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Abstract PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene’s reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

Abstract Background MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs ( T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR. Conclusions Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.

Epigenetic alterations, including aberrant DNA methylation, are now recognized as bone fide hallmarks of cancer, which can contribute to cancer initiation, progression, therapy responses and therapy resistance. Methylation of gene promoters can have a range of impacts on cancer risk, clinical stratification and therapeutic outcomes. We provide several important examples of genes, which can be silenced or activated by promoter methylation and highlight their clinical implications. These include the mismatch DNA repair genes MLH1 and MSH2 , homologous recombination DNA repair genes BRCA1 and RAD51C , the TERT oncogene and genes within the P15/P16/RB1/E2F tumour suppressor axis. We also discuss how these methylation changes might occur in the first place – whether in the context of the CpG island methylator phenotype or constitutional DNA methylation. The choice of assay used to measure methylation can have a significant impact on interpretation of methylation states, and some examples where this can influence clinical decision-making are presented. Aberrant DNA methylation patterns in circulating tumour DNA (ctDNA) are also showing great promise in the context of non-invasive cancer detection and monitoring using liquid biopsies; however, caution must be taken in interpreting these results in cases where constitutional methylation may be present. Thus, this review aims to provide researchers and clinicians with a comprehensive summary of this broad, but important subject, illustrating the potentials and pitfalls of assessing aberrant DNA methylation in cancer.

Abstract Background Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs. Conclusions Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 ( BRCA1/2) , four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p

The detection of methylated templates in cell-free DNA (cfDNA) is increasingly recognized as a valuable, non-invasive tool for diagnosis, monitoring and prognostication in a range of medical contexts. The importance of controlling pre-analytical conditions in laboratory workflows prior to cfDNA quantification is well-established. Significant variations in the recovery of DNA following processes such as cfDNA extraction and sodium bisulphite modification may confound downstream analysis, particularly when accurate quantification of templates is required. Given the wealth of potential applications for this emerging molecular technology, attention has turned to the requirement to recognize and minimize pre-analytical variables prior to cfDNA methylation analysis. We recently described the development of an approach using an exogenous DNA construct to evaluate the recovery efficiency of cfDNA following the extraction and bisulphite modification steps (CEREBIS). Here, we report our experience in the practical application of this technique in 107 consecutive patient plasma samples submitted for quantitative cfDNA methylation analysis. The mean recovery of cfDNA (as estimated using cerebis), following extraction and bisulphite modification, was 37% ± 7%. Nine (8.4%) of the 107 samples were found to be outside of control limits, where the recovery of cerebis indicated significant differences in the efficiency of the pre-analytical processing of these samples. Recognition of these out-of-control samples precluded subsequent molecular analysis. Implementation of data-driven quality control measures, such as the one described, has the potential to improve the quality of liquid biopsy methylation analysis, interpretation and reporting.

Background: Graft-derived cell-free DNA (gdcfDNA) analysis has shown promise as a non-invasive tool for monitoring organ health following solid organ transplantation. A number of gdcfDNA analysis techniques have been described; however, the majority rely on sequencing or prior genotyping to detect donor-recipient mis-matched genetic polymorphisms. Differentially methylated regions of DNA can be used to identify the tissue-of-origin of cell-free DNA (cfDNA) fragments. In this study, we aimed to directly compare the performance of gdcfDNA monitoring using graft-specific DNA methylation analysis and donor-recipient genotyping techniques in a pilot cohort of clinical samples from patients post-liver transplantation. Results: 7 patients were recruited prior to LT, 3 developed early, biopsy-proven TCMR in the first 6 weeks post-LT. gdcfDNA was successfully quantified in all samples using both approaches. There was a high level of technical correlation between results using the two techniques (Spearman testing, rs = 0.87, p < 0.0001). gdcfDNA levels quantified using the genotyping approach were significantly greater across all timepoints in comparison to the tissue-specific DNA methylation-based approach: e.g., day 1 post-LT median 31,350 copies/mL (IQR 6731–64,058) vs. 4133 copies/mL (IQR 1100–8422), respectively. Qualitative trends in gdcfDNA levels for each patient were concordant between the two assays. Acute TCMR was preceded by significant elevations in gdcfDNA as quantified by both techniques. Elevations in gdcfDNA, using both techniques, were suggestive of TCMR in this pilot study with a 6- and 3-day lead-time prior to histological diagnosis in patients 1 and 2. Conclusions: Both the graft-specific methylation and genotyping techniques successfully quantified gdcfDNA in patients post-LT with statistically significant concordance. A direct comparison of these two techniques is not only important from a technical perspective for orthogonal validation, but significantly adds weight to the evidence that gdcfDNA monitoring reflects the underlying biology. Both techniques identified LT recipients who developed acute TCMR, with several days lead-time in comparison to conventional diagnostic workflows. Whilst the two assays performed comparably, gdcfDNA monitoring based on graft-specific DNA methylation patterns in cfDNA offers major practical advantages over the donor-recipient genotyping, and hence enhances the potential to translate this emerging technology into clinical practice.

