Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer.

BACKGROUND: Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS: Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS: BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS: These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Assessment of structural chromosomal instability phenotypes as biomarkers of carboplatin response in triple negative breast cancer: the TNT trial.

BACKGROUND: In the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel. PATIENTS AND METHODS: Copy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS). RESULTS: Patients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm. CONCLUSIONS: Patients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting.

A decade of clinical development of PARP inhibitors in perspective.

Genomic instability is a hallmark of cancer, and often is the result of altered DNA repair capacities in tumour cells. DNA damage repair defects are common in different cancer types; these alterations can also induce tumour-specific vulnerabilities that can be exploited therapeutically. In 2009, a first-in-man clinical trial of the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib clinically validated the synthetic lethal interaction between inhibition of PARP1, a key sensor of DNA damage, and BRCA1/BRCA2 deficiency. In this review, we summarize a decade of PARP inhibitor clinical development, a work that has resulted in the registration of several PARP inhibitors in breast (olaparib and talazoparib) and ovarian cancer (olaparib, niraparib and rucaparib, either alone or following platinum chemotherapy as maintenance therapy). Over the past 10 years, our knowledge on the mechanism of action of PARP inhibitor as well as how tumours become resistant has been extended, and we summarise this work here. We also discuss opportunities for expanding the precision medicine approach with PARP inhibitors, identifying a wider population who could benefit from this drug class. This includes developing and validating better predictive biomarkers for patient stratification, mainly based on homologous recombination defects beyond BRCA1/BRCA2 mutations, identifying DNA repair deficient tumours in other cancer types such as prostate or pancreatic cancer, or by designing combination therapies with PARP inhibitors.

De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen International Expert Consensus Conference on the Primary Therapy of Early Breast Cancer 2017.

The 15th St. Gallen International Breast Cancer Conference 2017 in Vienna, Austria reviewed substantial new evidence on loco-regional and systemic therapies for early breast cancer. Treatments were assessed in light of their intensity, duration and side-effects, seeking where appropriate to escalate or de-escalate therapies based on likely benefits as predicted by tumor stage and tumor biology. The Panel favored several interventions that may reduce surgical morbidity, including acceptance of 2 mm margins for DCIS, the resection of residual cancer (but not baseline extent of cancer) in women undergoing neoadjuvant therapy, acceptance of sentinel node biopsy following neoadjuvant treatment of many patients, and the preference for neoadjuvant therapy in HER2 positive and triple-negative, stage II and III breast cancer. The Panel favored escalating radiation therapy with regional nodal irradiation in high-risk patients, while encouraging omission of boost in low-risk patients. The Panel endorsed gene expression signatures that permit avoidance of chemotherapy in many patients with ER positive breast cancer. For women with higher risk tumors, the Panel escalated recommendations for adjuvant endocrine treatment to include ovarian suppression in premenopausal women, and extended therapy for postmenopausal women. However, low-risk patients can avoid these treatments. Finally, the Panel recommended bisphosphonate use in postmenopausal women to prevent breast cancer recurrence. The Panel recognized that recommendations are not intended for all patients, but rather to address the clinical needs of the majority of common presentations. Individualization of adjuvant therapy means adjusting to the tumor characteristics, patient comorbidities and preferences, and managing constraints of treatment cost and access that may affect care in both the developed and developing world.

Assessment of microtubule-associated protein (MAP)-Tau expression as a predictive and prognostic marker in TACT; a trial assessing substitution of sequential docetaxel for FEC as adjuvant chemotherapy for early breast cancer.

