBC appear to benefit from the addition of capecitabine to adjuvant chemotherapy. Patients withBRCA1-like TNBC may also benefit. Additional research is needed to define the subgroup withinBRCA1-like TNBC patients who may not benefit from adjuvant capecitabine.

Preclinical studies have shown synergistic effects when combining PARP1/2 inhibitors and platinum drugs in BRCA1/2 mutated cancer cell models. After a formulation change of olaparib from capsules to tablets, we initiated a dose finding study of olaparib tablets bidaily (BID) continuously with carboplatin to prepare comparative studies in this patient group. Patients were included in a 3 + 3 dose‐escalation schedule: olaparib 25 mg BID and carboplatin area under the curve (AUC) 3 mg*min/mL d1/d22, olaparib 25 mg BID and carboplatin AUC 4 mg*min/mL d1/d22, followed by increasing dose‐levels of olaparib from 50 mg BID, 75 mg BID, to 100 mg BID with carboplatin at AUC 4 mg*min/mL d1/d22. After two cycles, patients continued olaparib 300 mg BID as monotherapy. Primary objective was to assess the maximum tolerable dose (MTD). Twenty‐four patients with a confirmed diagnosis of advanced cancer were included. Most common adverse events were nausea (46%), fatigue (33%) and platelet count decrease (33%). Dose‐level 3 (olaparib 75 mg BID and carboplatin AUC 4 mg*min/mL; n = 6) was defined as MTD. Fourteen out of 24 patients (56%) had a partial response as best response (RECIST 1.1). Systemic exposure of the olaparib tablet formulation appeared comparable to the previous capsule formulation with olaparib tablet AUC0‐12 of 16.3 μg/mL*h at MTD. Polymers of ADP‐ribose levels in peripheral blood mononuclear cells were reduced by 98.7% ± 0.14% at Day 8 compared to Day 1 for dose‐level 3. Olaparib tablets 75 mg BID and carboplatin AUC 4 mg*min/mL for two cycles preceding olaparib monotherapy 300 mg is a feasible and tolerable treatment schedule for patients with advanced cancer., What's new? Preclinical studies have shown synergistic effects when combining PARP1/2‐inhibitors and platinum drugs in BRCA1/2 mutated cancer cell models. This phase I trial of olaparib tablets combined with carboplatin in advanced cancer patients showed that the combination has an acceptable side‐effect profile. The maximum tolerable dose was olaparib tablets 75 mg BID and carboplatin AUC 4 mg*min/ml. The observed preliminary anti‐tumor activity was encouraging, with 58% of patients having a decrease in tumor volume of more than 30%. This study shows that the tablet formulation of olaparib can be administered safely in combination with carboplatin, compared to the previous capsule formulation.

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 BRCA-mutated 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.

