Torrent Suite Research Articles

Genomic Variation in Dengue Virus Non-Structural Protein 5 (NS5).

Objective: To identify the most frequently repeated mutations in the NS5 protein in DENV RNA isolated from dengue patients in the province Punjab of Pakistan.Methodology: The study was conducted at Aziz Fatimah Hospital and Allied Hospital Faisalabad during the 2022 dengue outbreak. The temporal sampling method was applied while selecting confirmed dengue patients from both hospitals, and written consent was taken. 120 blood samples were selected in total and after quantification only 23 samples were found fit for whole genome sequencing. Data was uploaded to Torrent Suite Server 4.10 for analysis. Epi Data Analysis, a WHO-developed software program, was used to calculate and summarize mutation frequencies. Excel analysis was used to check for flaws in the data.Results: There were a total of 199 mutations across the entire nonstructural protein 5 (NS5) sequence. NS5 mutations were found in 9 genomic isolates, with Sample (S) 17 having the highest number.Conclusion: The presence of large number of mutations in the structure of NS5 protein may be used in designing specific anti-viral against it, for better management of DENV infection in local settings in future.

Circular RNA hsa_circ_0002268 (PHACTR1) Is Specific to Gestational Diabetes Mellitus in a Polish Pregnant Population.

Gestational diabetes mellitus (GDM) is an intolerance of carbohydrate of any degree, which appears for the first time or is diagnosed during pregnancy. The objective of this study is to assess the differences in circular RNA (circRNA) in a Polish pregnant population with and without GDM. A total of 62 pregnant women, 34 with GDM and 28 controls, were enrolled in the study. Total RNAs were extracted from plasma and reverse transcription to complementary DNA (cDNA) was performed. A panel covering 271 amplicons, targeting both linear and circular as well as negative control gene transcripts, was used. Next-generation sequencing was used to evaluate the circRNA quantity. Data analysis was performed using the Coverage Analysis plugin in the Torrent Suite Software (Torrent Suite 5.12.3). A two-step normalization was performed by dividing each transcript read count by the total number of reads generated for the sample, followed by dividing the quantity of each transcript by β-actin gene expression. Both circular and linear forms of RNAs were independently evaluated. A total of 57 transcripts were dysregulated between pregnant women with GDM and controls. Most of the targets (n = 25) were downregulated (cut-off ratio below 0.5), and one target showed a trend toward strong upregulation (ratio 1.45). A total of 39 targets were positively correlated with fasting plasma glucose (FPG), but none of the tested targets were correlated with insulin, CRP or HOMA-IR levels. Among the pregnant women with gestational diabetes, the relative quantity of hsa_circ_0002268 (PHACTR1) was approximately 120% higher than among healthy pregnant women: 0.046 [0.022-0.096] vs. 0.021 [0.007-0.047], respectively, (p = 0.0029). Elevated levels of hsa_circ_0002268 (PHACTR1) might be specific to the Polish population of pregnant women with GDM, making it useful as a potential molecular biomarker in the management of GDM in Poland.

Mutational Landscape of Diffuse Large B-Cell Lymphoma (DLBCL) in Mexican Patients

