Germline Single Nucleotide Variants Research Articles

A method to comprehensively identify germline SNVs, INDELs and CNVs from whole exome sequencing data of BRCA1/2 negative breast cancer patients.

In the rapidly evolving field of genomics, understanding the genetic basis of complex diseases like breast cancer, particularly its familial/hereditary forms, is crucial. Current methods often examine genomic variants-such as Single Nucleotide Variants (SNVs), insertions/deletions (Indels), and Copy Number Variations (CNVs)-separately, lacking an integrated approach. Here, we introduced a robust, flexible methodology for a comprehensive variants' analysis using Whole Exome Sequencing (WES) data. Our approach uniquely combines meticulous validation with an effective variant filtering strategy. By reanalyzing two germline WES datasets from BRCA1/2 negative breast cancer patients, we demonstrated our tool's efficiency and adaptability, uncovering both known and novel variants. This contributed new insights for potential diagnostic, preventive, and therapeutic strategies. Our method stands out for its comprehensive inclusion of key genomic variants in a unified analysis, and its practical resolution of technical challenges, offering a pioneering solution in genomic research. This tool presents a breakthrough in providing detailed insights into the genetic alterations in genomes, with significant implications for understanding and managing hereditary breast cancer.

Whole-exome sequencing reveals an association of rs112065068 in TGOLN2 gene with distant metastasis of non-small cell lung cancer

Early prediction and prevention of recurring illness is critical for improving the survival rates of patients with non-small cell lung cancer (NSCLC). Previously, we demonstrated that the presence of premalignant epithelial changes in the small bronchi distant to the primary tumor is associated with NSCLC progression: isolated basal cell hyperplasia (iBCH) indicates a high risk of distant metastasis, BCH combined with squamous metaplasia (BCHSM) – a high risk of locoregional recurrence. Here, we aimed to identify germline single nucleotide variants (SNVs) and insertions and deletions (InDels) associated with distant metastasis and locoregional recurrence in cases with iBCH and BCHSM using whole-exome sequencing of 172 NSCLC patients. The rs112065068 of the TGOLN2 gene was identified only in iBCH patients and was associated with a high risk of distant metastasis (P < .001) and worse metastasis-free survival (HR = 4.19 (95 %CI 1.97–8.93); P < .001). This variant was validated in a group of 109 NSCLC patients using real-time PCR and Sanger sequencing analyses. To our knowledge, this study is the first to identify a germline variant associated with NSCLC distant metastasis.

Empirical Bayes single nucleotide variant-calling for next-generation sequencing data

One of the fundamental computational problems in cancer genomics is the identification of single nucleotide variants (SNVs) from DNA sequencing data. Many statistical models and software implementations for SNV calling have been developed in the literature, yet, they still disagree widely on real datasets. Based on an empirical Bayesian approach, we introduce a local false discovery rate (LFDR) estimator for germline SNV calling. Our approach learns model parameters without prior information, and simultaneously accounts for information across all sites in the genomic regions of interest. We also propose another LFDR-based algorithm that reliably prioritizes a given list of mutations called by any other variant-calling algorithm. We use a suite of gold-standard cell line data to compare our LFDR approach against a collection of widely used, state of the art programs. We find that our LFDR approach approximately matches or exceeds the performance of all of these programs, despite some very large differences among them. Furthermore, when prioritizing other algorithms’ calls by our LFDR score, we find that by manipulating the type I-type II tradeoff we can select subsets of variant calls with minimal loss of sensitivity but dramatic increases in precision.

Single-nucleotide variant calling in single-cell sequencing data with Monopogen.