Introduction/Background* Recently 3 Phase III first-line studies, PAOLA-1/ENGOT-ov25 (Ray-Coquard et al. NEJM 2019). PRIMA/ENGOT-ov26/GOG-3012 (Gonzales Martin et al. NEJM 2019) and VELIA/GOG-3005 (Coleman et al. NEJM 2019) have demonstrated that the addition of a PARP inhibitor (PARPi) to platinum-based therapy+/-bevacizumab improved progression-free survival (PFS) in advanced ovarian cancer (AOC) patients. The benefit was greater when the tumor was homologous recombination deficient (HRD) according to Myriad myChoice test, independently of BRCA status. The PAOLA-1 olaparib+bevacizumab maintenance regimen was approved in USA/Europe/Japan for HRD positive patients. The European initiative aims at evaluating various HRD tests on PAOLA-1 tumor samples to identify new reliable and feasible HRD tests Methodology The HRD initiative has 2 components; one based on artificial intelligence with various partners and the other, the European HRD ENGOT initiative (EHEI), is led by academic research laboratories (RL) from ENGOT groups. The HRD test evaluation protocol for the EHEI RL includes 3 phases. The phase 1 (2019/12) brought together European RL. Because non-BRCA Homologous Recombination Repair (HRR) mutations have not been found predictive of PARPi activity in PAOLA-1 (Pujade-Lauraine et al, SGO 2021)) RL tests based on these mutations were not selected for the next phases. Phase 2 evaluated the correlation between RL tests and the Myriad myChoice test on tumor samples from 85 PAOLA-1 BRCA wild type patients using the KAPPA statistics. Phase 3 is the final PFS evaluation on more than 350 additional patient samples. Result(s)* A total of 20 RL from 21 ENGOT groups participated to the EHEI phase 1. Half of them had a test mainly based on an HRR gene panel. Three RL did not pursue for various reasons (capacity, financial or regulatory). The remaining 7 RL from 6 countries (table 1) completed the phase 2 in May 2021 and may proceed to phase 3. Conclusion* The EHEI is a unique collaboration of European academic laboratories involved in gynecologic oncology translational research with the aim of providing a reliable biomarker (HRD) for selecting AOC patients who could benefit most from PARPi+/-bevacizumab in first-line therapy. HRD tests performance will be described after their phase 3 is completed.

Purpose: BRCA1-deficient breast cancers carry a specific DNA copy-number signature (“BRCA1-like”) and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors. Experimental Design: EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1–like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model. Results: The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1–like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents. Conclusions: Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.

The efficacy of programmed cell death protein 1 (PD-1) blockade in metastatic triple-negative breast cancer ( TNBC ) is low1–5, highlighting a need for strategies that render the tumor microenvironment more sensitive to PD-1 blockade. Preclinical research has suggested immunomodulatory properties for chemotherapy and irradiation6–13. In the first stage of this adaptive, non-comparative phase 2 trial, 67 patients with metastatic TNBC were randomized to nivolumab (1) without induction or with 2-week low-dose induction, or with (2) irradiation (3 × 8 Gy), (3) cyclophosphamide, (4) cisplatin or (5) doxorubicin, all followed by nivolumab. In the overall cohort, the objective response rate (ORR; iRECIST14) was 20%. The majority of responses were observed in the cisplatin (ORR 23%) and doxorubicin (ORR 35%) cohorts. After doxorubicin and cisplatin induction, we detected an upregulation of immune-related genes involved in PD-1–PD-L1 (programmed death ligand 1) and T cell cytotoxicity pathways. This was further supported by enrichment among upregulated genes related to inflammation, JAK–STAT and TNF-α signaling after doxorubicin. Together, the clinical and translational data of this study indicate that short-term doxorubicin and cisplatin may induce a more favorable tumor microenvironment and increase the likelihood of response to PD-1 blockade in TNBC. These data warrant confirmation in TNBC and exploration of induction treatments prior to PD-1 blockade in other cancer types. A pick-the-winner clinical trial design in patients with metastatic triple-negative breast cancer shows that immune induction with doxorubicin or cisplatin may improve clinical responses to PD-1 blockade and induce a more favorable tumor microenvironment.

Previously, we developed breast cancerThe detection rate of theThe newly trained classifiers detected most