Background. Diffuse Large B Cell Lymphoma (DLBCL) is the most common B-cell malignancy (1), recently classified with genomic sequencing and exome analysis into clusters with clinical implications (2,3). However, this analysis has not been informed neither in Mexican, or Latin-American population. The aim of this study was to describe the mutational landscape and its correlation with the clinical outcome in Mexican patients with DLBCL. Material and methods. A prospective, observational study of patients with diagnosis of DLBCL, attended at the National Cancer Institute in Mexico City. Inclusion criteria: patients > 18 years, diagnosis of DLBCL, without previous treatment and candidate to be treated with RCHOP. IRB approval was: Register number CEI/966/15. All signed informed consent. Clinical & pathological variables were analyzed. All patients were treated with 6 cycles of RCHOP. Clinical response was evaluated by PET-CT. Samples: DNA was extracted from paraffin-embedded tissue of the primary neoplasm using the kit Quick-DNA/RNA FFPE kit (Zymo Research). A custom panel (6,026 oligos) was used for the sequencing of coding regions and splicing sites of 79 genes associated with lymphomas. Therafter, the bioinformatic analysiswas carried out using Ion Torrent Suite Browser version 5.0 and Ion Reporter version 5.18. Variant calling was performed with Torrent Variant Caller. The following databases were used: Cancer Genome Interprteter (CGI), ClinVar (201706), COSMIC (v86), PolyPhen (v2.2.2), SIFT (v5.2.2), FATHMM (v2.1), gnomAD (r2.0.1), and the Variant Effect Predictor (v93.2). All variants had a depth greater than 500X. Statistical analysis. Descriptive statistics for clinical variables. Associations between clinical and pathological characteristics were assessed with the frequency of SNVs and InDels. Survival curves were estimated using the Kaplan-Meier method. The association of the overall survival (OS) and the recurrence-free survival (RFS) was evaluated with Log-Rank test. Statistical significance was set at p-value < 0.05. Cox proportional-hazard regression analysis was used to identify the variables predicting overall survival (OS). All statistical analyses were performed using R software (v4.2.2) (https://cran.r-project.org, accessed on 23rd May 2023) and SPSS version 25 software (IBM, Corp., Armonk, NY). Results We included 185 patients, median + SD age 59.3 + 14.1 years. Most (n=128, 69.2 %) were in ECOG score < 2, without B symptoms (n=131, 70.8 %), in advanced disease (n=134, 72.4%), and were germinal-center (GC) (n=126, 68.1 %), and 26 (14.1 %) were double hit lymphomas. At least a driver mutation was found in 110 cases (59.4 %). The genes with driver mutations most frequently were: TP53, EZH2, CREBBP, NOTCH1 and KMT2D. ( figure 1). The EZH2 mutations and KMT2D mutations were more frequent in female: ( EZH2 mutated [n=22/34, 64.7%], p=0.047]) ( KMT2D mutated, 75%, p=0.034), and had a higher frequency of bulky disease (63.3 %, p= 0.05). In contrast, MYD88 mutations were more frequent in male (MYD88 mutations: 76.9 %], p= 0.042). Response to R-CHOP treatment was: Complete (n= 129 (69.7%), partial (n=10, 5.4 %), or stable disease (n=3, 1.6 %); 26 patients (14 %) progressed during treatment. Median follow-up + SD was 41.99 + 21.4 months. The RFS was 60 % to 80 months and OS was 70 % to 84 months. ECOG >2 (p=0.002 [95 %CI: 1.52-3.97]), mutations at genes EZH2 (p=0.007 [95% CI: 0.193 -0.0.58), CREBBP (p=0.004 [95 % CI: 1.59 - 11.85]), and ARID1A (p= 0.027 [95 % CI: 1.34 - 131.45] were factors influencing on OS by univariate analysis. The multivariate analysis confirmed that an ECOG >2 (p=0.001 [95 CI: 1.46-3.34]) had a negative impact on OS. In contrast, EZH2 mutations (p=0.025 [95 % CI: 0.12 -0.86]) were a protective factor. Conclusions: This is the first approach to describe the genomic landscape in Latino-american population. The presence of EZH2 and KMT2 mutations were more frequent in female patients with mediastinal and bulky disease. After multivariate analysis, only the presence of EZH2 mutations was the only genomic factor influencing on OS. These results require to be confirmed by further studies.

Evaluation of the Thermo Fisher Oncomine Myeloid MRD DNA Assay on the Ion Torrent Platform

