Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Multi-omic features of oesophageal adenocarcinoma in patients treated with preoperative neoadjuvant therapy.

Oesophageal adenocarcinoma is a poor prognosis cancer and the molecular features underpinning response to treatment remain unclear. We investigate whole genome, transcriptomic and methylation data from 115 oesophageal adenocarcinoma patients mostly from the DOCTOR phase II clinical trial (Australian New Zealand Clinical Trials Registry-ACTRN12609000665235), with exploratory analysis pre-specified in the study protocol of the trial. We report genomic features associated with poorer overall survival, such as the APOBEC mutational and RS3-like rearrangement signatures. We also show that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. Transcriptomic analysis categorises patients into four immune clusters correlated with survival. The immune suppressed cluster is associated with worse survival, enriched with myeloid-derived cells, and an epithelial-mesenchymal transition signature. The immune hot cluster is associated with better survival, enriched with lymphocytes, myeloid-derived cells, and an immune signature including CCL5, CD8A, and NKG7. The immune clusters highlight patients who may respond to immunotherapy and thus may guide future clinical trials.

streammd: fast low-memory duplicate marking using a Bloom filter.

SUMMARY: Identification of duplicate templates is a common preprocessing step in bulk sequence analysis; for large libraries, this can be resource intensive. Here, we present streammd: a fast, memory-efficient, single-pass duplicate marker operating on the principle of a Bloom filter. streammd closely reproduces outputs from Picard MarkDuplicates while being substantially faster, and requires much less memory than SAMBLASTER. AVAILABILITY AND IMPLEMENTATION: streammd is a C++ program available from GitHub https://github.com/delocalizer/streammd under the MIT license.

Analysis of hereditary cancer gene variant classifications from ClinVar indicates a need for regular reassessment of clinical assertions.

The clinical classification of variants may change with new information, however, there is limited guidance on how often significant changes in variant classification occur. We used ClinVar to examine how variant classification changes over time. We developed a custom parser and accessed variant data from ClinVar between January 2015 and July 2021. The ClinVar-assigned "aggregate" classification of variants in 121 hereditary cancer genes was harmonized across releases to align to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology terms. Aggregate classification categories were grouped as: benign/likely benign (B/LB); likely pathogenic/pathogenic (LP/P); variant of uncertain significance (VUS); conflicting interpretations of pathogenicity (Conflicting); or Other. We profiled changes in aggregate variant classification between consecutive semi-annual ClinVar releases. The proportion of variants that changed aggregate classification between semi-annual ClinVar releases ranged from 0.6% to 6.4%. The most frequent changes were "VUS to conflicting," "other to LP/P," and "B/LB to Conflicting." A limited number of variants changed aggregate classification from "LP/P to B/LB," or vice versa. Our analysis indicates need for regular reassessment of clinical variant interpretations. The parser developed for this project will facilitate extraction of relevant interpretation data from ClinVar.

Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes.

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. SIGNIFICANCE: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711.

Comprehensive genomic and tumour immune profiling reveals potential therapeutic targets in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) has a poor overall survival with few treatment options. Whole genome sequencing (WGS) combined with the immune features of MPM offers the prospect of identifying changes that could inform future clinical trials. METHODS: We analysed somatic mutations from 229 MPM samples, including previously published data and 58 samples that had undergone WGS within this study. This was combined with RNA-seq analysis to characterize the tumour immune environment. RESULTS: The comprehensive genome analysis identified 12 driver genes, including new candidate genes. Whole genome doubling was a frequent event that correlated with shorter survival. Mutational signature analysis revealed SBS5/40 were dominant in 93% of samples, and defects in homologous recombination repair were infrequent in our cohort. The tumour immune environment contained high M2 macrophage infiltrate linked with MMP2, MMP14, TGFB1 and CCL2 expression, representing an immune suppressive environment. The expression of TGFB1 was associated with overall survival. A small subset of samples (less than 10%) had a higher proportion of CD8 T cells and a high cytolytic score, suggesting a 'hot' immune environment independent of the somatic mutations. CONCLUSIONS: We propose accounting for genomic and immune microenvironment status may influence therapeutic planning in the future.

Multiomic profiling of checkpoint inhibitor-treated melanoma: Identifying predictors of response and resistance, and markers of biological discordance.

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy.

qmotif: determination of telomere content from whole-genome sequence data.

Motivation: Changes in telomere length have been observed in cancer and can be indicative of mechanisms involved in carcinogenesis. Most methods used to estimate telomere length require laboratory analysis of DNA samples. Here, we present qmotif, a fast and easy tool that determines telomeric repeat sequences content as an estimate of telomere length directly from whole-genome sequencing. Results: qmotif shows similar results to quantitative PCR, the standard method for high-throughput clinical telomere length quantification. qmotif output correlates strongly with the output of other tools for determining telomere sequence content, TelSeq and TelomereHunter, but can run in a fraction of the time-usually under a minute. Availability and implementation: qmotif is implemented in Java and source code is available at https://github.com/AdamaJava/adamajava, with instructions on how to build and use the application available from https://adamajava.readthedocs.io/en/latest/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association.

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.

Considerations for using population frequency data in germline variant interpretation: Cancer syndrome genes as a model.

