Whole-exome Sequencing Of DNA Research Articles

Primary Upper Extremity Lymphedema Caused by a CELSR1 Variant

Primary lymphedema of the upper extremity is rare and often is associated with syndromic or generalized lymphedema. The purpose of the study was to identify novel causes for primary lymphedema of the arm. A 17-year-old healthy male with right upper extremity swelling since infancy was confirmed to have lymphedema by lymphoscintigraphy. He subsequently developed right leg disease in adolescence and his mother had lower extremity lymphedema as well. Whole-exome sequencing of genomic DNA from the subject identified a frameshift deletion resulting in a premature stop codon in exon 3 of the CELSR1 gene hg19: chr22:46,835,160_46,835,166del (c.4326_4332del; p.T1443Gfs*14). The variant was confirmed in the patient and his mother by Sanger sequencing. A novel variant in CELSR1 causes nonsyndromic upper extremity lymphedema; other variants in this gene previously have been associated with lower extremity disease.

Predictive Markers of Response to Neoadjuvant Durvalumab with Nab-Paclitaxel and Dose-Dense Doxorubicin/Cyclophosphamide in Basal-Like Triple-Negative Breast Cancer.

We examined gene expression, germline variant, and somatic mutation features associated with pathologic response to neoadjuvant durvalumab plus chemotherapy in basal-like triple-negative breast cancer (bTNBC). Germline and somatic whole-exome DNA and RNA sequencing, programmed death ligand 1 (PD-L1) IHC, and stromal tumor-infiltrating lymphocyte scoring were performed on 57 patients. We validated our results using 162 patients from the GeparNuevo randomized trial. Gene set enrichment analysis showed that pathways involved in immunity (adaptive, humoral, innate), JAK-STAT signaling, cancer drivers, cell cycle, apoptosis, and DNA repair were enriched in cases with pathologic complete response (pCR), whereas epithelial-mesenchymal transition, extracellular matrix, and TGFβ pathways were enriched in cases with residual disease (RD). Immune-rich bTNBC with RD was enriched in CCL-3, -4, -5, -8, -23, CXCL-1, -3, -6, -10, and IL1, -23, -27, -34, and had higher expression of macrophage markers compared with immune-rich cancers with pCR that were enriched in IFNγ, IL2, -12, -21, chemokines CXCL-9, -13, CXCR5, and activated T- and B-cell markers (GZMB, CD79A). In the validation cohort, an immune-rich five-gene signature showed higher expression in pCR cases in the durvalumab arm (P = 0.040) but not in the placebo arm (P = 0.923) or in immune-poor cancers. Independent of immune markers, tumor mutation burden was higher, and PI3K, DNA damage repair, MAPK, and WNT/β-catenin signaling pathways were enriched in germline and somatic mutations in cases with pCR. The TGFβ pathway is associated with immune-poor phenotype and RD in bTNBC. Among immune-rich bTNBC RD, macrophage/neutrophil chemoattractants dominate the cytokine milieu, and IFNγ and activated B cells and T cells dominate immune-rich cancers with pCR.

Satraplatin Demonstrates High Cytotoxic Activity Against Genetically Defined Lymphoid Malignancies

Satraplatin is an oral platinum analog with proven clinical efficacy and a more favorable toxicity profile, although with increased hematotoxicity, when compared to cisplatin. Hence, we carried out a systematic biomarker analysis to identify hematological malignancies with high susceptibility to satraplatin. Half-maximal inhibitory concentrations (IC50) for satraplatin and cisplatin were determined for 66 different cancer cell lines by CTG Luminescent Cell Viability Assay. In a second step, whole transcriptome RNA sequencing and whole-exome DNA sequencing technology followed by unbiased analysis of gene expression, gene mutation and copy number levels were performed and correlated with drug efficacy. Satraplatin was significantly more active against hematological malignancies compared to solid organ cancer. In addition, satraplatin showed a significantly more potent antiproliferative activity compared to cisplatin in most lymphoma cell lines achieving sub micromolar IC50 values. Single BCL2 apoptosis regulator (BCL2) gene mutation and 9p21 copy-number deletions including S-methyl-5'-thioadenosine phosphorylase (MTAP) deficiency were identified as key characteristics for high sensitivity to satraplatin. Satraplatin demonstrated a high cytotoxic activity in genetically well-defined hematological malignancies which is distinct from that of cisplatin. MTAP deficiency was identified as biomarker of enhanced satraplatin efficacy in hematological cancer-derived cell lines. These data in combination with the lipophilicity of satraplatin provide the rationale for targeting specific lymphatic entities such as primary central nervous system lymphoma and cutaneous T-cell lymphoma to improve clinical outcome.