Single-cell omics technologies enable molecular characterization of diverse cell types and states, but how the resulting transcriptional and epigenetic profiles depend on the cell's genetic background remains understudied. We describe Monopogen, a computational tool to detect single-nucleotide variants (SNVs) from single-cell sequencing data. Monopogen leverages linkage disequilibrium from external reference panels to identify germline SNVs and detects putative somatic SNVs using allele cosegregating patterns at the cell population level. It can identify 100 K to 3 M germline SNVs achieving a genotyping accuracy of 95%, together with hundreds of putative somatic SNVs. Monopogen-derived genotypes enable global and local ancestry inference and identification of admixed samples. It identifies variants associated with cardiomyocyte metabolic levels and epigenomic programs. It also improves putative somatic SNV detection that enables clonal lineage tracing in primary human clonal hematopoiesis. Monopogen brings together population genetics, cell lineage tracing and single-cell omics to uncover genetic determinants of cellular processes.

Mutations in coral soma and sperm imply lifelong stem cell renewal and cell lineage selection.

In many animals, the germline differentiates early in embryogenesis, so only mutations that accumulate in germ cells are inherited by offspring. Exceptions to this developmental process may indicate other mechanisms have evolved to limit the effects of deleterious mutation accumulation. Stony corals are animals that can live for hundreds of years and have been thought to produce gametes from somatic tissue. To clarify conflicting evidence about germline-soma distinction in corals, we sequenced high coverage, full genomes with technical replicates for parent coral branches and their sperm pools. We identified post-embryonic single nucleotide variants (SNVs) unique to each parent branch, then checked if each SNV was shared by the respective sperm pool. Twenty-six per cent of post-embryonic SNVs were shared by the sperm and 74% were not. We also identified germline SNVs, those that were present in the sperm but not in the parent. These data suggest that self-renewing stem cells differentiate into germ and soma throughout the adult life of the colony, with SNV rates and patterns differing markedly in stem, soma and germ lineages. In addition to informing the evolution of germlines in metazoans, these insights inform how corals may generate adaptive diversity necessary in the face of global climate change.

Deciphering the Somatic and Germline Structural Variation Landscape in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia By Whole Genome Optical Mapping