The identification of biomarkers of response to PARP inhibitors can enable selection of appropriate ovarian cancer patients for treatment. In this study, the authors report clinical results and exploratory biomarker analyses from the ARIEL2 phase 2 clinical trial on the safety and efficacy of the PARP inhibitor rucaparib in patients with recurrent ovarian cancers

A significant number of patients (pts) with metastatic melanoma do not respond to anti-programmed cell death 1 (PD1) therapies. Identifying predictive biomarkers therefore remains an urgent need. We retrospectively analyzed plasma DNA of pts with advanced melanoma treated with PD-1 antibodies, nivolumab or pembrolizumab, for five PD-1 genotype single nucleotide polymorphisms (SNPs): PD1.1 (rs36084323, G>A), PD1.3 (rs11568821, G>A), PD1.5 (rs2227981, C>T) PD1.6 (rs10204225, G>A) and PD1.9 (rs2227982, C>T). Clinico-pathological and treatment parameters were collected, and presence of SNPs correlated with response, progression free survival (PFS) and overall survival (OS). 115 patients were identified with a median follow up of 18.7 months (range 0.26 – 52.0 months). All were Caucasian; 27% BRAF V600 mutation positive. At PD-1 antibody commencement, 36% were treatment-naïve and 52% had prior ipilimumab. The overall response rate was 43%, 19% achieving a complete response. Overall median PFS was 11.0 months (95% CI 5.4 - 17.3) and median OS was 31.1 months (95% CI 23.2 - NA). Patients with the G/G genotype had more complete responses than with A/G genotype (16.5% vs. 2.6% respectively) and the G allele of PD1.3 rs11568821 was significantly associated with a longer median PFS than the AG allele, 14.1 vs. 7.0 months compared to the A allele (p=0.04; 95% CI 0.14 – 0.94). No significant association between the remaining SNPs and responses, PFS or OS were observed. Despite limitations in sample size, this is the first study to demonstrate an association of a germline PD-1 polymorphism and PFS in response to anti-PD-1 therapy in pts with metastatic melanoma. Extrinsic factors like host germline polymorphisms should be considered with tumor intrinsic factors as predictive biomarkers for immune checkpoint regulators.

Abstract Background Two germ line Fc‐γ receptor (FCGR) polymorphisms, rs1801274 [FCGR2A; His(H)131Arg(R)] and rs396991 [FCGR3A; Phe(F)158Val(V)], produce altered proteins through amino acid substitutions. We previously reported that the FCGR2A H/H genotype was associated with longer overall survival (OS) in cetuximab‐treated chemotherapy‐refractory patients with metastatic colorectal cancer. Here, we aimed to replicate and extend this finding in the Canadian Clinical Trials Group CO.20 trial. Methods After germ line DNA genotyping, polymorphic relationships with survival were assessed using log‐rank tests and hazard ratios (HR) from Cox proportional hazard models, adjusting for known prognostic factors. The dominant genetic inheritance model was used for the main analysis. Results Of 592 wild‐type KRAS patients treated with cetuximab, those with the FCGR2A H/H genotype (n = 165, 28%) had improved OS (HR: 0.66, P

Around 10% of high-grade serous ovarian carcinomas (HGSOC) harbor BRCA1 promoter methylation, but it is uncertain how it predicts response to PARP inhibition. Here, the authors show that homozygous BRCA1 methylation predicts response to rucaparib while heterozygous methylation of BRCA1 predicts resistance in HGSOC.

Abstract Background Male breast cancer (MBC) represents a poorly characterised group of tumours, the management of which is largely based on practices established for female breast cancer. However, recent studies demonstrate biological and molecular differences likely to impact on tumour behaviour and therefore patient outcome. The aim of this study was to investigate methylation of a panel of commonly methylated breast cancer genes in familial MBCs. Methods 60 tumours from 3 BRCA1 and 25 BRCA2 male mutation carriers and 32 males from BRCAX families were assessed for promoter methylation by methylation-sensitive high resolution melting in a panel of 10 genes (RASSF1A, TWIST1, APC, WIF1, MAL, RARβ, CDH1, RUNX3, FOXC1 and GSTP1). An average methylation index (AMI) was calculated for each case comprising the average of the methylation of the 10 genes tested as an indicator of overall tumour promoter region methylation. Promoter hypermethylation and AMI were correlated with BRCA carrier mutation status and clinicopathological parameters including tumour stage, grade, histological subtype and disease specific survival. Results Tumours arising in BRCA2 mutation carriers showed significantly higher methylation of candidate genes, than those arising in non-BRCA2 familial MBCs (average AMI 23.6 vs 16.6, p = 0.01, 45% of genes hypermethylated vs 34%, p

Abstract Background Determining the role of DNA methylation in various biological processes is dependent on the accurate representation of often highly complex patterns. Accurate representation is dependent on unbiased PCR amplification post bisulfite modification, regardless of methylation status of any given epiallele. This is highly dependent on primer design. Particular difficulties are raised by the analysis of CpG-rich regions, which are the usual regions of interest. Here, it is often difficult or impossible to avoid placing primers in CpG-free regions, particularly if one wants to target a specific part of a CpG-rich region. This can cause biased amplification of methylated sequences if the C is placed at those positions or to unmethylated sequences if a T is placed at those positions. Methods We examined the effect of various base substitutions at the cytosine position of primer CpGs on the representational amplification of templates and also examined the role of the annealing temperature during PCR. These were evaluated using methylation-sensitive high-resolution melting and Pyrosequencing. Results For a mixture of fully methylated and unmethylated templates, amplification using the C-, C/T (Y-) and inosine-containing primers was biased towards amplification of methylated DNA. The bias towards methylated sequences increased with annealing temperature. Amplification using primers with an A/C/G/T (N) degeneracy at the cytosine positions was not biased at the lowest temperature used but became increasingly biased towards methylated DNA with increased annealing temperature. Using primers matching neither C nor T was in the main unbiased but at the cost of poor PCR amplification efficiency. Primers with abasic sites were also unbiased but could only amplify DNA for one out of the two assays tested. However, with heterogeneous methylation, it appeared that both the primer type and stringency used have a minimal influence on PCR bias. Conclusions This is the first comprehensive analysis of base substitutions at CpG sites in primers and their effect on PCR bias for the analysis of DNA methylation. Our findings are relevant to the appropriate design of a wide range of assays, including amplicon-based next-generation sequencing approaches that need to measure DNA methylation.

(1) Background: Epithelial–mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial–mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RARβ, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/.

Detection of circulating tumor cells and micrometastases in patients with cancer should prove useful in determining prognosis and in planning and monitoring systemic therapies. We have developed immunomagnetic isolation of carcinoma cells followed by reverse transcription polymerase chain reaction (immunobead RT-PCR) as a method for identifying very small numbers of breast cancer cells in blood. The expression of cytokeratin 19 (K19) was used as the marker by which the isolated tumor cells were identified. The immunobead RT-PCR technique allowed detection of one tumor cell per 106 leukocytes in whole blood. Immunobead RT-PCR is a highly sensitive method of detecting cancer cells in a hematopoietic environment.

Hairy cell leukemia has been shown to be strongly associated with the BRAF V600E mutation. We screened 59 unenriched archived bone marrow aspirate and peripheral blood samples from 51 patients with hairy cell leukemia using high resolution melting analysis and confirmatory Sanger sequencing. The BRAF V600E mutation was detected in 38 samples (from 36 patients). The BRAF V600E mutation was detected in all samples with disease involvement above the limit of sensitivity of the techniques used. Thirty-three of 34 samples from other hematologic malignancies were negative for BRAF mutations. A BRAF K601E mutation was detected in a patient with splenic marginal zone lymphoma. Our data support the recent finding of a disease defining point mutation in hairy cell leukemia. Furthermore, high resolution melting with confirmatory Sanger sequencing are useful methods that can be employed in routine diagnostic laboratories to detect BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p

BACKGROUND: Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. METHODOLOGY/PRINCIPAL FINDINGS: To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). CONCLUSIONS/SIGNIFICANCE: We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.