The TACT trial is the largest study assessing the benefit of taxanes as part of adjuvant therapy for early breast cancer. The goal of this translational study was to clarify the predictive and prognostic value of Tau within the TACT trial. Tissue microarrays (TMA) were available from 3,610 patients. ER, PR, HER2 from the TACT trial and Tau protein expression was determined by immunohistochemistry on duplicate TMAs. Two parallel scoring systems were generated for Tau expression ('dichotomised' vs. 'combined' score). The positivity rate of Tau expression was 50 % in the trial population (n = 2,483). Tau expression correlated positively with ER (p < 0.001) and PR status (p < 0.001); but negatively with histological grade (p < 0.001) and HER2 status (p < 0.001). Analyses with either scoring systems for Tau expression demonstrated no significant interaction between Tau expression and efficacy of docetaxel. Contrary to the hypothesis that taxane benefit would be enriched in Tau negative/low patients, the only groups with a suggestion of a reduced event rate in the taxane group were the HER2-positive, Tau positive subgroups. Tau expression was seen to be a prognostic factor on univariate analysis associated with an improved DFS, independent of the treatment group (p < 0.001). It had no prognostic value in ER-negative tumours and the weak prognostic effect of Tau in ER-positive tumours (p = 0.02) diminished, when considering ER as an ordinal variable. On multivariable analyses, Tau had no prognostic value in either group. In addition, no significant interaction between Tau expression and benefit from docetaxel in patients within the PR-positive and negative subsets was seen. This is now the second large adjuvant study, and the first with quantitative analysis of ER and Tau expression, failing to show an association between Tau and taxane benefit with limited utility as a prognostic marker for Tau in ER-positive early breast cancer patients.

Patterns of metastasis in women with metachronous contralateral breast cancer.

BACKGROUND: The understanding of metastatic patterns after metachronous contralateral breast cancer (CBC) may help determine the biological nature of CBC. METHODS: A cohort of 8478 women with breast cancer treated at Guy's and St Thomas' NHS Foundation Trust between 1975 and 2006 were studied. Organ-specific 5-year cumulative incidence and incidence rate ratios were assessed for women diagnosed with unilateral breast cancer (UBC), CBC within 5 years and CBC more than 5 years of the initial diagnosis. RESULTS: Women diagnosed with CBC within 5 years had a higher incidence of metastases in all organs compared with UBC. Women with a short interval time to CBC developed metastasis more rapidly and were more likely to develop visceral and distant cutaneous metastases compared with bone metastasis. CONCLUSION: These findings explain poor prognosis of women with early occurring CBC and suggest that some of these CBCs are indicators of aggressive and/or systemic disease.

BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years.

BACKGROUND: Triple-negative (TN) tumours are the predominant breast cancer subtype in BRCA1 mutation carriers. Recently, it was proposed that all individuals below 50 years of age with TN breast cancer should be offered BRCA testing. We have evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer. METHODS: We undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history. RESULTS: BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England. CONCLUSION: Women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services.

CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help.

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40- lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.

The export of immunoglobulin D by human neoplastic B lymphocytes.

An investigation has been made into the ability of human neoplastic B lymphocytes expressing surface IgM and IgD to export IgD in culture. Cells that expressed surface Ig of the lambda light chain type frequently exported IgD (10/12 patients), whereas cells expressing surface Ig of the kappa light chain type exported no IgD, although most (8/11 patients) were able to export IgM. It appears, therefore, that in most of the 23 cases studied, cells synthesizing IgD with lambda light chains can both express and export IgD, whereas those synthesizing IgD kappa can only insert it into the surface membrane. This finding and the known preponderance of lambda in plasma IgD imply that the possession of a lambda chain facilitates the IgD secretory pathway, a conclusion that implicates a control mechanism subsequent to the surface/secretory dichotomy arising from different splicings of heavy chain messenger RNA.

Extracellular idiotypic immunoglobulin arising from human leukemic B lymphocytes.

The peripheral blood lymphocytes of nine out of nine patients with typical surface Ig-positive chronic lymphocytic leukemia but no paraprotein visible on serum electrophoresis have been shown by radioimmunoassay to export small amounts of pentameric IgM during culture (in the range of 2.4-7.2 ng/10(7) cells per h); three out of nine also exported monomeric IgD (0.7-1.4 ng/10(7) cells per h). Immunoglobulin turned over on the cell surface did not appear to contribute to material in the culture fluid, except possibly as vesicle-bound Ig. In three cases, which included two of the IgD producers, anti-idiotypic antibody raised against the cell surface Fab mu was used to demonstrate the idiotypic nature of the exported Ig. Anti-idiotypic antibody was also used to measure levels of idiotypic Ig in the sera of these three patients as a proportion of the total Ig. Total serum IgM was depressed in all three patients, and the idiotypic IgM represented 43%, 65%, and 96% of the IgM. The findings suggest that in typical chronic lymphocytic leukemia involving B lymphocytes, the export of a small amount of idiotypic Ig by the neoplastic cells in a common or even usual occurrence.

