Quantum chemical profiling of protein mutations via fragment-based DFT.

The completion of the Human Genome Project has unleashed a wealth of human genomics information, but it remains unclear how best to implement this information for the benefit of patients. The standard approach of biomedical research, with researchers pursuing advances in knowledge in the laboratory and, separately, clinicians translating research findings into the clinic as much as decades later, will need to give way to new interdisciplinary models for research in genomic medicine. These models should include scientists and clinicians actively working as teams to study patients and populations recruited in clinical settings and communities to make genomics discoveries-through the combined efforts of data scientists, clinical researchers, epidemiologists, and basic scientists-and to rapidly apply these discoveries in the clinic for the prediction, prevention, diagnosis, prognosis, and treatment of cardiovascular diseases and stroke. The highly publicized US Precision Medicine Initiative, also known as All of Us, is a large-scale program funded by the US National Institutes of Health that will energize these efforts, but several ongoing studies such as the UK Biobank Initiative; the Million Veteran Program; the Electronic Medical Records and Genomics Network; the Kaiser Permanente Research Program on Genes, Environment and Health; and the DiscovEHR collaboration are already providing exemplary models of this kind of interdisciplinary work. In this statement, we outline the opportunities and challenges in broadly implementing new interdisciplinary models in academic medical centers and community settings and bringing the promise of genomics to fruition.

Sequence Kernel Association Tests for the Combined Effect of Rare and Common Variants

Novel missense mutations in the tumor necrosis factor domain of Ectodysplasin-A cause non-syndromic tooth agenesis in two Chinese families.

TCL-495 Evaluation of Breast Implant-Associated Anaplastic Large Cell Lymphoma With Whole Exome and Genome Sequencing.

e20582 Background: Despite advances in therapies, the development of drug resistance in non-small cell lung cancer (NSCLC) poses a significant challenge. The emergence of neoantigen-based therapies offer a promising new frontier which enhances treatment efficacy and reduces associated costs. Methods: A total of 24 patients diagnosed with NSCLC were enrolled in this study. Primary tumor tissues and peripheral blood samples were collected from each patient. Paired whole exome and transcriptomic sequencing data were performed to identify somatic mutations and to quantify their respective expression levels. Somatic single nucleotide variants (SNVs) and small insertions or deletions (inDels) were detected using Strelka algorithm from whole exome sequencing (WES) data. Neoantigen prediction focused on human leukocyte antigens (HLA) type 1. HLA alleles were predicted using WES data encoding adjacent non-cancerous tissue samples, identifying four alleles that were present in at least 50% of the patients. Neoantigens were considered potentially immunogenic if their predicted median IC50 binding scores were ≤500nM while expressed transcripts per million (TPM) exceeds 1 in tumor samples. Results: Using systematic bioinformatics pipeline, we analyzed 24 NSCLC samples with matched tumor tissues and peripheral blood, and detected the 4677 mutations involving 2442 genes, including 2819 missense SNVs, 941 InDels, 229 gene fusion and 688 RNA alternative splicing events, 94.7% of which (4428 mutations involving in 2247 genes) were common with those reported in the TCGA-LUAD dataset. The average number of mutations was 194.8 in NSCLCs. We found that C>T/G>A transitions/ transversions were dominant. Missense mutations were the most frequent types of somatic mutation in the coding sequence regions. 98% of mutated genes were not detected simultaneously with DNA and RNA mutations. ADCK5 , CD151 and EIF3K were identified as potential new driver genes. Genomic variation profiling identified 2213 potential neoantigens with a median IC50 binding score of ≤500nM and a transcript expression level of >1 TPM. Among these, 41 were listed in databases TSNAdb, IEDB, and CTDatabase. A total of 14 genes harboring identical mutations were detected, with the identical mutations observed in over 50% of the patient cohort. Notably, mutations in 14 genes were either gene fusions or RNA alternative splicing events, and core antigenic sequences generated were found to be identical in the majority of patients. Of particular interest is the RPRD2 gene, where an RNA exitrons site was detected in all patient samples, along with the identification of the same neoantigen core sequence. Conclusions: Our analysis suggests that gene fusions and RNA splicing variations are more likely than point mutations to generate shared antigens, underscoring their importance for developing targeted immunotherapies.