Genetic Characteristics Associated With Drug Resistance in Lung Cancer and Colorectal Cancer Using Whole Exome Sequencing of Cell-Free DNA.

Circulating cell-free DNA (cfDNA) can be used to characterize tumor genomes through next-generation sequencing (NGS)-based approaches. We aim to identify novel genetic alterations associated with drug resistance in lung cancer and colorectal cancer patients who were treated with EGFR-targeted therapy and cytotoxic chemotherapy through whole exome sequencing (WES) of cfDNA. A cohort of 18 lung cancer patients was treated with EGFR TKI or cytotoxic chemotherapy, and a cohort of 37 colorectal cancer patients was treated with EGFR monoclonal antibody or cytotoxic chemotherapy alone. Serum samples were drawn before and after development of drug resistance, and the genetic mutational profile was analyzed with WES data. For 110 paired cfDNA and matched germline DNA WES samples, mean coverage of 138x (range, 52–208.4x) and 47x (range, 30.5–125.1x) was achieved, respectively. After excluding synonymous variants, mutants identified in more than two patients at the time of acquired resistance were selected. Seven genes in lung cancer and 16 genes in colorectal cancer were found, namely, APC, TP53, KRAS, SMAD4, and EGFR. In addition, the GPR155 I357S mutation in lung cancer and ADAMTS20 S1597P and TTN R7415H mutations in colorectal cancer were frequently detected at the time of acquired resistance, indicating that these mutations have an important function in acquired resistance to chemotherapy. Our data suggest that novel genetic variants associated with drug resistance can be identified using cfDNA WES. Further validation is necessary, but these candidate genes are promising therapeutic targets for overcoming drug resistance in lung cancer and colorectal cancer.

Whole-exome DNA sequencing in childhood anxiety disorders identifies rare de novo damaging coding variants.

Genetic factors contribute to the development of anxiety disorders, yet few risk genes have been previously identified. One genomic approach that has achieved success in identifying risk genes in related childhood neuropsychiatric conditions is investigations of de novo variants, which has yet to be leveraged in childhood anxiety disorders. We performed whole-exome DNA sequencing in 76 parent-child trios (68 trios after quality control) recruited from a childhood anxiety disorder clinic and compared rates of rare and ultra-rare de novo variants with 790 previously sequenced control trios (783 trios after quality control). We then explored overlap with risk genes for other neuropsychiatric conditions and enrichment in biologic pathways. Rare and ultra-rare de novo likely gene disrupting and predicted damaging missense genetic variants are enriched in anxiety disorder probands compared with controls (rare variant rate ratio 1.97, 95% confidence interval [CI]: 1.11-3.34, p = .03; ultra-rare variant rate ratio 2.59, 95% CI: 1.35-4.70, p = .008). These de novo damaging variants occur in individuals with a variety of childhood anxiety disorders and impact genes that have been associated with other neuropsychiatric conditions. Exploratory network analyses reveal enrichment of deleterious variants in canonical biological pathways. These findings provide a path for identifying risk genes and promising biologic pathways in childhood anxiety disorders by de novo genetic variant detection. Our results suggest the discovery potential of applying this approach in larger anxiety disorder cohorts to advance our understanding of the underlying biology of these common and debilitating conditions.

Molecular Correlates of Venous Thromboembolism (VTE) in Ovarian Cancer.

