Single Nucleotide Alterations Research Articles

High-resolution functional mapping of RAD51C by saturation genome editing

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.

Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin.

Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrBL208F mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Prediction of cancer driver genes and mutations: the potential of integrative computational frameworks.

The vast amount of available sequencing data allows the scientific community to explore different genetic alterations that may drive cancer or favor cancer progression. Software developers have proposed a myriad of predictive tools, allowing researchers and clinicians to compare and prioritize driver genes and mutations and their relative pathogenicity. However, there is little consensus on the computational approach or a golden standard for comparison. Hence, benchmarking the different tools depends highly on the input data, indicating that overfitting is still a massive problem. One of the solutions is to limit the scope and usage of specific tools. However, such limitations force researchers to walk on a tightrope between creating and using high-quality tools for a specific purpose and describing the complex alterations driving cancer. While the knowledge of cancer development increases daily, many bioinformatic pipelines rely on single nucleotide variants or alterations in a vacuum without accounting for cellular compartments, mutational burden or disease progression. Even within bioinformatics and computational cancer biology, the research fields work in silos, risking overlooking potential synergies or breakthroughs. Here, we provide an overview of databases and datasets for building or testing predictive cancer driver tools. Furthermore, we introduce predictive tools for driver genes, driver mutations, and the impact of these based on structural analysis. Additionally, we suggest and recommend directions in the field to avoid silo-research, moving towards integrative frameworks.

Ghrelin delays premature aging in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition that arises from a single nucleotide alteration in the LMNA gene, leading to the production of a defective lamin A protein known as progerin. The accumulation of progerin accelerates the onset of a dramatic premature aging phenotype in children with HGPS, characterized by low body weight, lipodystrophy, metabolic dysfunction, skin, and musculoskeletal age-related dysfunctions. In most cases, these children die of age-related cardiovascular dysfunction by their early teenage years. The absence of effective treatments for HGPS underscores the critical need to explore novel safe therapeutic strategies. In this study, we show that treatment with the hormone ghrelin increases autophagy, decreases progerin levels, and alleviates other cellular hallmarks of premature aging in human HGPS fibroblasts. Additionally, using a HGPS mouse model (LmnaG609G/G609G mice), we demonstrate that ghrelin administration effectively rescues molecular and histopathological progeroid features, prevents progressive weight loss in later stages, reverses the lipodystrophic phenotype, and extends lifespan of these short-lived mice. Therefore, our findings uncover the potential of modulating ghrelin signaling offers new treatment targets and translational approaches that may improve outcomes and enhance the quality of life for patients with HGPS and other age-related pathologies.

Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process.ResultsThis study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping.ConclusionsWe confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.

Comprehensive molecular profiling from liquid and tissue for analysing the somatic mutational landscape in an Indian breast cancer cohort.

e13080 Background: Breast cancer is a genetically and clinically heterogeneous entity, with high prevalence in the Indian population. The primary objective of this study was to characterize the somatic mutational landscape of metastatic breast cancer in Indian patients from tumor and liquid specimens. Methods: We performed molecular profiling of 341 tumor tissue specimens and 901 blood specimens from individual breast cancer patients, with paired tissue and blood processing in a sub cohort (n=101). Targeted NGS evaluating single nucleotide alterations, copy number alterations, and fusions was performed on these specimens. Results: In the cohort with tissue-based sequencing (n = 341), TP53 was the prevalently altered gene (55%). PIK3CA and ESR1 mutations were detected in 32% and 9%, respectively. BRCA1/2 alterations were detected in 10.8%, ARID1A in 7%, KEAP1 in 6%, NF1 in 6%, and ATR in 6%. ERBB2 amplification was observed in 10% of the cases. ESR1 gene fusions were detected in 3.7%. This is clinically relevant as along with other ESR1 resistance mutations, ESR1 fusions are also known as recurrent drivers of endocrine therapy resistance and are impervious to ER-targeted therapies. In a sub cohort comprising 255 specimens, TMB analysis showed a median TMB of 5 mut/Mb (range 1 - 88 mut/Mb), and 15.3% specimens with TMB-High (≥10 mut/mb). The ctDNA analysis (n = 901) revealed TP53 being the most altered (41%) gene, followed by PIK3CA (23%), ESR1(11%), EGFR (6%) and GNAS (4%). Acquired resistance mutations in ERBB2 were identified in ctDNA analysis liquid biopsy from two patients treated with HER2-targeted therapy and were absent in corresponding tissue samples. Mutations in ESR1 were more commonly seen in liquid biopsy than paired tissue, indicating this acquired resistance alteration being better captured in ctDNA than tissue due to tumor heterogeneity and tumor evolution. This indicates causative role for ESR1 mutations in endocrine resistance but not in disease pathogenesis. Conclusions: We report the largest liquid biopsy study reporting the genomic landscape from breast cancer cases of Indian origin along with tissue based molecular landscape. High concordance of liquid for PIK3CA alteration and higher sensitivity for detection of resistance conferring alterations like ESR1 and ERBB2 shows utility of liquid biopsy profiling. It reinforces the utility of liquid biopsy as effective tool that reveal clinically relevant information including mutational status and therapeutic options and seem more relevant in acquired resistance settings. [Table: see text]