484 Background: Molecular alterations and drivers of PSCC, an orphan malignancy, remain unclear. The Cancer Genome Atlas is not studying PSCC and the Catalogue of Somatic Mutations in Cancer has performed targeted analyses only. We report WES of PSCC tumors from a group of patients (pts). Methods: Freshfrozen macrodissected PSCC tumor tissue and adjacent normal tissue samples were procured from the Cooperative Human Tissue Network. DNA was isolated from tissue sections by phenol chloroform extraction. Exome capture was performed with the Agilent SureSelect clinical research exome kit and whole exome-seq was done on the Illumina HiSeq2500 with paired end 100bp chemistry. Raw sequence data in Fastq format were aligned to human reference genome and quantified, and compared by using a local instance of Galaxy (galaxy.uabgrid.uab.edu). These data were analyzed for mutations (SNPs) analysis, by Partek Genomic Suite/Flow(PGS, Partek, St. Louis, MO) for variance calling against human reference genome (hg19) as referenced to dbSNP; and copy number variants (cnv) by FishingCNV tool together with picard tools/samtools/GATK). We focused on missense mutations and amplifications among ≥ 2 tumor samples but not in normal samples as they may cause upregulation of gene/protein function, which may be therapeutically actionable. Results: PSCC tumors were available from 11 patients and adjacent normal tissue from 3 patients. The 10 most common genes with > 4 missense mutations among ≥ 2 tumor samples overall were the following in decreasing order of frequency: MUC4, HLA-DPA1, MUC16, XIRP2, SSPO, TTN, FCGBP, PABPC3, ALPK2 and MKI67. The top upstream transcriptional regulators were PIH1D3, PRDM5, PTK2, Coup-Tf and NBEAL2. When examining candidate actionable genes, recurrent missense alterations were seen in PIK3C2A and PIK3C2G. Additional analysis will study alterations in functional domains and cnv. Conclusions: WES identified a relatively high mutation burden in PSCC withrecurrent missense mutations in multiple genes, notably including the PI3K gene among potentially actionable genes. Validation of these findings and further study of downstream effects is required.

Genetic counseling in direct-to-consumer exome sequencing: a case report.

Exome Sequencing Studies Identify Mutations in STAB2 As a Genetic Risk for Venous Thromboembolic Disease

Application of Trusight Myeloid Panel on Whole Genome Amplified DNA in Myelodysplastic Syndrome Patients

253 Background: We conducted a phase-II study with selumetinib (AZD6244), a small molecule inhibitor of MEK1/2, in advanced biliary tract cancers (BTC). The observed preliminary activity was confirmed with another MEK inhibitor (MEK162) that was tested in a similar patient population. To assess for tumor-specific genetic variants that mediate sensitivity to MEK inhibition in BTC, we performed whole-exome sequencing in patients with an objective response including a complete response to selumetinib. Methods: Normal and tumor DNA from available formalin-fixed paraffin-embedded tissue from biopsies of primary or metastatic tumor from two patients who experienced an objective response underwent whole-exome sequencing. Raw sequence reads were processed with GATK workflow and tumor-specific variants were identified using MuTect and VarScan 2. Following, we assessed the functional consequences of the variants. Copy number changes and potential gene fusion events were also screened. We compared the findings to assess for any commonality between the two samples. Ingenuity Pathway Analysis was used to assess whether the identified somatic variants were intrinsic to the MAPK pathway. Results: 1169 and 628 tumor-specific variants were identified in the two tumor samples. Further analysis demonstrated a similar number of functional and novel variants between the two samples, which were 60 and 53, respectively. No common variants or detectable fusion events were observed between the two samples. Copy number changes were found in chromosomes 1, 5, 6, 9, 10, 12, 17, 20, 21 and 22 in sample one, and in chromosome 11 and 12 on sample two with no common findings. Several variants in genes associated with ERK signaling were present in each tumor sample. Conclusions: Although no common tumor-specific somatic changes of significance several genes associated with ERK signaling were identified in the patients with an objective response. Confirmatory studies investigating the role of the identified genes need to be further investigated.