Simple SummaryOvarian cancer is the most lethal form of gynecologic cancer. The prognosis is worse for patients with ovarian cancer who also develop a blood clot in their legs or lungs, known as a venous thromboembolism (VTE). Between 10-30% of women with ovarian cancer will develop a VTE but the mechanism behind this high incidence is not well understood. The aim of this study was to assess for the presence of tumor-specific molecular markers of VTE in order to better understand the relationship between ovarian cancer and VTE. We performed a comprehensive analysis assessing the proteomic and genomic profiles of 32 patients with ovarian cancer and identified that there are molecular differences in patients with a VTE compared to those without.Background: The incidence of venous thromboembolism (VTE) in patients with ovarian cancer is higher than most solid tumors, ranging between 10–30%, and a diagnosis of VTE in this patient population is associated with worse oncologic outcomes. The tumor-specific molecular factors that may lead to the development of VTE are not well understood. Objectives: The aim of this study was to identify molecular features present in ovarian tumors of patients with VTE compared to those without. Methods: We performed a multiplatform omics analysis incorporating RNA and DNA sequencing, quantitative proteomics, as well as immune cell profiling of high-grade serous ovarian carcinoma (HGSC) samples from a cohort of 32 patients with or without VTE. Results: Pathway analyses revealed upregulation of both inflammatory and coagulation pathways in the VTE group. While DNA whole-exome sequencing failed to identify significant coding alterations between the groups, the results of an integrated proteomic and RNA sequencing analysis indicated that there is a relationship between VTE and the expression of platelet-derived growth factor subunit B (PDGFB) and extracellular proteins in tumor cells, namely collagens, that are correlated with the formation of thrombosis. Conclusions: In this comprehensive analysis of HGSC tumor tissues from patients with and without VTE, we identified markers unique to the VTE group that could contribute to development of thrombosis. Our findings provide additional insights into the molecular alterations underlying the development of VTE in ovarian cancer patients and invite further investigation into potential predictive biomarkers of VTE in ovarian cancer.

Hornerin deposits in neuronal intranuclear inclusion disease: direct identification of proteins with compositionally biased regions in inclusions

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.

Novel PTCH1 and concurrent TP53 mutations in four patients with numerous non-syndromic basal cell carcinomas: The paradigm of oncogenic synergy.

There has been a significant increase in basal cell carcinoma (BCC) incidence, the most common cancer in humans and the age of presentation with the first diagnosis of BCC has decreased in past decades. In this study, we investigated the possibility of genetic markers that can lead to earlier and closer observation of patients at high risk for development of multiple BCCs. The overall goal is to decrease the morbidity and the economic burden of diagnosis and treatment of recurring and/or advanced BCCs. Four patients with numerous BCCs, some of them exceptionally large, were included in this study. A sample of representative BCCs, normal non-sun-exposed skin and blood samples were obtained from each patient. Whole-exome sequencing of DNA was conducted on all samples, and a series of bioinformatics filtering was performed to identify potentially pathogenic sequence variants. The analysis of the data resulted in detection of oncogenic mutations in PTCH1, two of which being novel, and concurrent mutations in TP53 in BCC tumours of all four patients. Such mutations may explain the numerous and postexcision recurring nature of the BCCs of exceptionally large size observed in all these patients, and they can be suggested to serve as a genetic marker for high-risk patients for early detection, prognostication and close follow-up.