Liquid and tissue profiling of somatic mutational landscape of non-small cell lung cancer from an Indian cohort.

e21202 Background: Circulating tumor DNA (ctDNA) encompasses spatial and temporal heterogeneity, tumor evolution, and is ideal for repeat sampling and predictive therapeutic course corrections whereas tissue is the first choice for molecular profiling. We report the mutational landscape of an Indian NSCLC cohort, with focus on liquid biopsy and value addition offered by paired tissue and liquid profiling. Methods: The study comprised 507 tumor tissue and 1271 liquid biopsies from NSCLC patients. Evaluation of single nucleotide alterations, copy number alterations and fusions was conducted using targeted next generation sequencing with paired tissue and liquid profiling in a sub cohort (n = 45). Results: In tissue based analysis (n = 507), EGFR-sensitizing mutations were the most common alterations, detected in 41.4% of samples. Among these, Exon 19 deletion was seen in 46.3%, Exon 21 L858R in 23.9%, Exon 20 insertion in 4.6%, Exon 18 G719X in 3.7% and compound mutations in 5%. TP53, KRAS and PIK3CA mutations were detected in 34.5%,13.4% and 6.3% respectively. MYC was commonly amplified (15.4%) gene followed by PIK3CA (4.1%), CCNE1 (3.3%), EGFR (2.9%), and MET (2.9%) amplifications. Tissue fusion analysis (n = 398) detected ALK in 6.3%, ROS1 in 3.3%, and RET in 1.8%. METEx14 was detected in 4.5%. Liquid biopsy (n = 1271) showed comparable mutation spectrum as tissue, with the EGFR gene being the most altered (32.9%). Mutations in TP53 were observed in 26.4%, KRAS in 7.4%, PIK3CA in 4.4% and BRAF in 3%. In EGFR positive individuals, EGFR T790M was detected at higher frequency in liquid (16.7%) than tissue (8.6%), indicating this acquired resistance alteration being better captured in liquid biopsy than tissue due to tumor heterogeneity and tumor evolution. 2 liquid biopsy samples identified additional KRAS driver mutations not seen in the paired tissue analysis, highlighting the utility of simultaneous tissue and liquid analysis. Concurrent actionable alterations were detected in 11% of tissue and 5% of liquid samples. Conclusions: We report the largest liquid biopsy study reporting the genomic landscape from NSCLC cases of Indian origin along with tissue based molecular landscape. Presence of alterations like KRAS and EGFR T790M in liquid but absence in concurrent tissue biopsy shows utility of paired tissue and liquid biopsy profiling. Higher incidence of targetable and co-existing alterations in the Indian population highlight the utility of broad molecular profiling for successful utilization of targeted therapies. [Table: see text]