Germline Mutations in RTEL1 cause Dyskeratosis Congenita

SummaryLung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening in vitro. This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.

Background: Understanding genetic aberrations in cancer has led to discoveries of new targets for cancer therapies. The genomic landscape of Hodgkin lymphoma (HL) has not been fully described. Methods: We performed targeted next generation sequencing (NGS) on 13 archival tumor samples from patients with relapsed/refractory HL treated in the phase I clinical trial with the mTOR inhibitor sirolimus and the HDAC inhibitor vorinostat using the Foundation One NGS panel (Foundation One, Foundation Medicine, MA). Subsequently, we performed whole exome and RNA sequencing on pre- and post-treatment tumor biopsies from 3 patients treated in the same study. Results: In archival samples from 13 HL patients tested using the Foundation One panel, a total of 21 gene aberrations across 13 genes were detected; 12 (92%) tumor samples had mutations in genes involved in immune response, apoptosis, and cell proliferation pathways (SOCS1, PIM1, MCL1, BRCA1, TP53, TNFAIP3, B2M, XPO1, BCL6) and 7 (54%) samples had mutations in DNA repair pathway genes (TP53, BRCA1, ATM, PIM1). In addition, whole exome and RNA sequencing of pre- and post-treatment tumor and germline (peripheral blood mononuclear cells) samples from HL patients treated with sirolimus and vorinostat (complete response, n = 1; stable disease for 3 months, n = 1; progression, n = 1) identified missense point mutations in key histone modification genes not included in the Foundation One panel, including HDAC8 (histone deacetylase 8), JMJD1C (jumonji domain containing 1C), and KDM2A (lysine-specific demethylase 2A) in the patient who progressed on therapy. This same patient additionally had a B2M missense mutation (M1R) affecting the same residue as the B2M event (M1I) identified in the archival cohort. Furthermore, there was a trend towards increased burden of molecular aberrations (median, 67 aberrations) in pre- and post- tumor samples of the patient who progressed compared to the other 2 patients (median, 6 aberrations), who did not progress. Conclusion: While analysis of additional HL specimens is needed, our data suggest that testing for molecular aberrations with NGS is feasible and somatic missense mutations in HDAC8, JMJD1C, and KDM2A may be associated with lack of clinical response to sirolimus and vorinostat. Citation Format: Winnie S. Liang, Bodour Salhia, Adrienne Helland, Shobana Sekar, Ignacio Garrido-Laguna, Michelle Fanale, Yasuhiro Oki, Jason R. Westin, R. Eric Davis, Funda Meric-Bernstam, Filip Janku. Identification of mutations in histone modification genes in Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-176. doi:10.1158/1538-7445.AM2015-LB-176

Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heterogeneity and the investigation of cancer evolution. Aims: By combining laser microdissection and single cell sequencing, we aimed to link tissue morphology and spatial information with sequencing data of the isolated cells. Materials & methods: In our preliminary study, we used fresh frozen tissue specimen of surgically resected material from a colorectal cancer (CRC) patient containing both cancerous and normal adjacent tissue (NAT). From part of the normal and cancerous tissue exome sequencing was performed in bulk (to assess somatic variants), while the other part was subjected to single cell sequencing. Fresh frozen tissues from both CRC and NAT were cryosectioned at -20°C with section thickness ranging from 16 to 25 µm to ensure that a layer of whole cells are present in the slides. Tissue slides were then scanned using a PANNORAMIC 1000 scanner (3DHISTECH Ltd.). After morphologic evaluation single normal colonocytes and cancerous cells were laser microdissected from the NAT and multiple CRC areas (invasive front, differentiated, non-differentiated cells) by using a CellCut Laser Microdissection system (MMI). The isolated cells were subjected to Repli-G Single Cell WGA Kit (Qiagen) and library preparation followed by whole exome sequencing (WES) on NextSeq 550 (Illumina). Blood sample was also collected before surgical treatment, cell-free DNA was isolated and exome sequencing was completed. Bioinformatic analysis was conducted using BaseSpace and GATK4 best practices. Common and unique variants were identified between cells and also compared with the bulk exom and cell free DNA sequencing results of the patient. Identified variants were further investigated using the oncoKB annotator. Results: Both healthy (1) and cancerous epithelial cells (3) were dissected and sequenced successfully. A median depth of coverage of 192 was achieved with a median of 43.3% of coverage of 50x or above in the target region compared to 73.9 and 52.8% 208 and 97.6% in tissue and plasma samples, respectively. Overall, we identified 105, likely oncogenic” and 2, predicted oncogenic” unique variants in the single cells using the oncoKB annotator. Among the cancerous single cells, we identified 10, likely oncogenic” and 1, predicted oncogenic” common variants (such as ARID4B, DNMT3B, MSH6 and HNF1A), while 15, 25 and 7, likely oncogenic” (such as CTLA4, MLH1, MSH2, CDK12, CDKN1B) variants were identified uniquely in the 3 cancerous cells. Conclusion: We were able to dissect, isolate and sequence single cells from CRC and NAT thus combining valuable morphologic information with sequencing data on a single cellular level with maintained spatial information. The distribution of variants among the single cells shows that it is a viable approach to investigate tumor heterogeneity and to link the morphologic phenotypes and genotypes of cancerous cells. Citation Format: William Jayasekara Kothalawala, Alexandra Kalmár, Gitta Szabó, Barbara Kinga Barták, Sára Zsigrai, Zsófia Brigitta Nagy, Ildikó Felletár, Krisztina Andrea Szigeti, István Takács, Béla Molnár. A histology based approach to spatial single cell analysis of colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1690.