Introduction Malignant myeloid diseases are a group of blood cancers that arise from genetic changes in hematopoietic cells in the bone marrow or peripheral blood. The presence of measurable residual disease (MRD), or detection of abnormal cells in the body following completion of therapy, is associated with a higher risk of disease relapse, and decreased overall survival (Schuurhuis 2018). Next-generation sequencing (NGS) can be leveraged as a high-sensitivity, minimally invasive method for detection of MRD. The Ion Torrent Oncomine Myeloid MRD Assay (Myeloid MRD) is a targeted NGS assay designed for the detection of single nucleotide variants (SNVs) and insertions/deletions in a curated selection of clinically relevant myeloid disease-related genes at low limits of detection. Assay performance was evaluated using normal and diseased clinical blood and bone marrow samples and NGS reference materials. Methods The Myeloid MRD DNA panel targets hotspot regions across 33 genes and uses Ion AmpliSeq HD technology with unique molecular tagging to suppress sequencing artifacts and improve analytical sensitivity. Briefly, DNA was extracted from whole blood or bone marrow samples and 120 ng of nucleic acid was used as input for the assay. The DNA was divided between 2 polymerase chain reactions (PCR), where it served as the template for target amplifications, before combining reaction products by sample. Library preparation followed the standard Ion AmpliSeq HD protocol, where amplicons were partially enzymatically digested, and a second round of amplification was performed using barcoded primers. Following bead purification, the final library was measured by quantitative PCR (qPCR) to inform final library loading onto the sequencer. Six libraries were pooled and sequenced on an Ion 550 chip using the Ion GeneStudio S5 System. Primary and secondary analyses were performed using the Torrent Suite software, and results were exported to the Ion Reporter software for variant calling. From the variant calling results, a probit model was utilized to determine the variant allele frequency (VAF) at which the assay achieved 90% power to call an expected variant, defining the limit of detection (LoD). Other parameters evaluated included analytical sensitivity, analytical specificity, positive predictive value (PPV), accuracy, and precision. Results Overall, consistent, high quality on-target reads were observed across 22 sequencing runs with a median Ion Sphere Particle (ISP) loading of 90%, usable sequence of 59%, and over 130 million total reads per chip. When evaluating individual samples according to the vendor-recommended number of mapped reads (> 10 million), 98.3% of samples tested passed quality control (QC). Additional sample QC metrics established during assay validation included thresholds of > 80% reads on-target, > 80% uniformity of base coverage, and a mean read length of at least 80 base pairs. The estimated LoD for the assay was 0.1% VAF for small variants (SNVs, insertions/deletions).Above the LoD, the assay demonstrated 96.03% sensitivity, 99.89% accuracy of base-calling, 100% specificity, and 98.83% PPV.Additionally,a comparison was performed usingmatched whole blood and bone marrow samples collected from patients with acute myeloid leukemia (AML) to evaluate the concordance of variant calling. It was determined that the same variants were identified across both sample types, although at higher allele frequency in bone marrow compared to whole blood. Conclusion This evaluation of the Myeloid MRD assay demonstrates that it is a precise and accurate assay with sensitivity that is acceptable for MRD analysis. Notably, variant detection was concordant between whole blood and bone marrow sample types collected from AML patients, with bone marrow providing improved sensitivity for low-frequency variant detection. The user-friendly workflow and integrated informatics pipeline offer opportunities for streamlined testing and interpretation of results. The Ion Torrent Myeloid MRD DNA assay is a powerful new tool for MRD detection across a growing myeloid disease target landscape.

Prognostic role of TP53 variants in the phase III study of FOLFIRI/cetuximab versus FOLFIRI/cetuximab followed by cetuximab (Cet) alone in first-line therapy of patients with RAS and BRAF wild-type (wt) metastatic colorectal cancer (mCRC) (ERMES study).