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

In Silico Post Translational Analysis of Functional Single Nucleotide Alterations in Human TERT Gene Associated with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) refers to a diverse assemblage of hematological malignancies that constitute clonal expansion of immature myeloid progenitor cells in the peripheral blood and bone marrow. TERT gene ensures telomeres maintenance, chromosome stability and prevention of malignancy. The TERT gene has several single nucleotide polymorphisms (SNPs) that have been linked to a number of diseases, including AML. Objective: To classify the harmful TERT gene mutations, and to analyze them using various computational approaches at structural, functional and translational expression levels Methods: National Centre for Biotechnology Information (NCBI) database was used to retrieve nsSNPs of TERT gene (Q53H, V170M, A184T, S255Y, A288V, H412Y, I540M, R631W) reported in AML and they were analyzed using various bioinformatics tools. Results: In this in silico analysis, it was observed that seven out of eight SNPs had a damaging effect; they could affect the protein stability, protein-protein interactions, hydrophobicity, protein folding, three-dimensional structure, secondary structure and conservation profile. 3D models were generated and validated by various tools and the structural effect of these alterations was observed on protein function that was destabilizing to the RNA folding, protein-protein interactions and other functionally associated proteins. Analysis of post translational modifications showed no significant effect of these mutations. Conclusions: These SNPs could be used in future as potential targets in disease diagnosis, biological markers and protein studies.

Identification of Single-nucleotide Alterations in MAP3K8 Gene in a Turkish Head and Neck Squamous Cell Carcinoma Patient Group

Identification of Single-nucleotide Alterations in MAP3K8 Gene in a Turkish Head and Neck Squamous Cell Carcinoma Patient Group

Circulating messenger RNA variants as a potential biomarker for surveillance of hepatocellular carcinoma.

Liver derived messenger ribonucleic acid (mRNA) transcripts were reported to be elevated in the circulation of hepatocellular carcinoma (HCC) patients. We now report the detection of high-risk mRNA variants exclusively in the circulation of HCC patients. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Our findings potentially offer a novel non-invasive platform for HCC surveillance and early detection. RNAseq analysis was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. Specimens from two cholangiocarcinoma (CCA) patients were also included in our study. HCC specific SNPs and Indels referred as "variants" were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants. The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. RNAseq analyses in tumors, normal liver tissue, plasma, and plasma derived EVs led to the detection of 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none of these were detected in normal liver, plasma of LC patients or normal healthy individuals. Our results demonstrate selective detection of concordant high-risk HCC-specific mRNA variants in free plasma, plasma derived EVs and tumors of HCC patients. The variants comprise of splicing, frameshift, fusion and single nucleotide alterations and correspond to cancer and tumor metabolism pathways. Detection of these high-risk variants in matching specimens from same subjects with an enrichment in circulating EVs is remarkable. Validation of these HCC selective ctmRNA variants in larger patient cohorts is likely to identify a predictive set of ctmRNA with high diagnostic performance and thus offer a novel non-invasive serology-based biomarker for HCC.

Discovering epistasis interactions in Alzheimer's disease using deep learning model

Alzheimer's disease (AD) is the most common form of dementia. Single Nucleotide Polymorphisms (SNPs) are single nucleotide alterations that can be used as genomic markers disclosing susceptibility to complex diseases like AD. Epistasis has long been significant for recognizing the function of genetic pathways and the evolutionary dynamics of difficult genetic systems. Discovering epistasis interactions holds a vital key to personalized medicine (PM). PM needs a better understanding of the relationship between human genetic data and complex diseases. In this proposed work, a deep neural network (DNN) is applied using SHapley Additive exPlanations (SHAP) to get top 20, 100, 300, and 500 ranking SNPs responsible for AD risk through epistasis interactions. Multi-locus interaction analysis is performed on these identified SNPs using Multifactor Dimensionality Reduction (MDR). This constructive induction algorithm is integrated with DNN for discovering epistasis interactions in a computationally effective method. The proposed framework is applied to Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset. The best accuracies are achieved using the top 500 SNPs, and the classification accuracies varied between 0.860 and 0.874 in the five-way interaction model. However, the classification accuracies of 2-way, 3-way, 4-way models varied between 0.663 and 0.670, 0.718 and 0.727, and 0.793 and 0.803, respectively. The results revealed that the reported accuracy scores of the proposed framework outperform the referenced literature work. The proposed framework presents high-ranked risk genes and promising epistasis interactions that may help in explaining the risk of AD.