Background: Tumor mutation burden (TMB) is emerging as a potential predictive biomarker for the efficacy of immune checkpoint inhibitors. TMB has traditionally been evaluated using whole exome sequencing (WES), an established method for analysis of genomic alterations that requires both tumor and germline DNA. In the phase 3 CheckMate 026 study (NCT02041533) of nivolumab vs chemotherapy in first-line non-small cell lung cancer (NSCLC), exploratory analyses were performed to determine the value of TMB as a predictive biomarker in 312 tumor samples using WES. Patients with baseline TMB in the upper tertile were associated with an increased objective response rate to nivolumab and longer median PFS, relative to patients with TMB in the lowest 2 tertiles and compared with chemotherapy-treated patients in the upper tertile (Carbone et al. N Engl J Med. 2017). Increasingly, targeted cancer gene panels, including the FoundationOne® (F1) assay, are being assessed as an alternative to WES for clinical use. To determine the feasibility of transitioning from WES to a potential in vitro diagnostic next-generation sequencing (NGS) platform for TMB analysis, we performed a study comparing values generated by WES with those generated by the F1 assay. Methods: Using the 2 hybridization-capture/NGS methods (WES and F1), TMB was assessed in formalin-fixed, paraffin-embedded NSCLC tumor samples. For WES, coding regions of 21,522 genes were analyzed, with TMB defined as the total number of missense mutations in the tumor exome. The F1 assay is a targeted gene panel of 315 cancer-related genes (Frampton et al. Nat Biotechnol. 2013), with TMB defined as the number of somatic mutations per megabase of sequenced tumor genome. Results: TMB data from 44 NSCLC samples were available on both F1 and WES platforms. We determined an empirical method to convert TMB values derived from WES into those derived from F1 to establish calibration between the 2 methodologies. TMB assessed by WES and by F1 was highly correlated (Spearman's r=0.9). As an additional test of concordance between the platforms, we examined agreement between the assays around the median WES value of 148 mutations. This value projects to 7.64 mutations per megabase on F1. The overall agreement between F1 and WES around these values was 86% (95% Wilson CI, 73-94). Positive and negative agreements between the 2 hybridization-capture/NGS methods were both 86% (95% Wilson CI, 67-95). Conclusion: Bridging TMB analysis by WES to F1 facilitates the transition of WES-derived biomarker data to the clinical in vitro diagnostic F1. This study demonstrates the feasibility of harmonization of TMB results across testing platforms, which provides alternative TMB testing options. Citation Format: Joseph D. Szustakowski, George Green, William J. Geese, Kim Zerba, Han Chang. Evaluation of tumor mutation burden as a biomarker for immune checkpoint inhibitor efficacy: A calibration study of whole exome sequencing with FoundationOne® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5528.