3544 Background: The ERMES study explored the optimal intensity of anti-EGFR-based first line therapy for RAS/BRAF wt mCRC once achieved disease control. Although the study did not demonstrate non-inferiority of maintenance with Cet alone versus standard treatment, preliminary results suggested that a strategy of de-escalation treatment with only Cet might be effective in selected patients. Conflicting results have been previously reported for the predictive and prognostic role of TP53 variants in mCRC patients receiving anti-EGFR monoclonal antibody therapy. Here we describe the role of TP53 mutations within the ERMES study. Methods: Patients with untreated RAS/BRAF wt mCRC were randomly assigned (1:1) to receive either FOLFIRI/Cet until PD/toxicity (arm A) or FOLFIRI/Cet for 8 cycles followed by Cet alone (arm B). Tumor tissue samples were tested using the Oncomine Solid Tumor DNA kit covering hot spot mutations in 22 genes, including KRAS, NRAS, BRAF and TP53. Variant calling was performed using the Variant Caller Ion Torrent suite 5.16 software. The prognostic value of TP53 variants was assessed in the intention-to-treat (ITT) patients’ population with available sequencing data. Results: Tumor tissue specimens were available for 418/593 (70.5%) patients of the ITT population. Testing failed in 29 cases, whereas KRAS/NRAS/BRAF V600E variants were found in 36/389 (9.2%) patients, who were enrolled on the basis of local testing. Progression free survival (PFS) data were not available for 4 cases. Therefore, the final population in analysis was 349 cases. TP53 variants were detected in 165/349 (47.3%) patients, of which 86/165 (52.1%) in arm A and 79/165 (47.9%) in arm B. The median PFS (mPFS) of TP53 wild type patients in the overall population was 9.4 months versus 10.4 months in the TP53 mutant subgroup (HR 0.8439; 95%CI, 0.673-1.058; p = 0.142). In arm A, the mPFS of TP53 wild type patients was 10.8 months versus 10.7 months in TP53 mutant patients, with an HR 1.053 (95%CI, 0.7582-1.462; p = 0.76). The mPFS of TP53 wild type patients in arm B was 8.3 months versus 10.2 months in the TP53 mutant cohort, with an HR 0.7 (95%CI, 0.5087-0.9657; p = 0.0298). No significant difference was observed for the overall response rate (ORR) in both arms between TP53 mutant and wild type patients. Conclusions: These preliminary data suggest a possible prognostic role of TP53 variants in RAS/BRAF wild type mCRC patients receiving first line FOLFIRI/cetuximab followed by Cet alone. Comprehensive genomic profiling is ongoing to identify genomic signatures associated with sensitivity to Cet.

Variability of <i>pgm</i>‑Region Genes in <i>Yersinia pestis</i> Strains from the Caspian Sandy and Adjacent Plague Foci

The aim of the study was to compare the nucleotide sequences of pgm‑region genes in Yersinia pestis strains isolated on the territory of the Caspian sandy and adjacent plague foci in 1925–2015. Materials and methods. 65 Y. pestis strains from the Caspian sandy and adjacent plague foci were used in the work. DNA isolation was performed using the PureLink Genomic DNA Mini Kit. Whole genome sequencing was conducted in Ion S5 XL System (Thermo Fischer Scientific). Data processing was carried out using Ion Torrent Suite software package 3.4.2 and NewblerGS Assembler 2.6. To compare the obtained sequences with the NCBI GenBank database, the Blast algorithm was used. The phylogenetic analysis was performed according to the data of whole genome SNP analysis based on 1183 identified SNPs. The search for marker SNPs was performed using the Snippy 4.6 program. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model. Results and discussion. The nucleotide sequences of pgm‑region genes of 65 Y. pestis strains from the Caspian sandy and adjacent plague foci have been assessed. Single nucleotide substitutions have been identified in Y. pestis strains from the Caspian sandy and Kobystan plain-foothill foci in the hmsR, astB, ybtS, ypo1944, ypo1943, ypo1936 genes, as well as a deletion of 5 bp in the ypo1945 gene, which is characteristic of strains of one of the phylogenetic lines of Y. pestis from the foci of Caucasus and Transcaucasia, isolated in 1968–2001. The data obtained can be used to differentiate Y. pestis strains from the Caspian sandy focus, as well as to establish the directions of microevolution of the plague pathogen in this region and adjacent foci.

