Somatic Loss Research Articles

Prevalence of cancer susceptibility variants in patients with multiple Lynch syndrome related cancers

Along with early-onset cancers, multiple primary cancers (MPCs) are likely resulting from increased genetic susceptibility; however, the associated predisposition genes or prevalence of the pathogenic variants genes in MPC patients are often unknown. We screened 71 patients with MPC of the stomach, colorectal, and endometrium, sequencing 65 cancer predisposition genes. A subset of 19 patients with early-onset MPC of stomach and colorectum were further evaluated for variants in cancer related genes using both normal and tumor whole exome sequencing. Among 71 patients with MPCs, variants classified to be pathogenic were observed in 15 (21.1%) patients and affected Lynch Syndrome (LS) genes: MLH1 (n = 10), MSH6 (n = 2), PMS2 (n = 2), and MSH2 (n = 1). All carriers had tumors with high microsatellite instability and 13 of them (86.7%) were early-onset, consistent with LS. In 19 patients with early-onset MPCs, loss of function (LoF) variants in RECQL5 were more prevalent in non-LS MPC than in matched sporadic cancer patients (OR = 31.6, 2.73–1700.6, p = 0.001). Additionally, there were high-confidence LoF variants at FANCG and CASP8 in two patients accompanied by somatic loss of heterozygosity in tumor, respectively. The results suggest that genetic screening should be considered for synchronous cancers and metachronous MPCs of the LS tumor spectrum, particularly in early-onset. Susceptibility variants in non-LS genes for MPC patients may exist, but evidence for their role is more elusive than for LS patients.

Haplotype-resolved germline and somatic alterations in renal medullary carcinomas

BackgroundRenal medullary carcinomas (RMCs) are rare kidney cancers that occur in adolescents and young adults of African ancestry. Although RMC is associated with the sickle cell trait and somatic loss of the tumor suppressor, SMARCB1, the ancestral origins of RMC remain unknown. Further, characterization of structural variants (SVs) involving SMARCB1 in RMC remains limited.MethodsWe used linked-read genome sequencing to reconstruct germline and somatic haplotypes in 15 unrelated patients with RMC registered on the Children’s Oncology Group (COG) AREN03B2 study between 2006 and 2017 or from our prior study. We performed fine-mapping of the HBB locus and assessed the germline for cancer predisposition genes. Subsequently, we assessed the tumor samples for mutations outside of SMARCB1 and integrated RNA sequencing to interrogate the structural variants at the SMARCB1 locus.ResultsWe find that the haplotype of the sickle cell mutation in patients with RMC originated from three geographical regions in Africa. In addition, fine-mapping of the HBB locus identified the sickle cell mutation as the sole candidate variant. We further identify that the SMARCB1 structural variants are characterized by blunt or 1-bp homology events.ConclusionsOur findings suggest that RMC does not arise from a single founder population and that the HbS allele is a strong candidate germline allele which confers risk for RMC. Furthermore, we find that the SVs that disrupt SMARCB1 function are likely repaired by non-homologous end-joining. These findings highlight how haplotype-based analyses using linked-read genome sequencing can be applied to identify potential risk variants in small and rare disease cohorts and provide nucleotide resolution to structural variants.

Role of miR-24 in Multiple Endocrine Neoplasia Type 1: A Potential Target for Molecular Therapy.

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant inherited multiple cancer syndrome of neuroendocrine tissues. Tumors are caused by an inherited germinal heterozygote inactivating mutation of the MEN1 tumor suppressor gene, followed by a somatic loss of heterozygosity (LOH) of the MEN1 gene in target neuroendocrine cells, mainly at parathyroids, pancreas islets, and anterior pituitary. Over 1500 different germline and somatic mutations of the MEN1 gene have been identified, but the syndrome is completely missing a direct genotype-phenotype correlation, thus supporting the hypothesis that exogenous and endogenous factors, other than MEN1 specific mutation, are involved in MEN1 tumorigenesis and definition of individual clinical phenotype. Epigenetic factors, such as microRNAs (miRNAs), are strongly suspected to have a role in MEN1 tumor initiation and development. Recently, a direct autoregulatory network between miR-24, MEN1 mRNA, and menin was demonstrated in parathyroids and endocrine pancreas, showing a miR-24-induced silencing of menin expression that could have a key role in initiation of tumors in MEN1-target neuroendocrine cells. Here, we review the current knowledge on the post-transcriptional regulation of MEN1 and menin expression by miR-24, and its possible direct role in MEN1 syndrome, describing the possibility and the potential approaches to target and silence this miRNA, to permit the correct expression of the wild type menin, and thereby prevent the development of cancers in the target tissues.