Aggregate population genomics data from large cohorts are vital for assessing germline variant pathogenicity. However, there are no specifications on how sequencing quality metrics should be considered, and whether exome-derived and genome-derived allele frequencies should be considered in isolation. Germline genome sequence data were simulated for nine read-depths to identify a minimum acceptable read-depth for detecting variants. gnomAD exome-derived and genome-derived datasets were assessed for read-depth, for six key cancer genes selected for variant curation by ClinGen expert panels. Non-Finnish European allele frequency (AF) or filter AF of coding variants in these genes, assigned into frequency bins using modified ACMG-AMP criteria, was compared between exome-derived and genome-derived datasets. A 30X read-depth achieved acceptable precision and recall for detection of substitutions, but poor recall for small insertions/deletions. Exome-derived and genome-derived datasets exhibited low read-depth for different gene exons. Individual variants were mostly assigned to non-divergent AF bins (>95%) or filter AF bins (>97%). Two major bin divergences were resolved by applying the minimal acceptable read-depth threshold. These findings show the importance of assessing read-depth separately for population datasets sourced from different short-read sequencing technologies before assigning a frequency-based ACMG-AMP classification code for variant interpretation.

Whole-genome sequencing of acral melanoma reveals genomic complexity and diversity.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.

Using whole-genome sequencing data to derive the homologous recombination deficiency scores.

The homologous recombination deficiency (HRD) score was developed using whole-genome copy number data derived from arrays as a way to infer deficiency in the homologous recombination DNA damage repair pathway (in particular BRCA1 or BRCA2 deficiency) in breast cancer samples. The score has utility in understanding tumour biology and may be indicative of response to certain therapeutic strategies. Studies have used whole-exome sequencing to derive the HRD score, however, with increasing use of whole-genome sequencing (WGS) to characterise tumour genomes, there has yet to be a comprehensive comparison between HRD scores derived by array versus WGS. Here we demonstrate that there is both a high correlation and a good agreement between array- and WGS-derived HRD scores and between the scores derived from WGS and downsampled WGS to represent shallow WGS. For samples with an HRD score close to threshold for stratifying HR proficiency or deficiency there was however some disagreement in the HR status between array and WGS data, highlighting the importance of not relying on a single method of ascertaining the homologous recombination status of a tumour.

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).

Running Genomic Analyses in the Cloud.

Genomic testing is rapidly moving into healthcare practice. However it comes with informatics challenges that the healthcare system has not previously faced - the raw data can be hundreds of gigabytes per test, the compute demands can be thousands of CPU hours, and the test can reveal deeply private health-srelated information that can have implications for anyone related to the person tested. While not a panacea, cloud computing has particular properties that can ameliorate some of these difficulties. This paper presents some of the key lessons learned while deploying a set of genomic analyses on cloud computing for Queensland Genomics.

Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.

Complex structural rearrangements are present in high-grade dysplastic Barrett's oesophagus samples.

BACKGROUND: Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett's oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals. METHODS: In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples. RESULTS: We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples. CONCLUSIONS: The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Whole genome sequencing of melanomas in adolescent and young adults reveals distinct mutation landscapes and the potential role of germline variants in disease susceptibility.

Cutaneous melanoma accounts for at least >10% of all cancers in adolescents and young adults (AYA, 15-30 years of age) in Western countries. To date, little is known about the correlations between germline variants and somatic mutations and mutation signatures in AYA melanoma patients that might explain why they have developed a cancer predominantly affecting those over 65 years of age. We performed genomic analysis of 50 AYA melanoma patients (onset 10-30 years, median 20); 25 underwent whole genome sequencing (WGS) of both tumor and germline DNA, exome data were retrieved from 12 TCGA AYA cases, and targeted DNA sequencing was conducted on 13 cases. The AYA cases were compared with WGS data from 121 adult cutaneous melanomas. Similar to mature adult cutaneous melanomas, AYA melanomas showed a high mutation burden and mutation signatures of ultraviolet radiation (UVR) damage. The frequencies of somatic mutations in BRAF (96%) and PTEN (36%) in the AYA WGS cohort were double the rates observed in adult melanomas (Q < 6.0 × 10-6 and 0.028, respectively). Furthermore, AYA melanomas contained a higher proportion of non-UVR-related mutation signatures than mature adult melanomas as a proportion of total mutation burden (p = 2.0 × 10-4 ). Interestingly, these non-UVR mutation signatures relate to APOBEC or mismatch repair pathways, and germline variants in related genes were observed in some of these cases. We conclude that AYA melanomas harbor some of the same molecular aberrations and mutagenic insults occurring in older adults, but in different proportions. Germline variants that may have conferred disease susceptibility correlated with somatic mutation signatures in a subset of AYA melanomas.

Germline and somatic variant identification using BGISEQ-500 and HiSeq X Ten whole genome sequencing.

Technological innovation and increased affordability have contributed to the widespread adoption of genome sequencing technologies in biomedical research. In particular large cancer research consortia have embraced next generation sequencing, and have used the technology to define the somatic mutation landscape of multiple cancer types. These studies have primarily utilised the Illumina HiSeq platforms. In this study we performed whole genome sequencing of three malignant pleural mesothelioma and matched normal samples using a new platform, the BGISEQ-500, and compared the results obtained with Illumina HiSeq X Ten. Germline and somatic, single nucleotide variants and small insertions or deletions were independently identified from data aligned human genome reference. The BGISEQ-500 and HiSeq X Ten platforms showed high concordance for germline calls with genotypes from SNP arrays (>99%). The germline and somatic single nucleotide variants identified in both sequencing platforms were highly concordant (86% and 72% respectively). These results indicate the potential applicability of the BGISEQ-500 platform for the identification of somatic and germline single nucleotide variants by whole genome sequencing. The BGISEQ-500 datasets described here represent the first publicly-available cancer genome sequencing performed using this platform.