Genomic characterisation of hormone receptor-positive breast cancer arising in very young women.

BACKGROUND: Very young premenopausal women diagnosed with hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+HER2-) early breast cancer (EBC) have higher rates of recurrence and death for reasons that remain largely unexplained. PATIENTS AND METHODS: Genomic sequencing was applied to HR+HER2- tumours from patients enrolled in the Suppression of Ovarian Function Trial (SOFT) to determine genomic drivers that are enriched in young premenopausal women. Genomic alterations were characterised using next-generation sequencing from a subset of 1276 patients (deep targeted sequencing, n = 1258; whole-exome sequencing in a young-age, case-control subsample, n = 82). We defined copy number (CN) subgroups and assessed for features suggestive of homologous recombination deficiency (HRD). Genomic alteration frequencies were compared between young premenopausal women (<40 years) and older premenopausal women (≥40 years), and assessed for associations with distant recurrence-free interval (DRFI) and overall survival (OS). RESULTS: Younger women (<40 years, n = 359) compared with older women (≥40 years, n = 917) had significantly higher frequencies of mutations in GATA3 (19% versus 16%) and CN amplifications (CNAs) (47% versus 26%), but significantly lower frequencies of mutations in PIK3CA (32% versus 47%), CDH1 (3% versus 9%), and MAP3K1 (7% versus 12%). Additionally, they had significantly higher frequencies of features suggestive of HRD (27% versus 21%) and a higher proportion of PIK3CA mutations with concurrent CNAs (23% versus 11%). Genomic features suggestive of HRD, PIK3CA mutations with CNAs, and CNAs were associated with significantly worse DRFI and OS compared with those without these features. These poor prognostic features were enriched in younger patients: present in 72% of patients aged <35 years, 54% aged 35-39 years, and 40% aged ≥40 years. Poor prognostic features [n = 584 (46%)] versus none [n = 692 (54%)] had an 8-year DRFI of 84% versus 94% and OS of 88% versus 96%. Younger women (<40 years) had the poorest outcomes: 8-year DRFI 74% versus 85% and OS 80% versus 93%, respectively. CONCLUSION: These results provide insights into genomic alterations that are enriched in young women with HR+HER2- EBC, provide rationale for genomic subgrouping, and highlight priority molecular targets for future clinical trials.

Basal expression of RAD51 foci predicts olaparib response in patient-derived ovarian cancer xenografts.

BACKGROUND: The search for biomarkers to evaluate ovarian cancer (OC) homologous recombination (HR) function and predict the response to therapy is an urgent clinical need to improve the selection of patients who could benefit from platinum- and olaparib (poly-ADP ribose polymerase inhibitors, PARPi)-based therapies. METHODS: We used a large collection of OC patient-derived xenografts (PDXs) (n = 47) and evaluated their HR status based on BRCA1/2 mutations, BRCA1 promoter methylation and the HRDetect score. RAD51 foci were quantified in formalin-fixed, paraffin-embedded untreated tumour specimens by immunofluorescence and the messenger RNA expression of 21 DNA repair genes by real-time PCR. RESULTS: Tumour HR deficiency predicted both platinum and olaparib responses. The basal level of RAD51 foci evaluated in geminin-positive/replicating cells strongly inversely correlated with olaparib response (p = 0.011); in particular, the lower the foci score, the greater the sensitivity to olaparib, while low RAD51 foci score seems to associate with platinum activity. CONCLUSIONS: The basal RAD51 foci score is a candidate predictive biomarker of olaparib response in OC patients as it can be easily translatable in a clinical setting. Moreover, the findings corroborate the importance of OC-PDXs as a reliable tool to identify and validate biomarkers of response to therapy.

ESMO recommendations on predictive biomarker testing for homologous recombination deficiency and PARP inhibitor benefit in ovarian cancer.

BACKGROUND: Homologous recombination repair deficiency (HRD) is a frequent feature of high-grade serous ovarian, fallopian tube and peritoneal carcinoma (HGSC) and is associated with sensitivity to PARP inhibitor (PARPi) therapy. HRD testing provides an opportunity to optimise PARPi use in HGSC but methodologies are diverse and clinical application remains controversial. MATERIALS AND METHODS: To define best practice for HRD testing in HGSC the ESMO Translational Research and Precision Medicine Working Group launched a collaborative project that incorporated a systematic review approach. The main aims were to (i) define the term 'HRD test'; (ii) provide an overview of the biological rationale and the level of evidence supporting currently available HRD tests; (iii) provide recommendations on the clinical utility of HRD tests in clinical management of HGSC. RESULTS: A broad range of repair genes, genomic scars, mutational signatures and functional assays are associated with a history of HRD. Currently, the clinical validity of HRD tests in ovarian cancer is best assessed, not in terms of biological HRD status per se, but in terms of PARPi benefit. Clinical trials evidence supports the use of BRCA mutation testing and two commercially available assays that also incorporate genomic instability for identifying subgroups of HGSCs that derive different magnitudes of benefit from PARPi therapy, albeit with some variation by clinical scenario. These tests can be used to inform treatment selection and scheduling but their use is limited by a failure to consistently identify a subgroup of patients who derive no benefit from PARPis in most studies. Existing tests lack negative predictive value and inadequately address the complex and dynamic nature of the HRD phenotype. CONCLUSIONS: Currently available HRD tests are useful for predicting likely magnitude of benefit from PARPis but better biomarkers are urgently needed to better identify current homologous recombination proficiency status and stratify HGSC management.