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common numerical and structural chromosomal alterations are high hyperdiploidy (HHD, 51-67 chromosomes) and t(12;21)(p13;q22) leading to ETV6::RUNX1 fusion. However, for a full-blown leukemia, secondary alterations are required. Furthermore, it is increasingly recognized that germline variants may enhance ALL susceptibility in ~4% of the patients (Bloom et al. 2019, Klco & Mullighan 2020). In large-scale genomic studies, the mutational landscape of somatic and germline single nucleotide variants and small indels has been well described in BCP-ALL. However, a comprehensive analysis of somatic and germline structural variants (SVs) in ETV6::RUNX1 and HHD tumors is still lacking. Recent feasibility studies showed that whole genome optical mapping (WGOM) is a novel method for reliable SV detection which could abrogate conventional cytogenetics and array based methods in the future (Neveling et al. 2021, Mantere et al. 2021). The aim of the study was to analyze the structural variation landscape in pediatric BCP-ALL patients by employing WGOM to gain significant insights on leukemia predisposition and development. Methods: We studied 60 pediatric patients diagnosed with ETV6::RUNX1 (n=30) or HHD (n=30) BCP-ALL with a median age at diagnosis of 3.9 (range 1.7-17) and a male to female ratio of 1.14:1. We collected tumor material at initial diagnosis and included fibroblast or remission material as matched non-tumor control of each patient. Ultra-high molecular weight DNA was extracted from 120 samples and labeled by a sequence specific enzyme. Fluorescently labeled DNA was analyzed using a Saphyr instrument (Bionano Genomics). We integrated comprehensive datasets of 60 matched tumor/non-tumor genomes to identify somatically acquired SVs >500bp. Corresponding data of standard methods as cyto- (karyotype, fluorescence in situ hybridization) and molecular genetics (SNP-array) of the leukemia samples were compared to WGOM data. In addition, the prevalence of rare germline SVs (<1% of 160 healthy control samples [Bionano] and minor allele frequency <0.01 in dbVar) occurring in known cancer predisposition genes was analyzed in the non-tumor samples. Results: WGOM was able to detect 94% (438/464) of previously observed events by conventional methods, including ETV6::RUNX1 and high hyperdiploid hallmark alterations in all BCP-ALL cases. Beyond that, we discovered 895 additional SVs with WGOM not detected by standard cyto- and molecular genetics. WGOM detected a median of 25 (range 0-70) somatic SVs secondary to the hallmark alterations in ETV6::RUNX1 tumors compared to 13 (range 0-28) SVs in HHD cases. We identified 60 recurrently altered regions occurring in ≥3 BCP-ALL cases including 12p13.2 (ETV6, 19/60), 9p13.2 (PAX5, 13/60), 9p21.3 (CDKN2A/B, 12/60), 11p12 (RAG2, 10/60), 20p12.2 (MKKS/SLXIP, 10/60) or 3q13.2 (CD200/BTLA, 8/60). We detected ETV6::RUNX1 subtype specific recurrent focal deletions in ATF7IP (10/30), BTG1 (10/30), NSD2 (5/30) and TBL1XR1 (4/30). Interestingly, we discovered recurrent SVs affecting putative novel genes involved in BCP-ALL leukemogenesis such as STAG2, GPN3 and UBA2 which are affected in ~10% of the BCP-ALL patients. Strikingly, WGOM revealed in total 9 novel potential in-frame fusion transcripts in both tumor subtypes, including a potential fusion of the cohesin complex members PDS5B::STAG2, arising through a t(X;13)(q25;q13.1) in an ETV6::RUNX1 patient (Figure 1). In addition, we detected 28 rare germline SVs targeting 22 known cancer predisposing genes in 35% (21/60) of the pediatric patients. 40% (9/22) of the affected genes were involved in hematopoietic or lymphoid organ development as ETV6, FANCA, IKZF1, ITCH or PTPRC. In 18% (11/60) of the children, the identified germline alteration potentially altered a coding region of a known cancer predisposing gene including a duplication of CIITA in an ETV6::RUNX1 patient. Conclusions: By applying the novel technology WGOM, we could detect a significant amount of previously hidden SVs in our BCP-ALL cohort in addition to conventional methods. We further generated a comprehensive landscape of somatic and germline SVs in childhood BCP-ALL, revealing recurrently altered regions and novel potential players in ALL leukemogenesis and predisposition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The genomic landscape across 474 surgically accessible epileptogenic human brain lesions.

Understanding the exact molecular mechanisms involved in the etiology of epileptogenic pathologies with or without tumor activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants (SNV) in low-grade epilepsy-associated tumors (LEAT; 7.92 ± 5.65 SNV) than in brain tissue from malformations of cortical development (MCD; 6.11 ± 4 SNV) or hippocampal sclerosis (HS; 5.1 ± 3.04 SNV). Tumor tissues also had the largest number of likely pathogenic variant carrying cells. LEAT had the highest proportion of samples with one or more somatic copy number variants (CNV; 24.7%), followed by MCD (5.4%) and HS (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among LEAT. For germline variant-associated MCD genes such as TSC2, DEPDC5, and PTEN, germline SNV were frequently identified within large loss of heterozygosity regions, supporting the recently proposed 'second hit' disease mechanism in these genes. We detect somatic variants in twelve established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the etiology of LEAT (e.g., BRAF) and MCD (e.g., SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with LEAT and NRAS Q61 mutated protein with a complex MCD characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21-q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical genetic analyses showed that the numbers of somatic SNV across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with LEAT. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening, and guides future directions for clinical implementation of epilepsy surgery genetics.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in KIT, ERBB2, KRAS, and TP53 in 30% of Thymic Carcinomas.