Abstract 448: High prevalence of pathogenic germline variants in patients with oncogene-driven non-small cell lung cancer

Abstract BACKGROUND: Preliminary data has highlighted inherited predisposition to lung cancer related to certain genes. The frequency of pathogenic germline variants (PGV) PGV in patients (pts) with lung cancer according to the presence of an oncogenic driver is unknown. We studied the PGV of genes predisposing to cancer in pts with non-small cell lung cancer (NSCLC), and the somatic molecular profile of lung tumors. METHODS: Retrospective study of whole exome sequencing (WES) from tissue biopsies performed in pts with advanced NSCLC enrolled, after signature of the inform consent, in the MOSCATO/MATCH-R trials between 2012 and 2018. Variants were considered as PGVs in the cancer predisposing genes (PMID: 29625052) if they satisfied the following criteria: (i) they had a “PASS” flag in HaplotypeCaller, (ii) were annotated as “Pathogenic” or “Likely Pathogenic” in ClinVar (PMID: 29165669) or InterVar (PMID: 28132688), or (iii) were truncating variants. Somatic driver mutations and Loss of Heterozygocity (LOH) of PGV harboring genes were further evaluated. The overlap to loss of heterozygocity regions was reported only when the variant allele frequency of the PGV was significantly higher than in the normal tissue. Cancer history, clinical and molecular data were retrospectively collected. The somatic mutations (m) in EGFR/BRAF/MET/HER2/KRAS and fusions in ALK/ROS1/RET were also considered for analysis. RESULTS: Among 134 pts, 48% were women, median age was 61 (range 24-83), 45% were nonsmokers, 74% had adenocarcinoma. The most common somatic oncogenic driver alterations were: EGFRm in 44 pts (33%), KRASm in 19 pts (14%), BRAFm in 12 pts (9%) and ALK in 12 pts (9%).PGV were found in 22 out of 152 (15%) cancer-predisposing genes; 4 pts had additional somatic mutations (2) or LOHs (2) in the same genes. 77% of PGVs were in genes which are part of DNA repair pathways including 3.6% nucleotide excision repair (ERCC1/2/3, XPA), 6.5% homologous recombination/Fanconi Anemia: (FANC/A/C/M/D2, BRCA1, RECQL), 2.1% base excision repair (MUTYH, NTHL1), while the others were represented by genes related to cell signaling and metabolism (NF1, MET, ELANE, PRDM9, TRIM37).In the 22 PGV-carriers, 68% had a somatic oncogene-driven alteration (15/22) : EGFRm (n=7; 5 ex19del, 2 ex21(L858R)), KRASm (n=3; 2 G12D, 1 G12V), METm (n=2), HER2m (n=1), ROS1 (n=1) and RET (n=1). PGV were observed in 16% of EGFRm (7/44), 67% of METm (2/3), 15% in KRASm (3/19), 33% of HER2m (1/3), 25% of ROS1 (1/4), 50% of RET (1/2); but no PGV was identified in pts with BRAFVm (12) or ALK (12). CONCLUSION: In our cohort, 15% of pts with NSCLC were PGV-carriers; 68% of PGV-carriers had oncogene-driven tumors, particularly with somatic EGFR mutations. PGV and oncogene-driven lung carcinogenesis need further evaluation. Citation Format: Laura Mezquita, Andrei Iurchenko, Jose Carlos Benitez, Maria Baz, Sergey Nikolaev, David Planchard, Felix Blanc-Durand, Mihaela Aldea, Patricia Martín-Romano, Yohann Loriot, Claudio Nicotra, Maud Ngocamus, Jean-Yves Scoazec, Stefan Michiels, Sophie Postel-Vinay, Julien Viot, Luc Friboulet, Antoine Italiano, Fabrice Andre, Christophe Massard, Jean-Charles Soria, Etienne Rouleau, Daniel Gautheret, Benjamin Besse. High prevalence of pathogenic germline variants in patients with oncogene-driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 448.