Background: Recent data demonstrate that patients with early-stage triple negative breast cancer (TNBC) benefit from escalating adjuvant treatment with capecitabine. However, since a substantial proportion of patients does not benefit, predictive biomarkers to select those individuals upfront are needed. Over half of all TNBCs have a BRCA1-like DNA copy number signature similar to the profile found in germline BRCA1-mutated breast cancers and indicative for homologous recombination deficiency. We evaluate this signature as a predictive biomarker for capecitabine benefit in archived specimens of the randomized controlled FinXX trial. Additionally, we compared the concordance of our DNA-based BRCA1-like classifier with the RNA-based NanoString BRCAness signature. Methods: Early-stage TNBC patients were randomized between adjuvant capecitabine-containing chemotherapy (TX+CEX: capecitabine plus docetaxel, followed by cyclophosphamide, epirubicin and capecitabine) and conventional adjuvant chemotherapy (T+CEF: docetaxel, followed by cyclophosphamide, epirubicin, and fluorouracil). Breast tumor BRCA1-like status was determined on low coverage, whole genome next-generation sequencing data using an established DNA comparative genomic hybridization algorithm. We used interaction analysis in proportional hazards models to evaluate whether benefit of adjuvant capecitabine-containing versus conventional chemotherapy differs between BRCA1-like and non-BRCA1-like tumors in early-stage TNBC patients.Results: For 129 (63.9%) of the 202 TNBC patients the BRCA1-like status could be determined. Thirty-five recurrences and 32 deaths occurred during a median follow-up of 10.7 years. The capecitabine effect on recurrence-free survival did not significantly differ between the 68 patients (52.7%) with a BRCA1-like tumor (HR 0.66, 95% CI 0.24-1.81) and others (HR 0.23, 95% CI 0.08-0.70, P interaction = 0.17), also after adjustment for clinico-pathological variables. Conclusions: In the FinXX trial, the BRCA1-like status was not associated with a differential benefit from capecitabine-containing adjuvant chemotherapy compared to conventional chemotherapy in the TNBC subgroup. Based on this study, it is unlikely that the BRCA1-like classifier can be used to distinguish patients who do and do not benefit from capecitabine-enriched standard adjuvant chemotherapy.

Effective treatment of invasive lobular carcinoma (ILC) of the breast is hampered by late detection, invasive growth, distant metastasis, and poor response to chemotherapy. Phosphoinositide 3-kinase (PI3K) signaling, one of the major druggable oncogenic signaling networks, is frequently activated in ILC. We investigated treatment response and resistance to AZD8055, an inhibitor of mammalian target of rapamycin (mTOR), in the K14-cre;Cdh1Flox/Flox;Trp53Flox/Flox (KEP) mouse model of metastatic ILC. Inhibition of mTOR signaling blocked the growth of primary KEP tumors as well as the progression of metastatic disease. However, primary tumors and distant metastases eventually acquired resistance after long-term AZD8055 treatment, despite continued effective suppression of mTOR signaling in cancer cells. Interestingly, therapeutic responses were associated with increased expression of genes related to antigen presentation. Consistent with this observation, increased numbers of tumor-infiltrating major histocompatibility complex class II-positive (MHCII+) immune cells were observed in treatment-responsive KEP tumors. Acquisition of treatment resistance was associated with loss of MHCII+ cells and reduced expression of genes related to the adaptive immune system. The therapeutic efficacy of mTOR inhibition was reduced in Rag1−/- mice lacking mature T and B lymphocytes, compared to immunocompetent mice. Furthermore, therapy responsiveness could be partially rescued by transplanting AZD8055-resistant KEP tumors into treatment-naïve immunocompetent hosts. Collectively, these data indicate that the PI3K signaling pathway is an attractive therapeutic target in invasive lobular carcinoma, and that part of the therapeutic effect of mTOR inhibition is mediated by the adaptive immune system.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor–positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice. These results should now be investigated in vivo before a prospective clinical trial. This material is based upon works supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL" and the SFi CDA Award 15/CDA/3438 Citation Format: O'Connor DP, Walsh L, Fan Y, Tarrant F, Chin S-F, Schouten P, Caldas C, Bernards R, Ni Chonghaile T, Gallagher WM. Bromodomain inhibitors for the treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-02.

Purpose: The BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial. Methods: Lymph node positive breast cancer patients were randomized to 3 × 3 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data. Results: 119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83 months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55–1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58–2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interaction = 0.094). Conclusions: The BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor–positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression was associated with poor recurrence-free survival (p With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice. These results should now be investigated in vivo before a prospective clinical trial. "This material is based upon works supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL". Citation Format: O'Connor DP, Walsh L, Tarrant F, Chin S-F, Schouten P, Linn S, Bernards R, Caldas C, Gallagher WM, ni Chonghaile T. Bromodomain inhibitors represent a rational therapeutic option for the treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-06.