Simple SummaryThe biology of thymic epithelial tumours (TETs), including thymomas and thymic carcinomas, and particularly the extent of molecular dysregulation, is poorly understood. Through next-generation sequencing of 15 genes implicated in common solid tumours in 53 TETs, we found a larger number of single nucleotide variants (SNVs) in thymic carcinomas than thymomas. About 30% of thymic carcinomas had at least one somatic pathogenic gene variant in TP53, ERBB2, KIT, or KRAS, whereas variants of uncertain clinical significance in KIT, ERBB2, and FOXL2 were found exclusively in thymomas. The presence of somatic pathogenic variants was non-significantly associated with shorter disease-free survival in thymic carcinoma patients. No somatic pathogenic or likely pathogenic SNV was found in thymomas. Importantly, we also evaluated germline SNVs, adding to the number of known genetic alterations in TETs and thereby enhancing our molecular understanding of these neoplasms.A better understanding of the molecular pathogenesis of thymic epithelial tumours (TETs) could revolutionise their treatment. We evaluated thymomas and thymic carcinomas by next-generation sequencing (NGS) of somatic or germline single nucleotide variants (SNVs) in genes commonly mutated in solid tumours. In total, 19 thymomas and 34 thymic carcinomas were analysed for nonsynonymous SNVs in 15 genes by targeted NGS (reference genome: hg19/GRCh37). Ten SNVs in TP53 (G154V, R158P, L194H, R267fs, R273C, R306 *, Q317 *), ERBB2 (V773M), KIT (L576P), and KRAS (Q61L) considered somatic and pathogenic/likely pathogenic were detected in 10 of 34 (29.4%) thymic carcinomas. No somatic SNVs confirmed as pathogenic/likely pathogenic were found in thymomas. Rare SNVs of uncertain or unknown functional and clinical significance, to our knowledge not reported previously in TETs, were found in ERBB2 (S703R), KIT (I690V), and FOXL2 (P157S) in 3 of 19 (16%) thymomas. The most frequent germline SNVs were TP53 P72R (94% TETs), ERBB2 I655V (40% TETs), and KIT M541L (9% TETs). No significant difference in median disease-free survival (DFS) was found between thymic carcinoma patients with and without pathogenic SNVs (p = 0.190); however, a trend toward a longer DFS was observed in the latter (16.0 vs. 30.0 months, respectively). In summary, NGS analysis of TETs revealed several SNVs in genes related to the p53, AKT, MAPK, and K-Ras signalling pathways. Thymic carcinomas showed greater genetic dysregulation than thymomas. The germline and rare SNVs of uncertain clinical significance reported in this study add to the number of known genetic alterations in TETs, thus extending our molecular understanding of these neoplasms. Druggable KIT alterations in thymic carcinomas have potential as therapeutic targets.

Validation of AmpliSeq NGS Panel for BRCA1 and BRCA2 Variant Detection in Canine Formalin-Fixed Paraffin-Embedded Mammary Tumors.

Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered (blood, fresh and FFPE tissues). Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.

Rare Germline Variants in Chordoma-Related Genes and Chordoma Susceptibility.

Simple SummaryChordoma is an extremely rare bone cancer that has not been fully characterized and few risk factors have been identified, highlighting the need for improving our understanding of the disease biology. Our study aims to identify chordoma susceptibility genes by investigating 265 genes involved in chordoma-related signaling pathways and other biological processes on germline DNA of 138 chordoma patients of European ancestry compared to internal control datasets and general population databases. Results were intersected with whole genome sequencing data from 80 skull-base chordoma patients of Chinese ancestry. Several rare loss-of-function and predicted deleterious missense variants were enriched in chordoma cases in both datasets, suggesting a complex model of pathways potentially involved in chordoma development and susceptibility, warranting further investigation in larger studies.Background: Chordoma is a rare bone cancer with an unknown etiology. TBXT is the only chordoma susceptibility gene identified to date; germline single nucleotide variants and copy number variants in TBXT have been associated with chordoma susceptibility in familial and sporadic chordoma. However, the genetic susceptibility of chordoma remains largely unknown. In this study, we investigated rare germline genetic variants in genes involved in TBXT/chordoma-related signaling pathways and other biological processes in chordoma patients from North America and China. Methods: We identified variants that were very rare in general population and internal control datasets and showed evidence for pathogenicity in 265 genes in a whole exome sequencing (WES) dataset of 138 chordoma patients of European ancestry and in a whole genome sequencing (WGS) dataset of 80 Chinese patients with skull base chordoma. Results: Rare and likely pathogenic variants were identified in 32 of 138 European ancestry patients (23%), including genes that are part of notochord development, PI3K/AKT/mTOR, Sonic Hedgehog, SWI/SNF complex and mesoderm development pathways. Rare pathogenic variants in COL2A1, EXT1, PDK1, LRP2, TBXT and TSC2, among others, were also observed in Chinese patients. Conclusion: We identified several rare loss-of-function and predicted deleterious missense variants in germline DNA from patients with chordoma, which may influence chordoma predisposition and reflect a complex susceptibility, warranting further investigation in large studies.