Pathogenic Germline Variants in Multiple Myeloma

BACKGROUND: There is growing evidence supporting inherited predisposition to multiple myeloma (MM). Epidemiologic studies have shown that 1st-degree relatives of MM patients (pts) have a 2-4 fold increase in risk of developing MGUS or MM. Genome-wide association studies (GWAS) have identified common SNPs as well as rare high-penetrance variants that collectively explain ~16% of the estimated heritability of multiple myeloma (PMID 30213928). Pathogenic/likely-pathogenic germline variants (PGV) in hereditary cancer genes (HCG) are common in adult cancer patients (~8%, PMID 29625052), but prevalence in MM is not known. The aim of our study is to investigate the occurrence of PGV in newly-diagnosed MM (NDMM), and to describe clinical characteristics & outcomes of carriers.METHODS: We analyzed MMRF CoMMpass data (version IA16) and identified 895 NDMM pts for whom whole-exome sequencing of germline DNA was available. We used the clinical annotation pipeline from Sema4, a CLIA/CAP certified genetic testing laboratory, to identify pts with PGV according to ACMG variant classification guidelines. We compared clinical characteristics & disease phenotypes of PGV carriers vs non-carriers. We used Chi-Square and Fisher's Exact tests to assess statistical significance, which we defined as a two-sided p value < 0.05. Logistic regression models were used for multivariate analyses. Kaplan-Meier method and Cox proportional-hazards models were used for uni- and multivariate survival analysis, respectively. Bonferroni method was used to account for multiple testing.RESULTS: We identified 83 PGV in 31 distinct HCG in 79 (8.8%) of 895 NDMM pts (83% European ancestry) [Figure 1A]. Most PGV involved DNA damage repair (DDR) genes (78%), and homologous recombination (HR) genes were the most commonly mutated (34%). PGV in CHEK2 were the most common (n=10, 1.1% of all MM pts). 2 pts carried PGV in TP53 and reported extensive family history of Li-Fraumeni-associated cancers (breast, sarcoma, gastric & melanoma). 6 pts had germline mismatch repair (MMR) gene defects (1:149, considerably higher than the estimated prevalence of Lynch syndrome in Western populations). 4 pts carried PGV in BRCA2 (previously identified in a family study as a potential MM predisposition gene, PMID 11904319). MM pts with a family history of hematologic malignancy (leukemia, lymphoma or MM) in a 1st or 2nd-degree relative were significantly more likely to carry PGV (22 vs 7.6%, OR=3.3, p<0.001), an association that remained significant in MVA (OR=4.1, p<0.001). CHEK2 variants emerged as leading drivers of this correlation (OR 18.2, 95% CI 4.1-75, adjusted p<0.01), & especially protein-truncating founder variant c.1100delC. Likelihood of being diagnosed w/ MM before age 40 was significantly higher in PGV carriers (6.3 vs 1.8%, OR=3.7, p=0.025). 25% of those younger than 40 y/o carried PGV, but none of these were in DDR-HR genes, a notable difference with other age groups (0 vs 41%, p=0.02). 2/6 MMR PGV were detected in pts diagnosed before age 40. In univariate survival analysis, DDR-PGV carriers had a significant PFS1 advantage over non-carriers (median 52 vs 35 months, p=0.008), as well as a non-significant OS advantage (p=0.08). PFS1 difference remained significant in MVA after adjusting for age, ISS stage, high-risk cytogenetics, treatment type & transplant status (OR 0.65, 95% CI 0.44-0.97, p=0.03) [Figure 1B].CONCLUSIONS: PGV in HCG were common (8.8%) in this large cohort of NDMM pts of predominantly European ancestry, especially in those with a family history of hematologic malignancy (1:4, with high prevalence of CHEK2 variants & particularly protein-truncating founder variant c.1100delC), and in those diagnosed before age 40 (1:4). Routine screening in high-prevalence subgroups might be warranted, as carriers may benefit from counseling and enrollment in early cancer detection programs. We observed a clinically and statistically significant PFS1 advantage in carriers of PGV in DDR genes, possibly due to increased sensitivity to MM therapies, a well-described phenomenon in other cancer types (PMID 33158305). Prospective validation of these findings is needed to better understand prognostic & therapeutic implications of PGV in MM. [Display omitted] DisclosuresChari: Karyopharm: Consultancy; Takeda Pharmaceutical Company: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Research Funding; Amgen: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy, Research Funding; Sanofi Genzyme: Consultancy; Oncopeptides: Consultancy; Antegene: Consultancy; Glaxosmithkline: Consultancy; Secura Bio: Consultancy. Richard: Karyopharm, Janssen: Honoraria. Richter: Sanofi: Consultancy; Antengene: Consultancy; Karyopharm: Consultancy; BMS: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau; X4 Pharmaceuticals: Consultancy; Oncopeptides: Consultancy; Adaptive Biotechnologies: Consultancy; Secura Bio: Consultancy; Astra Zeneca: Consultancy. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Genomic and Transcriptional Characterization of Primary Mediastinal Large B Cell Lymphoma