Whole-genome sequencing reveals clinically relevant insights into the aetiology of familial breast cancers.

BACKGROUND: Whole-genome sequencing (WGS) is a powerful method for revealing the diversity and complexity of the somatic mutation burden of tumours. Here, we investigated the utility of tumour and matched germline WGS for understanding aetiology and treatment opportunities for high-risk individuals with familial breast cancer. PATIENTS AND METHODS: We carried out WGS on 78 paired germline and tumour DNA samples from individuals carrying pathogenic variants in BRCA1 (n = 26) or BRCA2 (n = 22) or from non-carriers (non-BRCA1/2; n = 30). RESULTS: Matched germline/tumour WGS and somatic mutational signature analysis revealed patients with unreported, dual pathogenic germline variants in cancer risk genes (BRCA1/BRCA2; BRCA1/MUTYH). The strategy identified that 100% of tumours from BRCA1 carriers and 91% of tumours from BRCA2 carriers exhibited biallelic inactivation of the respective gene, together with somatic mutational signatures suggestive of a functional deficiency in homologous recombination. A set of non-BRCA1/2 tumours also had somatic signatures indicative of BRCA-deficiency, including tumours with BRCA1 promoter methylation, and tumours from carriers of a PALB2 pathogenic germline variant and a BRCA2 variant of uncertain significance. A subset of 13 non-BRCA1/2 tumours from early onset cases were BRCA-proficient, yet displayed complex clustered structural rearrangements associated with the amplification of oncogenes and pathogenic germline variants in TP53, ATM and CHEK2. CONCLUSIONS: Our study highlights the role that WGS of matched germline/tumour DNA and the somatic mutational signatures can play in the discovery of pathogenic germline variants and for providing supporting evidence for variant pathogenicity. WGS-derived signatures were more robust than germline status and other genomic predictors of homologous recombination deficiency, thus impacting the selection of platinum-based or PARP inhibitor therapy. In this first examination of non-BRCA1/2 tumours by WGS, we illustrate the considerable heterogeneity of these tumour genomes and highlight that complex genomic rearrangements may drive tumourigenesis in a subset of cases.

Mutational mechanisms of amplifications revealed by analysis of clustered rearrangements in breast cancers.

Background: Complex clusters of rearrangements are a challenge in interpretation of cancer genomes. Some clusters of rearrangements demarcate clear amplifications of driver oncogenes but others are less well understood. A detailed analysis of rearrangements within these complex clusters could reveal new insights into selection and underlying mutational mechanisms. Patients and methods: Here, we systematically investigate rearrangements that are densely clustered in individual tumours in a cohort of 560 breast cancers. Applying an agnostic approach, we identify 21 hotspots where clustered rearrangements recur across cancers. Results: Some hotspots coincide with known oncogene loci including CCND1, ERBB2, ZNF217, chr8:ZNF703/FGFR1, IGF1R, and MYC. Others contain cancer genes not typically associated with breast cancer: MCL1, PTP4A1, and MYB. Intriguingly, we identify clustered rearrangements that physically connect distant hotspots. In particular, we observe simultaneous amplification of chr8:ZNF703/FGFR1 and chr11:CCND1 where deep analysis reveals that a chr8-chr11 translocation is likely to be an early, critical, initiating event. Conclusions: We present an overview of complex rearrangements in breast cancer, highlighting a potential new way for detecting drivers and revealing novel mechanistic insights into the formation of two common amplicons.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Arterial tortuosity syndrome: clinical and molecular findings in 12 newly identified families.

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive connective tissue disease, characterized by widespread arterial involvement with elongation, tortuosity, and aneurysms of the large and middle-sized arteries. Recently, SLC2A10 mutations were identified in this condition. This gene encodes the glucose transporter GLUT10 and was previously suggested as a candidate gene for diabetes mellitus type 2. A total of 12 newly identified ATS families with 16 affected individuals were clinically and molecularly characterized. In addition, extensive cardiovascular imaging and glucose tolerance tests were performed in both patients and heterozygous carriers. All 16 patients harbor biallelic SLC2A10 mutations of which nine are novel (six missense, three truncating mutations, including a large deletion). Haplotype analysis suggests founder effects for all five recurrent mutations. Remarkably, patients were significantly older than those previously reported in the literature (P=0.04). Only one affected relative died, most likely of an unrelated cause. Although the natural history of ATS in this series was less severe than previously reported, it does indicate a risk for ischemic events. Two patients initially presented with stroke, respectively at age 8 months and 23 years. Tortuosity of the aorta or large arteries was invariably present. Two adult probands (aged 23 and 35 years) had aortic root dilation, seven patients had localized arterial stenoses, and five had long stenotic stretches of the aorta. Heterozygous carriers did not show any vascular anomalies. Glucose metabolism was normal in six patients and eight heterozygous individuals of five families. As such, overt diabetes is not related to SLC2A10 mutations associated with ATS.

Cardiac glycosides inhibit detubulation in amphibian skeletal muscle fibres exposed to osmotic shock.

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.