Whole-genome sequencing of phenotypically distinct inflammatory breast cancers reveals similar genomic alterations to non-inflammatory breast cancers

BackgroundInflammatory breast cancer (IBC) has a highly invasive and metastatic phenotype. However, little is known about its genetic drivers. To address this, we report the largest cohort of whole-genome sequencing (WGS) of IBC cases.MethodsWe performed WGS of 20 IBC samples and paired normal blood DNA to identify genomic alterations. For comparison, we used 23 matched non-IBC samples from the Cancer Genome Atlas Program (TCGA). We also validated our findings using WGS data from the International Cancer Genome Consortium (ICGC) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We examined a wide selection of genomic features to search for differences between IBC and conventional breast cancer. These include (i) somatic and germline single-nucleotide variants (SNVs), in both coding and non-coding regions; (ii) the mutational signature and the clonal architecture derived from these SNVs; (iii) copy number and structural variants (CNVs and SVs); and (iv) non-human sequence in the tumors (i.e., exogenous sequences of bacterial origin).ResultsOverall, IBC has similar genomic characteristics to non-IBC, including specific alterations, overall mutational load and signature, and tumor heterogeneity. In particular, we observed similar mutation frequencies between IBC and non-IBC, for each gene and most cancer-related pathways. Moreover, we found no exogenous sequences of infectious agents specific to IBC samples. Even though we could not find any strongly statistically distinguishing genomic features between the two groups, we did find some suggestive differences in IBC: (i) The MAST2 gene was more frequently mutated (20% IBC vs. 0% non-IBC). (ii) The TGF β pathway was more frequently disrupted by germline SNVs (50% vs. 13%). (iii) Different copy number profiles were observed in several genomic regions harboring cancer genes. (iv) Complex SVs were more frequent. (v) The clonal architecture was simpler, suggesting more homogenous tumor-evolutionary lineages.ConclusionsWhole-genome sequencing of IBC manifests a similar genomic architecture to non-IBC. We found no unique genomic alterations shared in just IBCs; however, subtle genomic differences were observed including germline alterations in TGFβ pathway genes and somatic mutations in the MAST2 kinase that could represent potential therapeutic targets.

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

Whole Exome Sequencing for the Identification of Mutations in CD8+ T-Cells.

Advances in next-generation sequencing and in particular whole exome sequencing (WES) have provided an innovative opportunity to perform a mutational screening of the entire coding region of the genome down to the single base, enhancing the discovery of causal mutations important for disease treatment and management. Recently, the accumulation of germline mutations in expanded CD8+ T-cells has been found to have a pathogenic significance in autoimmune diseases such as rheumatoid arthritis, and, on the other hand, this type of mutations may act in combination with newly acquired somatic mutations modulating tumorigenesis, evolution, and cancer recurrence determining the clinical outcome. Therefore, we describe a protocol for identifying and characterizing germline single nucleotide variants (SNVs) and small deletions (Indels) from next-generation WES data of CD8+ T-cells coming from patients with autoimmune diseases and comparing them to matching control samples. Conversely, the same protocol can be applied for identifying and characterizing germline SNVs from CD8+ T-cells isolated from tumor samples with a non-favorable clinical outcome compared to those from patients with a favorable clinical outcome used as controls.