Constitutive Activation of Ras and Inactivation of Ink4a/Arf Allele Induce a Fully Penetrant B-Cell Acute Lymphoblastic Leukemia Phenotype in Mice

Despite recent advances in treatment, precursor-B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging clinical entity. Recent genome-wide studies have uncovered frequent genetic alterations involving activating RAS pathway mutations and loss of the INK4A/ARF locus suggesting their important role in the pathogenesis, relapse, and chemoresistance of B-ALL. Therefore, to better understand the oncogenic mechanisms by which these alterations might promote B-ALL and to develop an in vivo preclinical model of relapsed B-ALL, we engineered mouse strains with induced somatic KrasG12D pathway activation and/or loss of Ink4a/Arf during early stages of B-cell development.Pathologic examination revealed significantly enlarged spleens and increased numbers of mononuclear cells in the spleens of 8-week-old KrasG12D mutated animals with or without Ink4a/Arf inactivation, but obvious histologic and IHC differences in spleens and lymph nodes between the mutant genotypes and CD19Cre/+ controls were not found. However, in agreement with the gross pathologic examination, an increase was noted by flow cytometry in splenic B220+ B-cells of mice with the KrasG12D mutation as compared with CD19Cre/+ and CD19Cre/+;Ink4a/ArfL/+ mice. In keeping with this, a significant increase in number of proliferating (Ki-67+) cells within spleen B-cell areas of CD19Cre/+;KrasG12D/+, and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice were observed in comparison with CD19Cre/+ controls and CD19Cre/+;Ink4a/ArfL/+ mice. TUNEL staining revealed fewer apoptotic cells within germinal center B-cell areas in CD19Cre/+;KrasG12D/+, CD19Cre/+;Ink4a/ArfL/+, and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice as compared with CD19Cre/+ controls. Next, to identify the biological processes underlying the observed phenotypical changes, we performed gene-set enrichment analysis of microarray expression data from mutant compared to control mice. In CD19+ B-cells from both CD19Cre/+;KrasG12D/+ and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice, marked upregulation of gene signatures related to proliferation, Ras-related signaling pathways, apoptosis, rewired cellular metabolism, DNA repair, and pre-B stage of B-cell development were observed in both CD19Cre/+;KrasG12D/+ and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice. Consistent with our previous observations, the proliferative signature in CD19+ B-cells from CD19Cre/+;Ink4a/ArfL/+ mice was less pronounced; instead, upregulation of several signaling pathways related to independence of external growth signals and retention of “stemness” was noted. Although constitutive activation of KrasG12D in B-cells induced prominent transcriptional changes that resulted in enhanced proliferation, it was insufficient by itself to induce development of a high-grade leukemia/lymphoma. Instead, in 40% of mice these engineered mutations promoted development of a clonal low-grade lymphoproliferative disorder resembling human extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue or lymphoplasmacytic lymphoma. Interestingly, loss of the Ink4a /Arf locus, apart from reducing the number of apoptotic B-cells broadly attenuated KrasG12D-induced transcriptional signatures. However, combinedKras activation and Ink4a/Arf inactivation cooperated functionally to induce a fully penetrant, highly aggressive B-ALL phenotype. Cross-species bioinformatic analyses consistently demonstrated that B-ALL tumors in the CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice recapitulated gene expression programs found in a poor-prognosis subgroups of human B-ALL that harbor BCR-ABL and CRLF2 rearrangements. Moreover, 90% of examined murine B-ALL tumors showed loss of the wild-type Ink4a/Arf locus without acquisition of common cooperating events, underscoring the role of Ink4a/Arf in restraining Kras-driven oncogenesis in the lymphoid compartment in our model.Taken together, the functional cooperation and the negative impact on survival of concurrent Ras mutation and Ink/Arf deletion in mice suggest that these changes may also have a functional impact in human B-ALL and should be considered in the course of disease classification, as well as for prognosis and choice of therapeutic strategy. DisclosuresSharpless:G1 Therapeutics: Equity Ownership; Pfizer: Other: NA; HealthSpan Diagnostics: Other: NA; Unity Biotechnology: Other: NA.