Comprehensive patient-level classification and quantification of driver events in TCGA PanCanAtlas cohorts.

There is a growing need to develop novel therapeutics for targeted treatment of cancer. The prerequisite to success is the knowledge about which types of molecular alterations are predominantly driving tumorigenesis. To shed light onto this subject, we have utilized the largest database of human cancer mutations–TCGA PanCanAtlas, multiple established algorithms for cancer driver prediction (2020plus, CHASMplus, CompositeDriver, dNdScv, DriverNet, HotMAPS, OncodriveCLUSTL, OncodriveFML) and developed four novel computational pipelines: SNADRIF (Single Nucleotide Alteration DRIver Finder), GECNAV (Gene Expression-based Copy Number Alteration Validator), ANDRIF (ANeuploidy DRIver Finder) and PALDRIC (PAtient-Level DRIver Classifier). A unified workflow integrating all these pipelines, algorithms and datasets at cohort and patient levels was created. We have found that there are on average 12 driver events per tumour, of which 0.6 are single nucleotide alterations (SNAs) in oncogenes, 1.5 are amplifications of oncogenes, 1.2 are SNAs in tumour suppressors, 2.1 are deletions of tumour suppressors, 1.5 are driver chromosome losses, 1 is a driver chromosome gain, 2 are driver chromosome arm losses, and 1.5 are driver chromosome arm gains. The average number of driver events per tumour increases with age (from 7 to 15) and cancer stage (from 10 to 15) and varies strongly between cancer types (from 1 to 24). Patients with 1 and 7 driver events per tumour are the most frequent, and there are very few patients with more than 40 events. In tumours having only one driver event, this event is most often an SNA in an oncogene. However, with increasing number of driver events per tumour, the contribution of SNAs decreases, whereas the contribution of copy-number alterations and aneuploidy events increases.

Retroperitoneal leiomyosarcoma in a female patient with a germline splicing variant RAD51D c.904-2A\u2009>\u2009T: a case report

BackgroundRAD51D (RAD51 paralog D) is an intermediate cancer susceptibility gene for primary ovarian cancer, including fallopian tube and peritoneal carcinomas and breast cancer. Although gynecological non-epithelial tumors such as uterine sarcomas are associated with genomic instability, including BRCA impairment, there is no clear evidence of the relationship between RAD51D variants and the risk of sarcoma development.Case presentationA Japanese woman in her 50s underwent multiple surgical resections and several regimens of chemotherapy for tumors that originated in the retroperitoneum and recurred in the peritoneum over a clinical course of approximately 4 years. The peritoneal tumor was histologically diagnosed as a leiomyosarcoma and was genetically identified to show a splice variant of RAD51D c.904-2A > T [NM_002878] through tumor profiling performed as a part of cancer precision medicine. The confirmatory genetic test performed after genetic counseling revealed that the RAD51D splicing variant detected in her tumor was of germline origin. In silico analyses supported the possible pathogenicity of the detected splice variant of RAD51D with a predicted attenuation in mRNA transcription and truncated protein production due to frameshifting, which was attributed to a single-nucleotide alteration in the splicing acceptor site at the 3′-end of intron 9 of RAD51D. Considering her unfavorable clinical outcome, which showed a highly aggressive phenotype of leiomyosarcoma with altered RAD51D, this case provided novel evidence for the relationship of a RAD51D splicing variant with malignant tumor development or progression. We report the findings of this rare case with possible involvement of the germline variant of RAD51D c.904-2A > T as a potential predisposing factor for malignant tumors, including leiomyosarcoma.ConclusionsWe present the findings of a case of leiomyosarcoma in the peritoneum of a female patient with a novel germline splicing variant of RAD51D as potential evidence for the pathogenicity of the variant and its involvement in the risk of sarcoma etiology and/or development. To the best of our knowledge, this is the first case report describing a leiomyosarcoma carrying a germline RAD51D splicing variant and elucidating its pathogenicity on the basis of computational prediction of the impairment of normal transcription and the presumed loss of functional protein production.