Germline Genetic Association between Stromal Interaction Molecule 1 (STIM1) and Clinical Outcomes in Breast Cancer Patients.

Among all cancers in women, breast cancer has the highest incidence. The mortality of breast cancer is highly associated with metastasis. Migration and malignant transformation of cancer cells have been reported to be modulated by store-operated calcium (SOC) channels, which control calcium signaling and cell proliferation pathways. Stromal interaction molecule 1 (STIM1) is a calcium sensor in the endoplasmic reticulum, triggering the activation of store-operated calcium signaling. However, the clinical relevance of STIM1 in breast cancer is still unclear. Here, we recruited 348 breast cancer patients and conducted a genetic association study to address this question. Four tagging germline single nucleotide variants (SNVs) in STIM1 were selected and RNA sequencing data of 525 breast cancer samples from The Cancer Genome Atlas (TCGA) database were evaluated. The results show that rs2304891 and rs3750996 were correlated with clinical stage of breast cancer. Expression quantitative trait loci (eQTL) analysis indicated that risk G allele of STIM1 contributed to the higher expression of STIM1. In addition, we found an increased risk of rs2304891 G allele and rs3750996 A allele in estrogen receptor (ER) positive and progesterone receptor (PR) positive patients. In conclusion, our results suggest that germline SNV, rs2304891 and rs3750996 as well as STIM1 expression are important biomarkers for the prediction of clinical outcomes in breast cancer patients.

Rare Tumor-Normal Matched Whole Exome Sequencing Identifies Novel Genomic Pathogenic Germline and Somatic Aberrations.

Whole exome sequencing (WES) of matched tumor-normal pairs in rare tumors has the potential to identify genome-wide mutations and copy number alterations (CNAs). We evaluated 27 rare cancer patients with tumor-normal matching by WES and tumor-only next generation sequencing (NGS) as a comparator. Our goal was to: 1) identify known and novel variants and CNAs in rare cancers with comparison to common cancers; 2) examine differences between germline and somatic variants and how that functionally impacts rare tumors; 3) detect and characterize alleles in biologically relevant genes-pathways that may be of clinical importance but not represented in classical cancer genes. We identified 3343 germline single nucleotide variants (SNVs) and small indel variants—1670 in oncogenes and 1673 in tumor suppressor genes—generating an average of 124 germline variants/case. The number of somatic SNVs and small indels detected in all cases was 523:306 in oncogenes and 217 in tumor suppressor genes. Of the germline variants, six were identified to be pathogenic or likely pathogenic. In the 27 analyzed rare cancer cases, CNAs are variable depending on tumor type, germline pathogenic variants are more common. Cell fate pathway mutations (e.g., Hippo, Notch, Wnt) dominate pathogenesis and double hit (mutation + CNV) represent ~18% cases.

Using sequential next-generation sequencing assays to identify germline cancer predisposition variants.

