RAD51 as a biomarker for homologous recombination deficiency in high-grade serous ovarian carcinoma: robustness and interobserver variability of the RAD51 test

The RAD51 test is emerging as a promising biomarker for the assessment of functional homologous recombination deficiency (HRD). Yet, the robustness and reproducibility of the immunofluorescence-based RAD51 test, in different academic laboratories, have not been systematically investigated. Therefore, we tested the performance of the RAD51 assay in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples in four European laboratories. Here, we confirm that subtle differences in staining procedures result in low variability of RAD51 and γH2AX scores. However, substantial variability in RAD51 scoring was observed in some samples, likely due to complicating technical and biological features, such as high RAD51 signal-to-noise ratio and RAD51 heterogeneity. These results support the need to identify and perform additional quality control steps and/or automating image analysis. Altogether, resolving technical issues should be a priority, as identifying tumours with functional HRD is urgently needed to guide the individual treatment of HGSOC patients. Follow-up studies are needed to define the key tissue quality requirements to assess HRD by RAD51 in FFPE tumour samples, as this test could help in guiding the individual treatment of HGSOC patients. MTG8 - Moleculaire pathologie van gynecologische tumorenMolecular tumour pathology - and tumour genetic

Clinical consequences of BRCA2 hypomorphism

Altres ajuts: Asociación Española contra el Cáncer (LABAE16020PORTT)Altres ajuts: Asociación Española contra el Cáncer (ERAPERMED2019-215)The tumor suppressor FANCD1/BRCA2 is crucial for DNA homologous recombination repair (HRR). BRCA2 biallelic pathogenic variants result in a severe form of Fanconi anemia (FA) syndrome, whereas monoallelic pathogenic variants cause mainly hereditary breast and ovarian cancer predisposition. For decades, the co-occurrence in trans with a clearly pathogenic variant led to assume that the other allele was benign. However, here we show a patient with biallelic BRCA2 (c.1813dup and c.7796 A > G) diagnosed at age 33 with FA after a hypertoxic reaction to chemotherapy during breast cancer treatment. After DNA damage, patient cells displayed intermediate chromosome fragility, reduced survival, cell cycle defects, and significantly decreased RAD51 foci formation. With a newly developed cell-based flow cytometric assay, we measured single BRCA2 allele contributions to HRR, and found that expression of the missense allele in a BRCA2 KO cellular background partially recovered HRR activity. Our data suggest that a hypomorphic BRCA2 allele retaining 37-54% of normal HRR function can prevent FA clinical phenotype, but not the early onset of breast cancer and severe hypersensitivity to chemotherapy

Homologous Recombination Deficiency Across Subtypes of Primary Breast Cancer

Purpose - Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. Methods - HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. Results - HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P Conclusion - The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD

Τι είναι η πατρίδα μας; [What is our fatherland?]

Ποια είναι η πατρίδα κάποιων; Ας δούμε το θέμα λίγο σαν πρόβλημα της φιλοσοφίας της γλώσσας: για ένα κατηγόρημα «Άγγλος» «Γάλλος», «Πορτογάλος», «Βέλγος», «Φλαμανδός», πώς να αποφασίσουμε ποια αντικείμενα (ποιοι άνθρωποι) εμπίπτουν σε ποιο κατηγόρημα; Έχουν τελικά νόημα αυτά τα κατηγορήματα; Η χρήση και κατάχρηση αυτών των κατηγορημάτων έχει αποτελέσει μια από τις κυριότερες πηγές δυστυχίας στη διάρκεια των δύο τελευταίων αιώνων και συνεχίζει να είναι στην ημερήσια διάταξη. Συνήθως εμφανίζεται στα πλαίσια αγώνων για την απόκτηση «ελευθερίας» σε αντιπαράθεση με την «καταπίεση», και σε παγκόσμιο επίπεδο και κοντύτερα μας: στη Μακεδονία, την Καταλονία, την Ιρλανδία, το Κουρδιστάν ... Νομίζω πώς αρκετά από αυτά βασίζονται σε λάθη

Olaparib and ceralasertib (AZD6738) in patients with triple-negative advanced breast cancer: Results from Cohort E of the plasmaMATCH trial (CRUK/15/010)

Purpose: Approximately 10% to 15% of triple-negative breast cancers (TNBC) have deleterious mutations in BRCA1 and BRCA2 and may benefit from PARP inhibitor treatment. PARP inhibitors may also increase exogenous replication stress and thereby increase sensitivity to inhibitors of ataxia telangiectasia and Rad3-related (ATR) protein. This phase II study examined the activity of the combination of PARP inhibitor, olaparib, and ATR inhibitor, ceralasertib (AZD6738), in patients with advanced TNBC. Patients and Methods: Patients with TNBC on most recent biopsy who had received 1 or 2 lines of chemotherapy for advanced disease or had relapsed within 12 months of (neo)adjuvant chemotherapy were eligible. Treatment was olaparib 300 mg twice a day continuously and celarasertib 160 mg on days 1–7 on a 28-day cycle until disease progression. The primary endpoint was confirmed objective response rate (ORR). Tissue and plasma biomarker analyses were preplanned to identify predictors of response. Results: 70 evaluable patients were enrolled. Germline BRCA1/2 mutations were present in 10 (14%) patients and 3 (4%) patients had somatic BRCA mutations. The confirmed ORR was 12/70; 17.1% (95% confidence interval, 10.4–25.5). Responses were observed in patients without germline or somatic BRCA1/2 mutations, including patients with mutations in other homologous recombination repair genes and tumors with functional homologous recombination deficiency by RAD51 foci. Conclusions: The response rate to olaparib and ceralasertib did not meet prespecified criteria for activity in the overall evaluable population, but responses were observed in patients who would not be expected to respond to olaparib monotherapy

Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics

© 2018 The Author(s). Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics

Chronic Allergic Inflammation Causes Vascular Remodeling and Pulmonary Hypertension in Bmpr2 Hypomorph and Wild-Type Mice

Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

BACKGROUND: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. PATIENTS AND METHODS: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. RESULTS: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. CONCLUSION: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors