Predicting tumour content of liquid biopsies from cell-free DNA.

BACKGROUND: Liquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample's tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy. RESULTS: We used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma. CONCLUSIONS: This proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies.

BnpC: Bayesian non-parametric clustering of single-cell mutation profiles.

MOTIVATION: The high resolution of single-cell DNA sequencing (scDNA-seq) offers great potential to resolve intratumor heterogeneity (ITH) by distinguishing clonal populations based on their mutation profiles. However, the increasing size of scDNA-seq datasets and technical limitations, such as high error rates and a large proportion of missing values, complicate this task and limit the applicability of existing methods. RESULTS: Here, we introduce BnpC, a novel non-parametric method to cluster individual cells into clones and infer their genotypes based on their noisy mutation profiles. We benchmarked our method comprehensively against state-of-the-art methods on simulated data using various data sizes, and applied it to three cancer scDNA-seq datasets. On simulated data, BnpC compared favorably against current methods in terms of accuracy, runtime and scalability. Its inferred genotypes were the most accurate, especially on highly heterogeneous data, and it was the only method able to run and produce results on datasets with 5000 cells. On tumor scDNA-seq data, BnpC was able to identify clonal populations missed by the original cluster analysis but supported by Supplementary Experimental Data. With ever growing scDNA-seq datasets, scalable and accurate methods such as BnpC will become increasingly relevant, not only to resolve ITH but also as a preprocessing step to reduce data size. AVAILABILITY AND IMPLEMENTATION: BnpC is freely available under MIT license at https://github.com/cbg-ethz/BnpC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational Analysis of DNA and RNA Sequencing Data Obtained from Liquid Biopsies.

Next-generation sequencing of DNA and RNA obtained from liquid biopsies of cancer patients may reveal important insights into disease progression and metastasis formation, and it holds the promise to enable new methods for noninvasive screening and clinical decision support. However, implementing liquid biopsy sequencing protocols is challenged by capturing circulating tumor cells or cell-free tumor DNA from blood samples, by amplifying genomic DNA and RNA in a reliable and unbiased manner, and by extracting biologically meaningful signals from the noisy sequencing data. In this chapter, we discuss computational methods for the analysis of DNA and RNA sequencing data obtained from liquid biopsies, addressing these challenges.

Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.

Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1-2 years. For each case, exome sequencing was performed on 2-5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma.

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients.

BACKGROUND: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. METHODS: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. RESULTS: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. CONCLUSION: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.

Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in the JAK3 gene.

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.

ctDNA monitoring using patient-specific sequencing and integration of variant reads.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Measurement of Plasma Cell-Free Mitochondrial Tumor DNA Improves Detection of Glioblastoma in Patient-Derived Orthotopic Xenograft Models.

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed. SIGNIFICANCE: These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.

Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients.

Glioma is difficult to detect or characterize using current liquid biopsy approaches. Detection of cell-free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole-genome sequencing (sWGS, at a coverage of < 0.4×) of cell-free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell-free DNA in CSF is different from that in plasma. This low-cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger-scale sequencing, such as by whole-exome and whole-genome sequencing.

Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients.

Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K3EDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in K3EDTA tubes, but were stable in BCT tubes. K3EDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between K3EDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis.

Dynamics of multiple resistance mechanisms in plasma DNA during EGFR-targeted therapies in non-small cell lung cancer.

Tumour heterogeneity leads to the development of multiple resistance mechanisms during targeted therapies. Identifying the dominant driver(s) is critical for treatment decision. We studied the relative dynamics of multiple oncogenic drivers in longitudinal plasma of 50 EGFR-mutant non-small-cell lung cancer patients receiving gefitinib and hydroxychloroquine. We performed digital PCR and targeted sequencing on samples from all patients and shallow whole-genome sequencing on samples from three patients who underwent histological transformation to small-cell lung cancer. In 43 patients with known EGFR mutations from tumour, we identified them accurately in plasma of 41 patients (95%, 41/43). We also found additional mutations, including EGFR T790M (31/50, 62%), TP53 (23/50, 46%), PIK3CA (7/50, 14%) and PTEN (4/50, 8%). Patients with both TP53 and EGFR mutations before treatment had worse overall survival than those with only EGFR Patients who progressed without T790M had worse PFS during TKI continuation and developed alternative alterations, including small-cell lung cancer-associated copy number changes and TP53 mutations, that tracked subsequent treatment responses. Longitudinal plasma analysis can help identify dominant resistance mechanisms, including non-druggable genetic information that may guide clinical management.

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

CVE: an R package for interactive variant prioritisation in precision oncology.

BACKGROUND: An increasing number of precision oncology programmes are being launched world-wide. To support this development, we present the Cancer Variant Explorer (CVE), an R package with an interactive Shiny web browser interface. RESULTS: Leveraging Oncotator and the Drug Gene Interaction Database, CVE offers exploration of variants within single or multiple tumour exomes to identify drivers, resistance mechanisms and to assess druggability. We present example applications including the analysis of an individual patient and a cohort-wide study, and provide a first extension of CVE by adding a tumour-specific co-expression network. CONCLUSIONS: The CVE package allows interactive variant prioritisation to expedite the analysis of cancer sequencing studies. Our framework also includes the prioritisation of druggable targets, allows exploratory analysis of tissue specific networks and is extendable for specific applications by virtue of its modular design. We encourage the use of CVE within translational research studies and molecular tumour boards. The CVE package is available via Bioconductor ( http://bioconductor.org/packages/CVE/ ).

Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma.

TP53 mutations are ubiquitous in high-grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low-grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged-amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain-of-function (GOF or nonsynonymous), loss-of-function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.