Structural and numerical Y chromosomal variations in elderly men identified through multiplex ligation-dependent probe amplification.

Human Y chromosomes frequently acquire structural and numerical alterations. Known alterations include germline copy-number variations (CNVs) in the azoospermia factor (AZF) region and somatic mosaic loss of the Y chromosome (mLOY). Here, we explored Y chromosomal variations in 160 Japanese men aged 75-90 years. Multiplex ligation-dependent probe amplification (MLPA) identified ten types of AZF-linked CNVs in 77 men and mLOY of various degrees in 37. Seventeen men carried both a CNV and mLOY. MLOY levels estimated by MLPA were closely correlated with those determined by droplet digital PCR. No association was found between AZF-linked CNVs and the frequency or levels of mLOY. These results emphasize the high frequency and large inter-individual variability of AZF-linked CNVs and mLOY, and demonstrate the usefulness of MLPA in the detection of these variations. More importantly, this study provides the first evidence that AZF-linked CNVs do not increase the risk of aging-related mLOY.

Use of comprehensive next-generation sequencing to identify pathogenic germline variants with therapeutic relevance in metastatic breast cancer.

10527 Background: Among patients with early-stage breast cancer, approximately 6-10% have a pathogenic germline variant (PGV) conferring inherited cancer predisposition. In contrast, few studies have explored the frequency and types of PGVs identified in patients with metastatic breast cancer (MBC); therefore, additional data is needed. Methods: From 2011-2020, 278 patients with MBC underwent fresh tumor biopsy and blood sample collection for paired tumor/normal DNA (targeted exome capture with analysis of 1700 genes) and RNA (tumor transcriptome) sequencing through the Michigan Oncology Sequencing (Mi-ONCOSEQ) program. Somatic and germline alterations were annotated and classified according to degree of clinical actionability with results returned to treating oncologists. Retrospective chart review was performed to determine if: 1) a PGV was identified prior to Mi-ONCOSEQ testing, 2) patients met National Comprehensive Cancer Network (NCCN) guideline criteria for genetic testing on the basis of personal or family cancer history and 3) patients received subsequent therapy informed by a PGV. Results: Forty-eight of the 278 patients (17.3%) had at least one PGV identified, with a total of 50 PGVs identified in this cohort. Only twelve of these PGVs (24%) had been identified prior to Mi-ONCOSEQ testing. The most frequent PGVs identified were in CHEK 2 (n = 9, 18%), MUTYH (n = 6, 12%), BRCA 1 (n = 5, 10%), BRCA2 (n = 5, 10%), ATM (n = 4, 8%) and PALB2 (n = 4, 8%). Somatic loss of heterozygosity events (LOH) occurred in 30 of the 50 cases with PGVs identified (60%). LOH events were observed in 83.3% of BRCA1, BRCA2, ATM and PALB2 PGVs, but were less frequently observed with CHEK2 (33.3%) and MUTYH (66.7%). Two hundred sixteen out of 278 patients (77.7%) in this cohort met NCCN criteria for genetic testing, although six patients with a PGV identified (CHEK2: n = 5; MUTYH: n = 1) did not meet NCCN criteria. Twenty-nine PGVs identified (58%) had potential therapeutic relevance and 11 patients (22.9%) received targeted therapy based on the PGV. Conclusions: The frequency of PGVs identified in this cohort is nearly double the frequency reported for patients with early-stage disease, suggesting that certain PGVs may confer worse prognosis. CHEK2, the most frequently identified PGV, was less likely to have an identifiable LOH event. The direct role of CHEK2 PGVs in tumor pathogenesis is uncertain, but other mechanisms of silencing the wild type allele must be considered. Despite the majority of patients meeting NCCN criteria for genetic testing, those with PGVs in CHEK2 were less reliably identified by this mechanism. The majority of PGVs identified were of potential therapeutic relevance, supporting the recommendation for genetic testing in all patients with MBC.