Multiplexed functional genomic analysis of 5\u2019 untranslated region mutations across the spectrum of prostate cancer

The functional consequences of genetic variants within 5’ untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5’ UTR mutations in human prostate cancer. We show that 5’ UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5’ UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5’ UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5’ UTRs are functional in cancer.

Abstract 25: Reversion patterns in homologous recombination repair genes in metastatic cancer patients profiled by cell-free DNA

Abstract Background: Inactivating mutations in homologous recombination repair (HRR) genes are associated with a response to PARP inhibitors and platinum-based therapies. However, secondary reversion mutations in HRR genes remain a major mechanism of resistance. We sought to characterize the prevalence and spectrum of these reversions in advanced cancer patients in BRCA1/2 and other HRR genes, such as ATM and CDK12. Methods: We analyzed cfDNA from >75,000 late-stage cancer patients previously tested as part of routine clinical care using Guardant360®, a 73- to 74-gene panel. This assay detects single-nucleotide variants (SNVs), indels, fusions, and copy-number alterations in cfDNA with a reportable range of ≥0.04%, 0.02%, 0.04%, and ≥2.12 copies, respectively. Secondary reversion mutations were identified by re-analysis using a bespoke HRR deficiency (HRD) Module, which annotates SNV/indels and calls multi-exon deletions and copy-number loss with a 95% limit of detection of ≥ 0.1% and 10 to 25% tumor fraction, respectively. Results: Among >20,000 breast, ovarian, prostate and pancreatic cancer patients, deleterious germline or somatic mutations were detected in BRCA1 (2.7%), BRCA2 (4.8%), ATM (2.9%) and CDK12 (1.7%). The majority of reversions occurred in BRCA1/2-mut (8.7%), compared to 0.07% of CDK12-mut and none in ATM. Reversions showed selective gene and allele representation. In breast cancer, reversions in BRCA2-mut patients were twice as frequent as those in BRCA1-mut (12.9% vs 5.5%, p-value=0.0046), while the near opposite was true for ovarian (7.6% vs 12.8%, p-value=0.73). In pancreatic cancer (>7,500 patients), reversions presented exclusively in BRCA2-mut (8.4%, n=131 vs 0 in BRCA1-mut, n=63). CDK12 reversions were relatively rare: 1 detected in >5,000 prostate cancer patients. Across all genes, the majority of reversions were small indels while 18% of reversions removed the exon entirely. Pathogenic hotspots BRCA2 codons A938 and S1982 - accounted for ≥10% of reversions and were more likely to revert compared to other deleterious sites (p-value<1.0×10-6). Overall, 16.0% of BRCA1/2 mutant breast, ovarian, pancreatic, prostate patients reverted, consistent with previous reports (Lin et al. 2018). Conclusions: cfDNA has been previously shown to detect BRCA1/2 germline, somatic and polyclonal reversion mutations acquired over PARP and platinum-based therapy. We demonstrate that the Guardant HRD Module can robustly identify these reversions in the cfDNA of metastatic patients. We emphasize that reversions were frequent among BRCA1/2-mut breast, ovarian, prostate and pancreatic cancers, with preferential enrichment based on gene and tumor type. Understanding resistance patterns may aid in the treatment selection for progressing patients. Further work is needed to define these ideal treatment options. Citation Format: Yu Fu, Jennifer Yen, Alexander Aartyomenko, Sante Gnerre, Caroline Weiport, Elena Helman, Arielle Yablonovitch, Stephen Pettitt, Christopher Llord, Andrew Tutt, Rebecca Nagy, Justin Odegaard, Darya Chudova, AmirAli Talasaz. Reversion patterns in homologous recombination repair genes in metastatic cancer patients profiled by cell-free DNA [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 25.