Introduction: Primary mediastinal large B-cell lymphoma (PMBL) is a rare non-Hodgkin lymphoma subtype that occurs predominantly in young adults, with an overall favorable prognosis. The cell of origin is presumed to be thymic medullary B-cells and the gene expression profile of PMBL is similar to classic Hodgkin lymphoma. Recent studies have begun unravelling the genomic alterations underlying PMBL. Frequent, recurrent mutations (e.g. B2M, TNFAIP3, SOCS1, STAT6, GNA13) have been reported, but most of the studies have analyzed a small number of cases. To gain further insights into disease biology, we recruited 63 cases of PMBL as part of the Atlas of Blood Cancer Genomes (ABC-G) initiative, a consortium consisting of 25 institutions.Methods: Formalin-fixed paraffin-embedded (FFPE) biopsies and clinical data were collected. All cases were subjected to centralized review by an experienced panel of hematopathologists to ensure accurate diagnosis. Whole-exome DNA and RNA sequencing was performed using the Illumina platform and the DNA and RNA reads aligned to the GRCh38 genome and transcriptome respectively. Exonic variants were filtered using a set of paired normal samples and population-based databases to identify putative driver mutations, which were then aggregated at the gene level. Mutational analysis was performed on 56 samples that passed quality filtering and expression analysis on 45 samples. RNAseq data was normalized using DESeq2.Results: The cohort included samples from 16 males and 24 females, with a median age of 33 years (range 16 - 72) at the time of diagnosis. The majority of patients were treated with R-CHOP (47%) or R-EPOCH (43%), with 93% of patients surviving through the end of follow-up (median follow-up: 60.1 months). Besides the known recurrent mutations involving the JAK-STAT (STAT6 -21%, SOCS1 - 26%), NFKB (TNFAIP3 - 27%, NFKB1A - 7%), immune escape (B2M - 20%, LTB - 11%, IRF8 - 9%, IRF4 -9%), and chromatin modification (ZNF217 - 16%, CREBBP - 11%, KMT2D -11%) pathways , we discovered recurrent somatic variants in novel candidate driver genes in this disease, including NOTCH4 (7%), DICER1 (11%), MCL1 (7%), amongst others. EZH2, EP300, and XPO1 mutations were not detected. CIITA mutations and fusions were observed in 14% and 11% of cases, respectively, with novel partner genes (IGHA2, IGHG1, CDC6) detected in 67% of the fusion positive cases. Copy number alterations included gains at 2p16.1 (REL - 20%) and 9p24.2 (JAK2/PDL1/PDL2 - 24%), as well as loci not previously implicated in PMBL, 8q24.3 and 9q34.3 (each in 20%). Of note, CIITA alterations and 9p24 gains were virtually mutually exclusive, highlighting diverse mechanisms of immune escape in this entity. The transcriptomes of cases harboring CIITA alterations demonstrated differential enrichment of genes involved in protein glycosylation. The PMBLs in our series showed significant enrichment of the reported PMBL genetic classifier score, compared to nodal diffuse large B cell lymphoma (DLBCL) (p=0.0003). Finally, the gene expression profile of thymic B cells was more similar to that of PMBL than nodal DLBCL (p=0.0144).Conclusions: Our study, representing one of the largest comprehensive genomic and transcriptomic analyses of PMBL, expands the mutational landscape of PMBL, provides evidence for biologically distinct disease subsets and suggests an origin of PMBLs from thymic B-cells. DisclosuresHsi: AbbVie: Research Funding; Eli Lilly: Research Funding; Cytomx: Honoraria; Seattle Genetics: Honoraria. McKinney: BTG: Consultancy; Celgene: Consultancy, Research Funding; Epizyme: Consultancy; Genetech: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Molecular Templates: Consultancy, Research Funding; Nordic Nanovector: Research Funding; Novartis: Research Funding; Pharmacyclics: Consultancy; Verastem: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau. Jaye: Stemline Therapeutics: Honoraria. Cohen: Genentech, Takeda, BMS/Celgene, BioInvent, LAM, Astra Zeneca, Novartis, Loxo/Lilly: Research Funding; Janssen, Adaptive, Aptitude Health, BeiGene, Cellectar, Adicet, Loxo/Lilly, AStra ZenecaKite/Gilead: Consultancy. Behdad: Lilly: Speakers Bureau; Roche/Foundation Medicine: Speakers Bureau; Thermo Fisher: Speakers Bureau. Dave: Data Driven Bioscience: Current equity holder in publicly-traded company.

PIEZO1 Mutation May Determine Early Onset of Clinical Manifestation of Anemia of Myelodysplastic Syndromes

BACKGROUNDAnemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether “young”, not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIMWe considered a cohort of 21 consecutive MDS cases with age at diagnosis < 60 years and anemia, referred to our MDS Unit for a second opinion. METHODSAfter bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTSFive of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF >25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSIONOnset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 “young” MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of “young” MDS. DisclosuresSanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Controversy on the management of patients carrying RET p.V804M mutation.