Accumulation of genome-wide somatic loss of heterozygosity (LOH) as a prominent feature of advanced malignant soft tissue tumors and association with the BRCAness status, suppression of immune responses, and lower survival rates.

11533 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Recent genomic analysis revealed widespread CNA and the cumulative burden of cancer-related pathogenic germline mutations/variants, their clinical and therapeutic significance were unknown. Methods: We recruited 155 patients with advanced malignant soft tissue tumors (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) with confirmed metastasis/recurrence and information on familial cancer burden. Whole exome sequencing was performed in both blood and tumor samples as described in 2018ASCO. The copy number of BRCA2 gene was determined by the MLPA method. Tumor immune microenvironment was assessed by immunohistochemistry. The MSI status was analysed by PCR. Results: We analyzed the LOH status in 595 COSMIC genes and found that genome-wide accumulation of somatic LOH of polygenic germline mutations/variants. Patients with more than 33% LOH genes (n=102) in the total of somatic and LOH mutations showed significantly lower OS rates compared with those (n=53) with less LOH genes (5-year survival rates; 49 vs 75%, p=0.010), which constitute 78% of LMS (n=78/100) and 26% of LPS (n=5/19). Total of 41 patients (26%, n=41/155) including 33 LMS (33%, n=33/100) showed LOH in the BRCA2 locus with hemizygous VUS. Those patients with BRCA2 LOH (n=41) showed significantly lower OS rates compared with those without BRCA2 LOH (n=114) (5-year survival rates; 43 vs. 64%, p=0.019). Neither TMB nor the MSI status was associated with LOH. In contrast, accumulation of somatic LOH (mean LOH values of 71.7 vs. 15.7%) was clearly and negatively associated with CD8+T-cell immune infiltrates (T-cells; 44±23 vs 555±180/mm2, n=7, p=0.016), CD20+B-cell accumulation in tertiary lymphoid structures (TLS) (TLS; 0.57±0.43 vs. 20.1±6.1/tumor, n=7, p=0.008) and low levels of neutrophil-to-lymphocyte ratio (NLR) (NLR; 3.63±0.45 vs. 1.71±0.17, n=7, p=0.002), hallmarks of the immunological response to tumors. Conclusions: This study suggests that in advanced malignant soft tissue tumors, accumulation of genome-wide LOH of germline mutations/variants is associated with the BRCAness status and suppression of the immune responses to tumors, and thus influences therapeutic response and survival of the patients.

Clinical Validation of Whole Genome Sequencing for Cancer Diagnostics

Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures, resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content. Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall; 98.5%) and precision (positive predictive value; 97.8%) for detection of somatic single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0%; EGFR and MET) as well as somatic complete loss (100%; CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (human papillomavirus), an accuracy of 100% compared with standard testing. In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay.

Pancreatic Neuroendocrine Neoplasms in Multiple Endocrine Neoplasia Type 1.

Pancreatic neuroendocrine tumors (pNETs) are a rare group of cancers accounting for about 1–2% of all pancreatic neoplasms. About 10% of pNETs arise within endocrine tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1). pNETs affect 30–80% of MEN1 patients, manifesting prevalently as multiple microadenomas. pNETs in patients with MEN1 are particularly difficult to treat due to differences in their growth potential, their multiplicity, the frequent requirement of extensive surgery, the high rate of post-operative recurrences, and the concomitant development of other tumors. MEN1 syndrome is caused by germinal heterozygote inactivating mutation of the MEN1 gene, encoding the menin tumor suppressor protein. MEN1-related pNETs develop following the complete loss of function of wild-type menin. Menin is a key regulator of endocrine cell plasticity and its loss in these cells is sufficient for tumor initiation. Somatic biallelic loss of wild-type menin in the neuroendocrine pancreas presumably alters the epigenetic control of gene expression, mediated by histone modifications and DNA hypermethylation, as a driver of MEN1-associated pNET tumorigenesis. In this light, epigenetic-based therapies aimed to correct the altered DNA methylation, and/or histone modifications might be a possible therapeutic strategy for MEN1 pNETs, for whom standard treatments fail.