Predictive biomarkers can guide treatment decisions in breast cancer. Many studies are undertaken to discover and translate these biomarkers, yet few biomarkers make it to practice. Before use in clinical decision making, predictive biomarkers need to demonstrate analytical validity, clinical validity and clinical utility. While attaining analytical and clinical validity is relatively straightforward, by following methodological recommendations, the achievement of clinical utility is extremely challenging. It requires demonstrating three associations: the biomarker with the outcome (prognostic association), the effect of treatment independent of the biomarker, and the differential treatment effect between the prognostic and the predictive biomarker (predictive association). In addition, economical, ethical, regulatory, organizational and patient/doctor-related aspects are hampering the translational process. Traditionally, these aspects do not receive much attention until formal approval or reimbursement of a biomarker test (informed by Health Technology Assessment (HTA)) is at stake, at which point the clinical utility and sometimes price of the test can hardly be influenced anymore. When HTA analyses are performed earlier, during biomarker research and development, they may prevent further development of those biomarkers unlikely to ever provide sufficient added value to society, and rather facilitate translation of the promising ones. Early HTA is particularly relevant for the predictive biomarker field, as expensive medicines are under pressure and the need for biomarkers to guide their appropriate use is huge. Closer interaction between clinical researchers and HTA experts throughout the translational research process will ensure that available data and methodologies will be used most efficiently to facilitate biomarker translation.

BRCA1-deficient breast cancers carry a specific DNA copy-number signature ("EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles asThe highest EZH2 expression was found in BRCA1-associated tumors harboring aOur findings demonstrate that EZH2 is expressed at significantly higher levels in

Background: The BRCA1-like copy number (CN) profile can be used as a biomarker to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break (DSB)-inducing chemotherapy. In addition, a BRCA1-like gene expression classifier, derived from the BRCA1-like CN profile, can predict which patient group will achieve an increased pathological complete remission rate on a standard neoadjuvant regimen complemented with carboplatin/veliparib. A non-myeloablative, dose-dense schedule of epirubicin, paclitaxel and cyclophosphamide (ETC) is used in the clinic to treat stage III patients. Since ETC contains an intensified dose of DNA DSB-inducing cyclophosphamide, we tested the BRCA1-like CN profile as a predictive biomarker for ETC benefit in the GAIN trial. Methods: The GAIN trial was a prospective, multi-center, non-blinded, randomized phase III trial. Eligibility comprised histologically confirmed invasive breast cancer with at least one positive axillary or internal mammary lymph node and no signs of distant metastases. The allocated adjuvant treatment was intensified chemotherapy with sequential E (150 mg/m2), T (225 mg/m2) and C (2500, after amendment 2000 mg/m2) each 3 cycles every 2 weeks (ETC) or concurrent E (112.5 mg/m2) and C (600 mg/m2) for 4 cycles every 2 weeks followed by 10 cycles of weekly T (67.5 mg/m2) combined with 4 cycles of capecitabine (2000mg/m2) on day 1-14 in a 3-weekly cycle (EC-TX). Only the triple negative patients were used for these analyses. For samples with good quality DNA extracted from formalin-fixed paraffin-embedded tumor tissue, a library was prepared and sequenced on an Illumina HiSeq2000 platform. Copy number estimates were extracted from the sequence data by normalizing GC-content and mappability corrected read counts to the average read count. These CN profiles were classified as either BRCA1-like or non-BRCA1-like using a previously established shrunken centroid classifier with an established cut-off. Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method, the difference in survival was analysed using log-rank tests. Multivariate analyses were done by generating Cox regression models with important prognostic factors. Results: Out of 424 triple negative patients, 166 patients with available tissue and a tumour cell content of at least 60% were analysed for BRCA1-like status and included in the analyses (classified as BRCA1-like: n=122). Based on clinicopathological characteristics, these patients were similar to the total group of triple negative participants. In the BRCA1-like patients, there was no significant difference in DFS or OS between ETC and EC-TX (log-rank tests n.s.). In accordance, Cox regression models confirmed these findings. Conclusion: In contrast to the predictive value of the BRCA1-like profile in myeloablative chemotherapy, it could not predict survival benefit for a non-myeloablative, non-platinum-/veliparib-containing regimen in this study population. An intensified dose of cyclophosphamide resulted in similar outcomes in BRCA1-like patients as addition of capecitabine to standard chemotherapy. These results help to define the appropriate application of the BRCA1-like profile as a predictive biomarker. Citation Format: van Rossum AGJ, Schouten PC, Weber KE, Nekljudova V, Denkert C, von Minckwitz G, Karn T, Möbus VJ, Linn SC, Loibl S, Marmé F. BRCA1-like profile as predictive biomarker in non myeloablative chemotherapy (GAIN study). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-28.