RET p.V804M is classified as a moderate risk mutation for familial medullary thyroid cancer (FMTC). There is a significant controversy on the management of patients carrying this mutation. We describe a family incidentally discovered to have this mutation and review the literature on RET p.V804M mutation. The proband was born to first-degree relative parents. He was noticed to have hypertrophy of some parts of the body and vascular skin changes. Whole-exome sequencing of DNA extracted from a skin biopsy showed a mutation in the PIK3CA (c.3132T>G, p.ASN1044LYS). This variant was not found in DNA extracted from blood. This confirmed the diagnosis of CLOVES syndrome (Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi and Scoliosis, skeletal or spinal anomalies). Another incidentally found mutation in the skin biopsy and blood sample was RET p.V804M. Although there was no family history of MTC or MEN 2 syndromes, family screening revealed RET p.V804M mutation and FMTC in the proband's father, paternal grandmother, one sister, and one aunt. There was significant interfamilial heterogeneity in the age of presentation and pathology. A review of literature showed that RET p.V804M mutation is a moderate risk mutation associated with late-onset FMTC, usually at middle to old age. Despite the controversy and the heterogeneous presentation of patients with RET p.V804M mutation, our study and review of the literature suggest that this seemingly "low" risk mutation is associated with late-onset but potentially aggressive MTC. This indicates the need for follow-up and timely intervention based on calcitonin level elevation.

P-061: Single-cell whole-exome DNA sequencing traces clonal trajectory in paired evolution of MGUS to multiple myeloma

<h3>Background</h3> Accurate measurement of heterogeneity and reconstruction of evolutionary pathways are key to decipher clonal dynamics in tumour origins. Although bulk population genetic sequencing data has been used extensively to understand the life history of tumours, sequencing single cell DNA is likely to provide a much higher resolution of clonal dynamics and yield new insights into competing cellular populations with different genotypes. This is of significant importance when clonal evolution from a benign state to malignant disease is being considered, such as in the clonal evolution of MGUS to multiple myeloma (MM). <h3>Methods</h3> To this end, we further refined a multiple displacement amplification-based single cell whole-exome sequencing (scWES) protocol and applied it to examine MM cells (2 cases), and in one of these cases, probed evolution from a pre-existing MGUS stage in a paired setting. Single tumour cells were isolated from CD138+CD38+ fluorescence-activated cell sorted populations and CD3+ for T cells as germline controls. We developed a novel computational pipeline to accurately call somatic SNVs/indels and copy number aberration (CNA) events in the form of loss-of-heterozygosity (LOH) and gains in each single cell, identified drivers at the genome-wide level and reconstructed the tumour phylogeny while estimating and accounting for allelic dropout (N=176 single cells, 25× each cell). <h3>Results</h3> By focusing on the patient that allowed comparison of single cells at pre- and post-malignancy states, we show that pre-malignant subclones can be readily identified, and strikingly that malignant cells arise and expand from one of the MGUS subclones in the combined tumour phylogeny. <h3>Conclusions</h3> These results suggest that this scWES based method is a pragmatic approach to investigate clonal dynamics by allowing: 1) the analysis of genetic heterogeneity at the genome-wide level (much higher resolution than targeted panels), and 2) at low sequencing costs across large set of cells when compared with whole genome sequencing-based methods.

De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

[Figure: see text].

Using DNA sequencing data to quantify T cell fraction and therapy response.

The immune microenvironment influences tumour evolution and can be both prognostic and predict response to immunotherapy1,2. However, measurements of tumour infiltrating lymphocytes (TILs) are limited by a shortage of appropriate data. Whole-exome sequencing (WES) of DNA is frequently performed to calculate tumour mutational burden and identify actionable mutations. Here we develop T cell exome TREC tool (T cellExTRECT), a method for estimation of T cell fraction from WES samples using a signal from T cell receptor excision circle (TREC) loss during V(D)J recombination of the T cell receptor-α gene (TCRA (also known as TRA)). TCRA T cell fractioncorrelates with orthogonal TIL estimates and is agnostic to sample type. Blood TCRA T cell fraction is higher in females than in males and correlates with both tumour immune infiltrate and presence of bacterial sequencing reads. Tumour TCRA T cell fraction is prognostic in lung adenocarcinoma. Using a meta-analysis of tumours treated with immunotherapy, we show that tumour TCRA T cell fraction predicts immunotherapy response, providing value beyond measuring tumour mutational burden. Applying T cell ExTRECT to a multi-sample pan-cancer cohort reveals a high diversity of the degree of immune infiltration within tumours. Subclonal loss of 12q24.31-32, encompassing SPPL3, is associated with reduced TCRA T cell fraction. T cellExTRECT provides a cost-effective technique to characterize immune infiltrate alongside somatic changes.

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

SummaryAging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Processing and Analysis of Tissue Samples from Esophageal Cancer Patients in an African Setting.