Constitutional 2p16.3 deletion including MSH6 and FBXO11 in a boy with developmental delay and diffuse large B-cell lymphoma

We describe a case of a boy with neurodevelopmental delay and a diffuse large B-cell lymphoma (DLBCL) in whom we discovered a germline de novo 2p16.3 deletion including MSH6 and part of the FBXO11 gene. A causative role for MSH6 in cancer development was excluded based on tumor characteristics. The constitutional FBXO11 deletion explains the neurodevelopmental delay in the patient. The FBXO11 protein is involved in BCL-6 ubiquitination and BCL-6 is required for the germinal center reaction resulting in B cell differentiation. Somatic loss of function alterations of FBXO11 result in BCL-6 overexpression which is a known driver in DLBCL. We therefore consider that a causative relationship between the germline FBXO11 deletion and the development of DLBCL in this boy is conceivable.

Comprehensive Molecular Characterization and Response to Therapy in Fumarate Hydratase-Deficient Renal Cell Carcinoma.

Fumarate hydratase-deficient renal cell carcinoma (FH-RCC) is a rare, aggressive form of RCC associated with hereditary leiomyomatosis and RCC syndrome. Evidence for systemic therapy efficacy is lacking. We studied clinical and genomic characteristics of FH-RCC, including response [objective response rate (ORR)] to systemic therapies and next-generation sequencing (NGS). Patients with metastatic FH-RCC, defined by presence of pathogenic germline or somatic FH mutation plus IHC evidence of FH loss, were included. A total of 28 of 32 included patients (median age 46; range, 20-74; M:F, 20:12) underwent germline testing; 23 (82%) harbored a pathogenic FH germline variant. Five (16%) were negative for germline FH mutations; all had biallelic somatic FH loss. Somatic NGS (31/32 patients) revealed co-occurring NF2 mutation most frequently (n = 5). Compared with clear-cell RCC, FH-RCC had a lower mutation count (median 2 vs. 4; P < 0.001) but higher fraction of genome altered (18.7% vs. 10.3%; P = 0.001). A total of 26 patients were evaluable for response to systemic therapy: mTOR/VEGF combination (n = 18, ORR 44%), VEGF monotherapy (n = 15, ORR 20%), checkpoint inhibitor therapy (n = 8, ORR 0%), and mTOR monotherapy (n = 4, ORR 0%). No complete responses were seen. Median overall and progression-free survival were 21.9 months [95% confidence interval (CI): 14.3-33.8] and 8.7 months (95% CI: 4.8-12.3), respectively. Although most FH-RCC tumors are due to germline FH alterations, a significant portion result from biallelic somatic FH loss. Both somatic and germline FH-RCC have similar molecular characteristics, with NF2 mutations, low tumor mutational burden, and high fraction of genome altered. Although immunotherapy alone produced no objective responses, combination mTOR/VEGF therapy showed encouraging results.

Molecular basis of ETV6-mediated predisposition to childhood acute lymphoblastic leukemia

AbstractThere is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Roles of the BAP1 Tumor Suppressor in Cell Metabolism.

BRCA1-associated protein 1 (BAP1) is emerging as an intensively studied cancer-associated gene. Germline mutations in BAP1 lead to a cancer syndrome, and somatic loss is found in several cancer types. BAP1 encodes a deubiquitinase enzyme, which plays key roles in cell-cycle regulation, cell death, and differentiation. Recent studies have demonstrated that BAP1 is also involved in several aspects of cellular metabolism, including metabolic homeostasis, glucose utilization, control of ferroptosis, and stress response. A better knowledge of the metabolic roles of cancer-associated genes is important to understanding tumor initiation and progression, as well as highlighting potential therapeutic avenues. With this review, we summarize the current knowledge regarding BAP1-mediated regulation of metabolic activities that may support new strategies to treat BAP1-mutated cancers.