Purpose: The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics. Experimental Design: Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort. Results: Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001). Conclusions: HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.

Mutational load varies widely between malignancies and has been used as a proxy for the immunological foreignness of human cancers. However, without well-defined reference points it is difficult to determine which human tumors can be considered sufficiently foreign to the T-cell-based immune system. We established a neo-antigen prediction pipeline that processes single nucleotide variants, indels and gene fusion events and established its precision in identifying T-cell-recognized antigens. We used this pipeline to benchmark immunological foreignness of human cancers against pathogens for which T-cell control has been established. We demonstrate that up to 50% of tumors, spanning 25 sites of origin, are more foreign than these pathogen benchmarks. In addition, we demonstrate that the neo-antigen repertoire of treatment-naive tumors is not detectably influenced by immune editing. These data suggest that immunotherapeutic strategies that enhance activity of the endogenous T-cell compartment may be of value for a large fraction of human cancers.

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10–15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination‐mediated DNA repair and deficiency results in genomic instability. BRCA1‐mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1‐like or non‐BRCA1‐like. BRCA1 mutation, promoter methylation, BRCA1‐like status and genome‐wide expression data was determined for 112 TN breast cancer samples with long‐term follow‐up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1‐like and non‐BRCA1‐like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty‐five percent of tumors classified as BRCA1‐like. The functions of genes significantly up‐regulated in BRCA1‐like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1‐like (P, Highlights Triple negative breast cancers subdivide into 2 groups: BRCA1‐like, non‐BRCA1‐like.In a retrospective analysis, patients with BRCA1‐like tumors have a worse outcome.Mutation and gene expression patterns reveal potential therapy targets in subgroups.