BRCA1 dysfunction in sporadic basal-like breast cancer.

Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age- and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P=0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P=0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (P<0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (P<0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer.

Triple negative tumours: a critical review.

Breast cancer is a heterogeneous disease that encompasses several distinct entities with remarkably different biological characteristics and clinical behaviour. Currently, breast cancer patients are managed according to algorithms based on a constellation of clinical and histopathological parameters in conjunction with assessment of hormone receptor (oestrogen and progesterone receptor) status and HER2 overexpression/gene amplification. Although effective tailored therapies have been developed for patients with hormone receptor-positive or HER2+ disease, chemotherapy is the only modality of systemic therapy for patients with breast cancers lacking the expression of these markers (triple-negative cancers). Recent microarray expression profiling analyses have demonstrated that breast cancers can be systematically characterized into biologically and clinically meaningful groups. These studies have led to the re-discovery of basal-like breast cancers, which preferentially show a triple-negative phenotype. Both triple-negative and basal-like cancers preferentially affect young and African-American women, are of high histological grade and have more aggressive clinical behaviour. Furthermore, a significant overlap between the biological and clinical characteristics of sporadic triple-negative and basal-like cancers and breast carcinomas arising in BRCA1 mutation carriers has been repeatedly demonstrated. In this review, we critically address the characteristics of basal-like and triple-negative cancers, their similarities and differences, their response to chemotherapy as well as strategies for the development of novel therapeutic targets for these aggressive types of breast cancer. In addition, the possible mechanisms are discussed leading to BRCA1 pathway dysfunction in sporadic triple-negative and basal-like cancers and animal models for these tumour types.

Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification.

Women heterozygous for mutations in the breast-cancer susceptibility genes BRCA1 and BRCA2 have a highly elevated risk of developing breast cancer [1]. BRCA1 and BRCA2 encode large proteins with no sequence similarity to one another. Although involvement in DNA repair and transcription has been suggested, it is still not understood how loss of function of these genes leads to breast cancer [2]. Embryonic fibroblasts (MEFs) derived from mice homozygous for a hypomorphic mutation (Brca2(Tr2014)) within the 3' region of exon 11 in Brca2 [3], or a similar mutation (Brca2(Tr)) [4], proliferate poorly in culture and overexpress the tumour suppressor p53 and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These MEFs have intact p53-dependent DNA damage G(1)-S [3] [4] and G(2)-M checkpoints [4], but are impaired in DNA double-strand break repair [3] and develop chromosome aberrations [4]. Here, we report that Brca2(Tr2014/Tr2014) MEFs frequently develop micronuclei. These abnormal DNA-containing bodies were formed through both loss of acentric chromosome fragments and by chromosome missegregation, which resulted in aneuploidy. Absence of Brca2 also led to centrosome amplification, which we found associated with the formation of micronuclei. These data suggest a potential mechanism whereby loss of BRCA2 may, within subclones, drive the loss of cell-cycle regulation genes, enabling proliferation and tumourigenesis.

Radiobiology of breast cancer.

Advances in molecular and cellular biology are transforming our understanding of breast cancer and promise the same for radiotherapy over the next few years. At the clinical level, the molecular basis of fractionation dependency and other tumour and normal tissue responses are likely to become clearer. More importantly, they will become useful in the clinic, where molecular characterisation of the patient and tumour will start to determine therapeutic options. Although many of the fundamental processes are only amenable to study in laboratory systems, the power of array-based technologies makes it possibly to address highly relevant questions in the clinic, using functional imaging and/or tissue biopsies. To help clinical oncologists exploit these opportunities in translational research, some aspects of the molecular and cellular basis of radiotherapy are described below in their relation to breast cancer.