LSD1/KDM1A Inactivation Causes Hereditary Food-Dependent Cushing’s Syndrome

Purpose: To investigate the genetic cause of Food-dependent Cushing’s syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the GIP receptor (GIPR). Germline ARMC5 alterations have been reported in about 25 % of PBMAH index cases but are absent in patients with FDCS. Methods: A multi-omics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNAseq, SNP array, methylome, miRNome, exome sequencing). Results: The integrative analysis revealed three molecular groups with different clinical features: G1, PBMAH due to ARMC5 inactivating variants; G2, with GIPR ectopic expression and FDCS; and G3, with a less severe phenotype. Exome sequencing revealed germline truncating variants of LSD1/KDM1A in the G2 group, constantly associated with a somatic loss of the LSD1/KDM1A wildtype allele on 1p, leading to a loss of LSD1/KDM1A expression both at mRNA and protein levels (p=1.2x10-12 and p<0.01, respectively). Subsequently, LSD1/KDM1A pathogenic variants were identified in 4/4 additional index cases with FDCS. Conclusion: LSD1/KDM1A inactivation explains about 90 % of FDCS PBMAH. Genetic screening for ARMC5 and LSD1/KDM1A can be offered now for the majority of PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.

Gene Network Analysis Reveals Association between Somatic Loss of Y and Genes Involved in Hemostasis

Background and Hypothesis: &#x0D; Somatic loss of the Y chromosome (LOY) is associated with diagnoses of various cancers, Alzheimer’s disease, and cardiovascular events. In the case of cardiovascular disorders, no mechanistic link to LOY has been elucidated. To explore this relationship, we utilized Weighted Gene Co-Expression Analysis (WGCNA), a powerful technique that can reveal associations between modules of co-expressed genes and clinical traits. We hypothesized WGCNA would reveal a module that illuminates the relationship between LOY and cardiovascular disease. &#x0D; &#x0D; Methods: &#x0D; Information about 403 male participants was obtained from Alzheimer’s Disease Neuroimaging Initiative (ADNI), including Genome Wide Association Study (GWAS), microarray, and patient demographic data. GWAS data was obtained from each participant within one year of collecting gene expression data. Using GWAS data for each participant, a median log R ratio was calculated for probes in the male specific region of the Y (MSY), which showed detectable LOY in 16 participants. Microarray data was used for WGCNA. Genes with correlated expression levels were organized into modules which were tested for associations with age, Alzheimer’s disease status, APOE-e4 allele status, and LOY. Ensembl IDs were used to investigate Gene Ontology (GO) enrichment in the GO database. &#x0D; &#x0D; Results: &#x0D; One module was associated with LOY (p=0.001). GO enrichment revealed significant representation of 47 GO terms within this module. Highly enriched GO terms included “platelet aggregation” (FDR = 8.05 x 10-6), “blood coagulation, fibrin clot formation” (FDR= 0.0328), “regulation of megakaryocyte differentiation” (FDR=3.01 x 10-5), and other terms related to the vascular response to injury. &#x0D; &#x0D; Potential Impact: &#x0D; WGNCA revealed a module associated with LOY that is related to vascular injury response. While LOY is associated with cardiovascular events, more work is needed to explore its association with biomarkers for cardiovascular disease. Our findings warrant more investigation into the relationship between LOY and cardiovascular dysfunction in males.

MBCL-21. GERMLINE ELONGATOR MUTATIONS IN SONIC HEDGEHOG MEDULLOBLASTOMA

BACKGROUNDOur previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes.METHODSCase-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB.RESULTSGermline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response.CONCLUSIONSWe identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.

ATRT-21. RHABDOID PREDISPOSITION SYNDROME: REPORT OF MOLECULAR PROFILES AND TREATMENT APPROACH IN THREE CHILDREN WITH SYNCHRONOUS ATYPICAL TERATOID/RHABDOID TUMOR AND MALIGNANT RHABDOID TUMOR