Background Preclinical studies revealed that the combination of platinum compounds and olaparib is additive and possibly even synergistic in cell models with BRCA1 or -2 mutations. Early clinical trials suggested high benefit of olaparib with induction carboplatin in BRCA1 and -2 mutation carrier enriched populations. However, there is no evidence yet that carboplatin-olaparib has a superior benefit-risk compared to current standard therapy in advanced breast cancer in BRCA1 and -2 mutation carriers. Trial design We initiated a phase-I/II study due to an olaparib formulation change from capsule to tablet. During phase-I a traditional 3+3 dose escalation study is performed. Carboplatin will be dose escalated in 1 step from AUC 3 to AUC 4 with a constant olaparib dose of 25 mg BID. Olaparib is then dose escalated in 3 steps to 50, 75 and 100 mg BID until > 1/6 patients develop a DLT, the previous safe dose-level will be determined the MTD. After the MTD is established a randomised phase-II trial will be initiated where patients are randomised between standard capecitabine 1250 mg/m2 BID day 1-14, q day 22 or 2 cycles carboplatin-olaparib followed by olaparib monotherapy 300mg BID. After progression, patients in the experimental arm receive capecitabine, all other patients receive physicians choice of paclitaxel, vinorelbine or eribulin at standard dose. A compassionate use program with olaparib is available for patients in the standard arm after progression on second line treatment. Eligibility criteria In phase-II patients with histological or cytological proof of advanced BRCA1 or -2 mutated HER2 negative breast cancer are eligible if they are ≥18 years, have measureable disease according to RECIST 1.1 criteria, a WHO performance status of 0–2, a life expectancy ≥ 3 months and a negative pregnancy test. Pretreatment should contain an anthracycline and/or taxane in the (neo)adjuvant setting, unless not indicated. In the advanced setting only hormonal pre-treatment is allowed. Minimal laboratory values ANC ≥ 1.5 x 109 /L, Hb ≥ 6.2 mM (no transfusions in the last 28 days), platelet count ≥ 100 x 109 /L, serum bilirubin < 1.5 x ULN, ASAT and ALAT < 2.5 x ULN and a serum creatinine < 1.5 x ULN or creatinine clearance ≥ 50 mL/min. Aims In phase-1 we establish the MTD for treatment in phase-II where we study progression free survival on first line treatment(PFS1) compared with standard of care capecitabine. Statistical methods Toxicity analysis in phase-I can take place after all patients completed their 28 day DLT period. A total of 104 events in 110 patients on first line treatment need to be observed in phase-II to detect a clinical meaningful improvement in median PFS1 in the experimental arm from 4 to 7 months, assuming an accrual of 2 years and a follow-up of ≥6 months, providing a power of 80% (two-sided significance level of 5%). An interim analysis for futility and efficacy will be performed when 52 events have been observed. Accrual It is expected that 15-20 patients are needed in phase-I, inclusion is due around November 2015. Phase-II will be multicentre and is expected to start accrual December 2015. Citation Format: Dackus GMHE, Schouten PC, Geenen JJ, Marchetti S, Sonke GS, Linn SC, Schellens JHM. A phase I followed by a randomized phase II trial of two cycles carboplatin-olaparib followed by olaparib monotherapy versus capecitabine in BRCA-1 or -2 mutated Her2 negative advanced breast cancer as first line treatment (REVIVAL study). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-10.

Background: Patients with BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments.Methods: A diagnostic gene expression signature (BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher's exact test, and the relative performance between arms (biomarker × treatment interaction, likelihood ratio p Results: We developed a gene expression signature to identify BRCA1-like status. In the I-SPY 2 neoadjuvant setting the BRCA1ness signature associated significantly with response to V-C (p = 0.03), but not in the control arm (p = 0.45). We identified a significant interaction between BRCA1ness and V-C (p = 0.023) after correcting for HR.Conclusions: A genomic-based BRCA1-like signature was successfully translated to an expression-based signature (BRC1Aness). In the I-SPY 2 neoadjuvant setting, we determined that the BRCA1ness signature is capable of predicting benefit of V-C added to standard chemotherapy compared to standard chemotherapy alone.Trial registration: I-SPY 2 TRIAL beginning December 31, 2009: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2), NCT01042379.

In the version of this article originally published, there was an error in Fig. 3j. A label on the heatmap read "TGF-α signaling via NF-κB". It should have read "TNF-α signaling via NF-κB". The error has been corrected in the HTML and PDF versions of this article.