Abstract 3750: Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology

Abstract Background: DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA hypermethylation events co-occur in groups of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Here we designed and performed a first step of validation of a novel next-generation sequencing (NGS) targeted methylation panel that can simultaneously evaluate the methylation levels of multiple cancer-related genes. Methods: The WG00446_MET_A AmpliSeq™ Methylation Panel for Cancer Research was designed using the Ion AmpliSeq technology to amplify 152 regions with an amplicon size range of 125-175bp, that covers a total of 1107 promoter and intragenic CpGs of 18 cancer-related genes. The performance of the panel was assessed by running in duplicate commercially available fully methylated and unmethylated control human gDNA samples and a variable mixture of them to simulate 10%, 25%, 75%, and 90% of global methylation. The libraries (manual preparation using the Ion AmpliSeq Plus Library Kit) were loaded on 530 chips and run on the GeneStudio S5 Sequencer (Thermo Fisher Scientific). The sequencing output was analyzed using the methylation_analysis plugin, available on Ion Torrent Suite v5.16 (Thermo Fisher Scientific). Results: The WG00446_MET_A panel allowed to run 6 samples per 530 chips to reach an observed mean target depth >2,000X (observed reads on-targets: >80%; average number of mapped reads: >450,000/sample). The conversion efficiency, determined by spiking-in Unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed % of global methylation for all CpGs were >98% and <2% for fully methylated and unmethylated gDNA samples, respectively. The observed results for the variable mixtures were in agreement with what expected. Conclusions: The methylation-specific NGS analysis using the WG00446_MET_A panel represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. It will offer the opportunity to design a more robust algorithm for disease prediction for those cancer patients with a low quantity of biological material available. The orthogonal testing on DNA extracted by FFPE and frozen samples is ongoing with a subset of amplicons from the WG00446_MET_A panel. Citation Format: Federico Pio Fabrizio, Stefano Castellana, Angelo Sparaneo, Tommaso Mazza, Flavia Centra, Domenico Trombetta, Vito Michele Fazio, Lucia Anna Muscarella. Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3750.

DIPG-36. The brain-gut-microbiota axis to predict outcome in pediatric diffuse intrinsic pontine glioma

OBJECTIVE: Diffuse intrinsic pontine glioma (DIPG) is a rare childhood brain tumour with poor prognosis. Radiotherapy (RT) remains the only palliative intervention. In this scenario, it is essential to investigate tumor and patient microinvironment. Microbiota plays a critical role in human health and a functional link between the central nervous system and gut microbiota has been reported: the microbiota-gut-brain axis. With these premises, we have investigated the gut microbiota in DIPG. METHODS: A cohort of 18 patients was enrolled and we collected stool specimens at diagnosis (pre-RT) and after radiotherapy. Microbiota content was analysed through 16S rRNA sequencing on IONS5xl; base calling and demultiplexing were performed by Torrent Suite (ThermoFisher). Association to progression free survival (PFS) and overall survival (OS) were assessed by cox-regression univariate analyses and differential abundance (DA) analyses identified differences following RT. RESULTS: The Firmicutes/Bacteroidetes (F/B) ratio in pre-RT specimens has a median of 0.757 (range= 0.243-1.19). Having PFS as endpoint and at family classification level, an increased risk of disease progression was disclosed for Flavobacteriaceae and Bacillales with HR of 1.57 (p=0.00913) and 1.57 (p=0.215), respectively. The anaerobic bacteria Synergistaceae is found related to a decreased risk of progression with HR 0.662 (p=0.00718). These findings were also confirmed with OS as endpoint. DA analyses pointed out a number of families belonging to Firmicutes and Bacteroidetes phyla highly differentiated (log2|fold-change|>2) between pre- and post-RT. CONCLUSIONS: This study represents the first report of the microbiota-gut-brain axis role in DIPG patients. The F/B ratio, a parameter of normal intestinal homeostasis, show the presence of inflammatory patterns and some family members are associated to increased disease progression. These preliminary findings needs further validations. However, 16S microbiota has the potential ability to stratify patients and probiotic administration could restore a normal F/B ratio.