BACKGROUNDRhabdoid predisposition syndrome is characterized by germline alterations in SMARCB1 or SMARCA4, leading to synchronous or metachronous central nervous system (CNS) and extra-CNS rhabdoid tumors. Rare survivors have been reported to date.METHODSWe describe the molecular profiling and treatment regimen of three patients with synchronous atypical teratoid/rhabdoid tumor (ATRT) and malignant rhabdoid tumor of the kidney (MRT-K). All patients underwent radical nephrectomy of the kidney, and gross total resection of the primary CNS tumor was achieved for two patients. An intensive chemotherapy regimen was administered; an induction phase based on the modified Third Intergroup Rhabdomyosarcoma Study (IRS-III) for ATRT followed by a consolidation phase with three cycles of high-dose chemotherapy and autologous hematopoietic progenitor cell rescue, without irradiation. All three patients were enrolled on an institutional comprehensive genomic profiling protocol.RESULTSA germline focal 22q deletion, including SMARCB1, was detected in two patients, while the third patient had a maternally-inherited heterozygous frameshift variant in SMARCB1. Somatic loss of heterozygosity of 22q was identified in all patients, resulting in biallelic inactivation of SMARCB1. Divergent tumor subgroups were described using DNA methylation. The three MRT-K samples were classified as MYC subtype. One ATRT was classified as SHH while the other as TYR. One patient is currently three years off-therapy without evidence of disease, while the other two patients have completed the consolidation phase without recurrent disease.CONCLUSIONMolecular profiling of CNS and extra-CNS rhabdoid tumors revealed different epigenetic subgroups. An intensive multimodal therapeutic approach without irradiation may achieve prolonged survival.

Common germline-somatic variant interactions in advanced urothelial cancer

The prevalence and biological consequences of deleterious germline variants in urothelial cancer (UC) are not fully characterized. We performed whole-exome sequencing (WES) of germline DNA and 157 primary and metastatic tumors from 80 UC patients. We developed a computational framework for identifying putative deleterious germline variants (pDGVs) from WES data. Here, we show that UC patients harbor a high prevalence of pDGVs that truncate tumor suppressor proteins. Deepening somatic loss of heterozygosity in serial tumor samples is observed, suggesting a critical role for these pDGVs in tumor progression. Significant intra-patient heterogeneity in germline-somatic variant interactions results in divergent biological pathway alterations between primary and metastatic tumors. Our results characterize the spectrum of germline variants in UC and highlight their roles in shaping the natural history of the disease. These findings could have broad clinical implications for cancer patients.

The rs78378222 prevalence and the copy loss of the protective allele A in the tumor tissue of diffuse large B-cell lymphoma.

BackgroundRare single nucleotide polymorphisms (SNPs) are likely to be a crucial genetic factor for human diseases, including cancer. rs78378222 is rare SNP in 3′-untranslated region (UTR) of TP53 gene leading to disturbance of 3′-end mRNA processing. The frequency of rs78378222 varies in several studied populations. The meta-analysis of 34 genome-wide association studies indicated that rs78378222 was significantly associated with an increased risk of cancer overall. Bioinformatic analysis indicates that somatic loss of the protective A allele of rs78378222 occurs in the tumor tissue of some malignant. The goal of the current study is to document the rs78378222 prevalence and evaluate the copy loss status of the protective allele A in the tumor tissue of patients with diffuse large B-cell lymphoma (DLBCL).MethodsTotal DNA was isolated from FFPE-samples and peripheral blood of patients with DLBCL and comparable in age and sex controls. rs78378222 genotyping was performed by the PCR-RFLP method using restriction endonuclease HindIII. Direct Sanger’s sequencing was used to confirm the presence of C allele of the rs78378222. The search for TP53 gene mutations was carried out by Sanger’s direct sequencing method, according to the IARC protocol.ResultsThe result of genotyping of 136 DNA samples from DLBCL tumor tissue suggested that frequency of the rs78378222 was 11/136 (8.1%). Rare allele C frequency was 11/272 (4.2%). A total of 5/11 DLBCL rs78378222 heterozygous samples had the heterozygosity loss in the TP53 gene. Only one of these cases was combined with TP53 gene mutations which have proven oncogenic potential—p.Arg196Gln, other four cases have not mutations in the coding regions of gene.ConclusionsAt the stages of DLBCL initiation or progression a loss of the protective allele A of rs78378222 occurs. Further efforts are needed to study possible molecular mechanisms underlying somatic alterations in DLBCL in this region of the TP53 3′-UTR as well as functional studies to illustrate how the presents of rs78378222 may affect tumor progression of lymphoma.