1581 Background: Next-generation sequencing (NGS) increasingly guides clinical care in hematological malignancies by identifying DNA mutations that change dynamically over time. Clinical samples contain variable numbers of malignant and non-malignant cells. So, careful interpretation is required to determine if a particular variant is somatic, germline, or clonal hematopoietic in origin. Methods: The University of Chicago uses a targeted NGS assay of ~1200 genes, reporting 150 as a clinical test. We aimed to identify individuals with hereditary predisposition by detecting persistent variants on sequential assays regardless of disease state. Results: 943 NGS assays from July 2017 – Feb. 2020 on 711 patients [ages 1 mo – 95 yrs, median 65 yrs] were included. 2,320 variants in 33 genes were identified with 144 patients having the same variant identified on more than one assay. Single nucleotide variants (SNVs) with variant allele frequency (VAF) ≥ 0.3 were prioritized. The first candidate gene identified with potential germline SNVs was CSF3R. 28 unique SNVs in CSF3R were found, 14 were confirmed as germline, 6 somatic, and 8 were unconfirmed due to lack of available tissue. At least 2 confirmed germline CSF3R variants were likely deleterious based on functional testing. Sequential SNVs were quantified using the coefficient of variation, characterizing each by change in VAF over time. Using a worst-case-scenario analysis, in which unconfirmed variants were not counted as germline, a computer algorithm was designed to identify potential germline variants (specificity 0.89, PPV 0.75). Via an iterative method, the algorithm compares new assays to a pool of previously reported tests, flagging patients with potential germline mutations so that biopsies may be studied in the lab, records reviewed, and referrals placed to genetic counselors. To date, 61 patients with 89 likely germline variants have been identified. Known hereditary hematological malignancy genes, such as ATM, ASXL1, CHEK2, DDX41, TSC1, and RUNX1, had the most variants identified. Limitations include the challenge in distinguishing variants that do not change over time, reliance on a targeted NGS panel, and normalizing VAF data prior to analysis. Conclusions: These data highlight the utility of NGS of bone marrow and peripheral blood samples to identify patients suspected of having germline DNA variants. In addition to identifying known predisposition syndromes, one may discover new inherited cancer syndromes and help guide clinical practice in real time.

Exploration of germline variants responsible for adverse events of crizotinib in anaplastic lymphoma kinase-positive non-small cell lung cancer by target-gene panel sequencing

ObjectivesCrizotinib is a standard treatment for advanced anaplastic lymphoma kinase (ALK)- or ROS1-fusion-gene-positive non-small cell lung cancer; however, serious adverse events (AEs), including elevated alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and interstitial lung disease (ILD), develop occasionally. Here, we evaluated relationships between clinically significant crizotinib-associated AEs and germline variations. Materials and MethodsDNA obtained from 75 patients allowed selection of 147 genes according to function, exon identification and sequencing, and determination of germline single nucleotide variants (SNVs). Correlations between clinically significant AEs and presence of germline variants were estimated by Fisher’s exact test. ResultsWe defined clinically significant AEs as grade 4 hematological toxicity, grade ≥3 non-hematological toxicity, and any grade of ILD. These AEs were observed in 26 patients (35%), with elevated AST/ALT (15%) the most common, followed by neutropenia (5%), ILD (4%), and thromboembolic events (4%). Nonsynonymous SNVs in epoxide hydrolase 1 (EPHX1) [odds ratio (OR): 3.86; p = 0.0009) and transcription factor 7-like 2 (TCF7L2) (OR: 2.51; p = 0.025) were associated with the presence of clinically significant AEs. ConclusionNonsynonymous EPHX1 and TCF7L2 SNVs might be associated with clinically significant crizotinib-associated AEs. These data indicated that target-gene sequencing could be feasible for predicting anticancer-agent toxicity, and that germline multi-gene information might be useful for predicting patient-specific AEs to promote precision medicine.

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.

SNVSniffer: an integrated caller for germline and somatic single-nucleotide and indel mutations.

BackgroundVarious approaches to calling single-nucleotide variants (SNVs) or insertion-or-deletion (indel) mutations have been developed based on next-generation sequencing (NGS). However, most of them are dedicated to a particular type of mutation, e.g. germline SNVs in normal cells, somatic SNVs in cancer/tumor cells, or indels only. In the literature, efficient and integrated callers for both germline and somatic SNVs/indels have not yet been extensively investigated.ResultsWe present SNVSniffer, an efficient and integrated caller identifying both germline and somatic SNVs/indels from NGS data. In this algorithm, we propose the use of Bayesian probabilistic models to identify SNVs and investigate a multiple ungapped alignment approach to call indels. For germline variant calling, we model allele counts per site to follow a multinomial conditional distribution. For somatic variant calling, we rely on paired tumor-normal pairs from identical individuals and introduce a hybrid subtraction and joint sample analysis approach by modeling tumor-normal allele counts per site to follow a joint multinomial conditional distribution. A comprehensive performance evaluation has been conducted using a diversity of variant calling benchmarks. For germline variant calling, SNVSniffer demonstrates highly competitive accuracy with superior speed in comparison with the state-of-the-art FaSD, GATK and SAMtools. For somatic variant calling, our algorithm achieves comparable or even better accuracy, at fast speed, than the leading VarScan2, SomaticSniper, JointSNVMix2 and MuTect.ConclusionsSNVSniffers demonstrates the feasibility to develop integrated solutions to fast and efficient identification of germline and somatic variants. Nonetheless, accurate discovery of genetic variations is critical yet challenging, and still requires substantially more research efforts being devoted. SNVSniffer and synthetic samples are publicly available at http://snvsniffer.sourceforge.net.