Molecular Characterization of Muellerian Tumors of the Urinary Tract.

In the 2016 WHO classification of genitourinary tumors Muellerian tumors of the urinary tract (MTUT) comprise clear cell adenocarcinomas and endometrioid carcinomas. Since these rare tumors remained understudied, we aimed to characterize their molecular background by performing DNA- and RNA-based targeted panel sequencing. All tumors (n = 11) presented single nucleotide alterations (SNVs), with ARID1A mutations being the most prevalent (5/11, 45%). Besides frequent ARID1A mutations, loss of ARID1A protein is not a suitable marker since protein expression is (partly) preserved also in mutated cases. Copy number alterations (CNVs) were found in 64% of cases (7/11), exclusively gene amplifications. Interestingly, a functionally relevant RSPO2 gene fusion/microdeletion was discovered in the endometrioid adenocarcinoma case. Comparing our findings with mutational profiles of other tumor entities, absence of TERT promoter mutations argues for a non-urothelial origin. No similarities were also found between MTUT and kidney cancers while parallels were observed for specific SNVs with endometrial carcinomas. In conclusion, immunohistochemical PAX8-positivity and lack of TERT promoter mutations could serve as key diagnostic features in difficult cases. Thus, understanding the molecular background of these tumors helps to refine treatment options and offers the possibility of targeted therapies in cases where needed.

Cytogenomics of Deschampsia P. Beauv. (Poaceae) Species Based on Sequence Analyses and FISH Mapping of CON/COM Satellite DNA Families.

The genus Deschampsia P. Beauv. (Poaceae) involves a group of widespread polymorphic species, and many of them are highly tolerant to stressful environmental conditions. Genome diversity and chromosomal phylogeny within the genus are still insufficiently studied. Satellite DNAs, including CON/COM families, are the main components of the plant repeatome, which contribute to chromosome organization. For the first time, using PCR-based (Polymerase Chain Reaction) techniques and sequential BLAST (Basic Local Alignment Search Tool) and MSA (Multiple Sequence Alignment) analyses, we identified and classified CON/COM repeats in genomes of eleven Deschampsia accessions and three accessions from related genera. High homology of CON/COM sequences were revealed in the studied species though differences in single-nucleotide alteration profiles detected in homologous CON/COM regions indicated that they tended to diverge independently. The performed chromosome mapping of 45S rDNA, 5S rDNA, and CON/COM repeats in six Deschampsia species demonstrated interspecific variability in localization of these cytogenetic markers and facilitated the identification of different chromosomal rearrangements. Based on the obtained data, the studied Deschampsia species were distinguished into karyological groups, and MSA-based schematic trees were built, which could clarify the relationships within the genus. Our findings can be useful for further genetic and phylogenetic studies.

Concomitant inhibition of B7-H3 and PD-L1 expression by a novel and synthetic microRNA delivers potent antitumor activities in colorectal tumor models.

The families of miR-34 and miR-449 share the same seed region. However, the members showed differential effects on the expression of B7-H3 and PD-L1 in HCT-116 cells. Using miR-34a as a template, the non-seed region was modified by nucleotide alteration, yielding four synthetic microRNA (miRNA) analogs. Among those, NS-MX3, with a base alteration from G to C at the 18th locus of miR-34a, showed the most potent inhibition on both B7-H3 and PD-L1 expression. Subsequent investigations demonstrated that NS-MX3 had a broad anti-proliferation activity against several colorectal tumor cell lines and its antitumor effect was consistently reflected by tumor growth inhibition (TGI) in the HCT-116 xenograft model. In addition, NS-MX3 displayed a synergistic effect on TGI when combined with bevacizumab or regorafenib. Further analysis revealed that the superior antitumor activity of NS-MX3 was correlated to concomitant suppression of both B7-H3 and PD-L1 expression in tumor tissues. Taken together, the present study indicates that the non-seed region of miRNAs plays an important role in the regulation of checkpoint genes, thus showcasing single nucleotide alteration of the non-seed region as a promising approach to discover and develop novel immunotherapies.