Although infectious diseases continue to present a major health care problem in Africa, the incidence of cancer is increasing rapidly on the African continent and this merits an increased investment in cancer research in low to medium resource settings. Esophageal squamous cell carcinoma (ESCC) has a high incidence in Eastern and Southern Africa, with late clinical presentation and a very poor prognosis. There is limited research on the molecular pathology of this cancer in Africa, partly as a result of a lack of infrastructure for biobanking and sample processing in many African countries. The aim of this study was to establish a practical and robust workflow to collect, store, and process esophageal cancer samples such that both the tissue architecture and quality of the samples would be preserved and suitable for future genomic research. We developed a workflow that allows storage of fresh biopsy tissue in sterile Eppendorf tubes containing RNAlater, an efficient RNAse inhibitor. We collected 142 ESCC biopsy samples and showed that storage in RNAlater for up to 18 months did not alter tissue morphology, thus allowing histologic assessment by experienced pathologists and determination of tumor content in each biopsied sample. DNA and RNA extracted from tissue samples was assessed for purity, molecular size, and yield. The quantity and quality of nucleic acids obtained were suitable for genomic applications, and whole-exome sequencing of DNA from tumor tissues produced sequence data with a high proportion of both usable reads and correct base calling. We conclude that this workflow may be applicable to a wide range of malignancies for future genomic research in low-resource settings.

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3′ UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Sudden cardiac death due to ARVC in the young: molecular autopsy by whole exome sequencing of DNA from dried blood spots (DBS) collected at birth

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Uppsala University Hospital (ALF) and Marcus Börgström Foundation Background The post-mortem investigation of cases with sudden cardiac death (SCD) is critical for risk stratification and adequate care of surviving relatives. Genetic testing is limited by access to DNA from the proband, as blood samples are not routinely collected at the time of death, or DNA of poor quality obtained from formalin-fixed paraffin-embedded (FFPE) tissues. Whole exome sequencing (WES) on minute amounts of DNA has opened up for the possibility to use DNA from Dried Blood Spots (DBS). Purpose To evaluate the feasibility of WES on DNA from archived DBS collected at the time of newborn screening in cases of SCD. Methods Through National Health Databases and Registries, we identified all cases of SCD in the young (&amp;lt;35 y.o.a.) in Sweden with a post-mortem diagnosis of ARVC between 2000 and 2010 (n = 22). Clinical records, medical and family histories, and autopsy findings were collected. Surviving parents were invited to participate. DNA was extracted from samples in National Biobanks such as DBS collected at birth (n = 19), formalin-fixed paraffin-embedded (FFPE) heart tissues (n = 8) from autopsies, and frozen blood samples (n = 3). Patient and parental DNA samples (when available) underwent WES. DNA- and sequencing-quality check (QC) metrics were compared between two different sequencing platforms and specimen types. We analysed 392 cardiomyopathy and arrhythmia genes and variants were classified as pathogenic or likely pathogenic according to the criteria from the American College of Medical Genetics. Results A higher quality but a lower yield of DNA (7.3 – 140 ng) was obtained from DBS when compared to FFPE samples (515 – 3065 ng). However, 100% of DBS vs. 62,5% of FFPE samples passed QC before sequencing. The average mean target depth for DBS samples (160X) was similar to that in blood samples (155X) and higher than FFPE (82X). Furthermore, &amp;gt;97% of target regions were covered with read depths &amp;gt;30X in DNA from DBS and blood compared to 88% for FFPE samples (Min 63%; Max 97%). Analysis of WES data uncovered clinically relevant variants in 12 out of 19 families (63%), of which four were located in ARVC genes. In six cases, the molecular autopsy identified another arrhythmogenic syndrome. Additionally, we identified one SCD with hemochromatosis and one with myotonic dystrophy as a possible contributing cause. In two additional families, variants of unknown significance fulfilled criteria for further segregation analysis. The concordance of positive results between different tissue samples was 100%. Conclusions DNA obtained from DBS has a quality comparable to that of blood and is a reliable source for WES-based molecular autopsy that may improve the diagnostic yield in cases of SCD. In a proportion of cases diagnosed post-mortem with ARVC, our molecular autopsy analysis pointed to a different diagnosis, highlighting its importance for the correct medical care of surviving relatives.

Abstract PD13-01: Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases

Abstract BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains &amp;lt; 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR &amp;lt; 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p &amp;lt; 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.