Abstract P6-06-02: Germline CDH1 mutations in lobular carcinoma in situ

Abstract BACKGROUND: Germline CDH1 mutations are responsible for the increased risk of both gastric cancer and invasive lobular breast cancer (ILC) in families with hereditary diffuse gastric cancer syndrome; yet germline CDH1 mutations in women with ILC without a family history (FH) of gastric cancer are rare. Lobular carcinoma in situ (LCIS) is both a risk factor and non-obligate precursor of ILC and recent data suggest that germline CDH1 mutations may be present in up to 8% of patients with bilateral LCIS +/- ILC; raising questions about the role of genetic testing in this context. The purpose of this study was to determine the frequency of germline CDH1 mutations in a large prospectively followed cohort of patients with pathologically confirmed bilateral LCIS. METHODS: Patients with a biopsy proven history of LCIS, entering surveillance or presenting for surgery (prophylactic or therapeutic mastectomy), between 2005 and 2013 were prospectively identified and enrolled in IRB approved protocols at Memorial Sloan-Kettering Cancer Center for the collection of tissue and/or germline DNA (IRB 01-135, 99-030). All biopsies were reviewed to confirm LCIS and mastectomy specimens were subject to extensive sampling of all quadrants. Cases with confirmed bilateral LCIS were chosen for the primary analysis. Cases where bilateral mastectomy tissue sampling confirmed only unilateral LCIS were included for comparison. Germline DNA was anonymized and analyzed for CDH1 mutations using targeted capture sequencing with baits for all exons of CDH1 on HiSeq2000. Germline single nucleotide variants were called using GATK HaplotypeCaller and insertions/deletions by Varscan and Scalpel. Mutations were manually inspected using the Integrative Genomics Viewer (IGV). Clinical data were abstracted prior to anonymization. RESULTS: Germline DNA was available for 114 patients; 78 underwent bilateral mastectomy for breast cancer (BC), 8 chose prophylactic mastectomy and 28 patients with biopsy proven bilateral LCIS were identified in surveillance. Following mastectomy, tissue sampling confirmed bilateral LCIS in 67/86 (78%) patients, and ruled out bilateral LCIS in 19 patients; yielding 95 patients with bilateral LCIS for the primary analysis. Median age at LCIS diagnosis for bilateral and unilateral cases respectively was 48yrs (range 36-70) and 44 yrs (range 38-63). One patient with bilateral LCIS also reported a FH of gastric cancer. Pathogenic germline CDH1 mutations (D72N (missense) and E35* (nonsense)) were identified in 2/95 (2%) patients with bilateral LCIS, one of whom also had invasive breast cancer (ILC). A germline CDH1 mutation was not identified in the patient with bilateral LCIS and a FH of gastric cancer, nor were CDH1 mutations identified among the 19 patients with unilateral LCIS. CONCLUSIONS: In this cohort of 95 patients with pathologically documented bilateral LCIS +/- BC, the overall frequency of CDH1 germline mutations was 2%; considerably lower than previously reported. To our knowledge this is the largest series to address this question and these findings do not support germline testing for CDH1 mutations in women with bilateral LCIS. Citation Format: Reyes SA, Sakr RA, Schizas M, Towers R, Park AY, Ng CKY, Weigelt B, Reis-Filho JS, King TA. Germline CDH1 mutations in lobular carcinoma in situ. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-06-02.