3118 Background: The PARP-inhibitor olaparib has single-agent activity in BRCA mutated breast and ovarian cancer. Preclinical studies show synergistic effects when combining PARP-inhibitors and platinum drugs in BRCA1/2 mutated cancer cell models. A formulation change from olaparib capsules to tablets initiated a new dose finding study of olaparib tablets BID continuously with carboplatin. Methods: Patients were included in a 3+3 dose-escalation schedule in the following dose-levels: olaparib 25mg BID and carboplatin AUC 3 d1/d22, olaparib 25mg BID and carboplatin AUC 4 d1/d22, olaparib 50mg BID and carboplatin AUC4 d1/d22, olaparib 75mg and carboplatin AUC 4 d1/d22 and olaparib 100mg BID and carboplatin AUC 4 d1/d22. After two cycles patients continued olaparib 300mg BID as monotherapy. Primary objective was to assess the Maximum Tolerable Dose (MTD). Secondary objectives were to investigate the preliminary response rate, pharmacodynamics and systemic exposure. Results: In total 24 patients were included with breast cancer (n = 18), ovarian cancer (n = 3), melanoma (n = 1), colorectal cancer (n = 1) and esophageal cancer (n = 1). Nineteen out of 24 patients had a germline BRCA mutation (79%). Most common AEs were nausea (46%), fatigue (33%) and platelet count decrease (33%). The majority of AEs (83%) were grade 1/2 in severity. Because two dose-limiting toxicities (consisting of ≥ 7 days dose delay of cycle 2 or missing ≥ 5 doses of olaparib due to hematologic toxicity) occurred in dose-level 4, dose-level 3 (olaparib 75mg and carboplatin AUC 4; n = 6 patients) was determined to be the MTD. Fourteen out of 24 patients (56%) had a partial response as best response, according to RECIST 1.1. Systemic exposure of the olaparib tablet formulation appeared comparable to the previous capsule formulation with an olaparib tablet AUC0-14 of 16.3 μg/ml*h at MTD. PARP activity in PBMCs was decreased by 98.7% ± 0.14% at day eight compared to day one for dose-level 3. Conclusions: Olaparib tablets 75mg BID and carboplatin AUC 4 for two cycles preceding olaparib monotherapy is a feasible and tolerable treatment schedule with encouraging clinical antitumor activity. Clinical trial information: NCT02418624.

The development of drug-biomarker combinations holds promise for tailoring treatment for patients with cancer on the basis of the biology of the tumor exemplified by estrogen receptor expression and blockade and human epidermal growth factor receptor 2 targeting. Approximately 85% of breast cancers have a target for therapy, but triple-negative breast cancers (TNBCs) do not. Although trials have focused on identifying the most efficacious anticancer drug (regimen) for this subtype, it may be that predictive biomarkers are required to guide treatment choices because of the intrinsic heterogeneity of TNBCs. Molecular characterizations have demonstrated a strong association between TNBCs and BRCA1 mutations. Preclinical mechanistic insight indicated that tumor cells with a defect in BRCA1 have impaired homologous recombination (HR), the only error-free pathway of repair of interstrand crosslinks. Cells with impaired HR display sensitivity to agents that induce such lesions. In the general breast cancer population, platinum agents that cause interstrand crosslinks are not preferred over other treatment regimens. However, given the possibility that TNBCs are enriched for such a targetable HR deficiency (HRD), platinum has attracted renewed interest as has been shown in recently published articles. Furthermore, TNBCs may be enriched for BRCA1 mutations and also for a larger group of nonmutated tumors that exhibit HRD. The use of platinum in TNBCs with biomarker analyses has been investigated in the two articles that accompany this editorial. Telli et al neoadjuvantly treated patients with triple-negative or BRCA1/ 2-associated breast cancer with a regimen containing gemcitabine, carboplatin, and iniparib in the PrECOG 0105 trial. Overall, a pathologic complete remission rate of 36% and acceptable toxicity of the regimen were reported. Isakoff et al conducted a phase II trial with cisplatin or carboplatin in patients with metastatic TNBC (TBCRC009; Platinum for Triple-Negative Metastatic Breast Cancer and Evaluation of p63/p73 as a Biomarker of Response). The overall response rate was 25%. Both studies reproduce previous findings of efficacy of a platinum-based regimen or platinum alone in TNBC. However, only a subset of patients in these trials derived clinical benefit. Therefore, secondary investigations may help identify biomarkers that associate with a preferential benefit from use of a platinum regimen, which may help guide therapy decisions. Both studies report their analysis of BRCA germline mutation status as well as potential biomarkers for assessing HRD, specifically the HRD loss of heterozygosity (HRD-LOH) score and the HRD large-scale transition (HRD-LST) score. Both markers evaluate so-called genomic scars as signatures of aberrations that can be observed from singlenucleotide polymorphism array–based sequencing or similar technologies and that are associated with defects in error-free repair of interstrand crosslinks. The HRD-LOH score counts the numbers of LOH regions larger than 15 MB but smaller than chromosome size, whereas the HRD-LST score counts the number of breaks between adjacent segments of at least 10 MB. Cutoffs were trained to best separate tumors that had a known deleterious aberration in BRCA1/2 from those that did not. In both studies, patients with a BRCA germline mutation had higher response rates than the general cohort, and the HRD scores highly correlated with BRCA1/2 germline mutations and response rates. In addition, Telli et al identified BRCA1 methylation in nonmutated tumors with high HRD-LOH score, which suggests that these scores identify a subgroup of patients who could benefit from therapy that targets their HRD. Unfortunately, single-arm phase II studies do not allow proper evaluation of a biomarker as a predictor of response to therapy, and they offer limited insight on how to exploit the observed association with outcome of BRCA-associated biomarkers (Fig 1). Thus, the studies by Telli et al and Isakoff et al join several other articles published over the last decade that have reported on the potential promise of BRCA-associated HRD biomarkers but did not provide definitive data on clinical utility. It has been established that the optimal biomarker trial design requires biomarker-positive and -negative patients and patients who have been treated with and without the therapy of interest to evaluate both prognostic and predictive signals. If not included in the phase II design, such a control group could be added. Hence, the lack of a control arm in these two new studies is indeed a major limitation of their findings. In contrast, the TNT trial (Triple Negative Breast Cancer Trial, recently reported as an abstract) was able to more appropriately evaluate the use of HRD biomarkers, at least in patients with advanced disease. In that trial, patients with locally advanced or metastatic TNBC were randomly assigned to therapy with carboplatin at area under the [concentration-time] curve 6 or standardof-care docetaxel. Of great interest, among the 376 patients in that study, 29 were known to be BRCA1 or BRCA2 mutation carriers (including 13 who had estrogen receptor–positive, HER2-negative disease). Prespecified analyses specifically addressed populations JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 33 NUMBER 17 JUNE 1

Specific clinical questions rise when patients, who are diagnosed with breast cancer, are at risk of carrying a mutation in BRCA1 and -2 gene due to a strong family history or young age at diagnosis. These questions concern topics such as 1. Timing of genetic counseling and testing, 2. Choices to be made for BRCA1 or -2 mutation carriers in local treatment, contralateral treatment, (neo)adjuvant systemic therapy, and 3. The psychological effects of rapid testing. The knowledge of the genetic status might have several advantages for the patient in treatment planning, such as the choice whether or not to undergo mastectomy and/or prophylactic contralateral mastectomy. The increased risk of developing a second breast cancer in the ipsilateral breast in mutation carriers, is only slightly higher after primary cancer treatment, than in the general population. Prophylactic contralateral mastectomy provides a substantial reduction of contralateral breast cancer, although only a small breast cancer specific survival benefit. Patients should be enrolled in clinical trials to investigate (neo)-adjuvant drug regimens, that based on preclinical and early clinical evidence might be targeting the homologous recombination defect, such as platinum compounds and PARP inhibitors. If rapid testing is performed, the patient can make a well-balanced decision. Although rapid genetic counseling and testing might cause some distress, most women reported this approach to be worthwhile. In this review the literature regarding these topics is evaluated. Answers and suggestions, useful in clinical practice are discussed.