TBIO-01. Mutation Profiling of Paediatric Solid Tumours in a Cohort of South African Patients

BACKGROUND: The incidence of childhood cancer incidence is increasing gradually in low-middle income countries, such as South Africa. Globally, there is an extensive range of familial- and hereditary-cancer syndromes, where underlying germline variants increase the likelihood of developing cancer in childhood. Next-Generation Sequencing (NGS) technologies have been key in determining the occurrence and genetic contribution of germline variants to paediatric cancer development. We aimed to design and evaluate a candidate gene panel, specific to inherited cancer-predisposing genes to provide a comprehensive insight into the contribution of germline variants to childhood cancer. METHODS: 32 paediatric patients (aged 0-18 years) diagnosed with a malignant tumour were recruited and biological samples were obtained. After quality control, DNA was sequenced using an ion Ampliseq 50 candidate gene panel design and Ion Torrent S5 technologies. Sequencing variants were called using Ion Torrent Suite software and were subsequently annotated using Ion Reporter and Ensembl's VEP. High priority variants were manually analysed using tools such as MutationTaster, SIFT-INDEL and VarSome. Putative identified candidates were validated via Sanger Sequencing. RESULTS: The patients studied had a variety of cancers, the most common being nephroblastoma (13), followed by osteosarcoma (4) and astrocytoma (3). We identified 10 pathogenic / likely pathogenic variants in 10 patients, most of which were novel. CONCLUSIONS: According to literature, we expected ~10% of our patient population to harbour pathogenic or likely pathogenic germline variants, however we reported about 3 times (~30%) more than we expected. Majority of the identified variants are novel; this may be because this is the first study of its kind in an understudied South African population

Rate of Clonal Hematopoiesis in Patients with Venous Thromboembolism

BackgroundVenous thromboembolism (VTE) is the third most common cardiovascular disease with an incidence over 1% in the elderly. About 50% of VTE is unprovoked, which is associated with higher rates of recurrent VTE and complications such as post-thrombotic syndrome. Clonal hematopoiesis (CH) refers to recurrent somatic mutations associated with hematologic malignancies that are present in otherwise healthy individuals. CH is more common with increased age, occurring in more than 10% of people over the age of 70 and has been associated with an increased risk of coronary artery disease. The rates of CH in patients with VTE or complications arising from this is not known.MethodsAdult patients (age ≥ 18) with VTE were enrolled at the Ohio State University Thrombosis Clinic between 2017 and 2020. Clinical data was obtained at the time of routine clinic follow-up or ad hoc, and a research blood sample was collected with routine blood work. DNA extracted from peripheral blood mononuclear cells were analyzed for the presence of CH via error corrected next generation sequencing (NGS) using the Ion Gene Studio S5 System. A custom AmpliSeq panel IAH150087 was designed to detect CH, which included whole gene sequencing and hotspots in 25 common genes associated with myeloid malignancies, including DNMT3A, TET2, ASXL1, among others, with a sequencing depth of up to 50,000X. Data was collected and analyzed using Ion Torrent S5/GS-S5 Instrument with Torrent Suite 5.12.0 version. Final analysis of sequence data was performed using combination of software: Variant Caller v.5.12.10-1, IGV5.01 (0) and Ion Reporter v.5.12.3.0. The hg19 reference sequence was used to assess for deviation. CH was defined as a variable allele frequency ≥ 2%. Two sample t-test and chi-square test were used to compare patients with and without CH for continuous and categorical variables, respectively.ResultsA total of 167 patients are included the study. Median age is 60.3 years and 55% of patients are female. 80% of patients are white. The majority of patients have unprovoked VTE (81%); 23 patients (13.8%) have VTE associated with cancer. 84% of patients were treated with a direct oral anticoagulant. Over the median follow-up duration of 400 days, 7 patients had recurrent VTE and 19 patients had a total of 21 bleeding events (7 major bleeding and 14 clinically relevant non-major bleeding [CRNMB] by ISTH criteria).Overall, 22 patients (13.2%) have detectable CH. Patients with CH are significantly older than those without CH (mean age 70 vs 59, p <0.0001). There is a trend towards increased CH rate in male patients (17% of male patients vs 10% of female patients with CH). There are no other differences in baseline characteristics among patients with and without CH, including type or location of VTE or cancer status. Among the 144 VTE patients without cancer, the median age is 60 years old and the CH rate is also 13.2%. In this group, the mean age of patients with CH is 12 years older than those without CH (70.5 years vs 58.4 years, p <0.0001). No other significant baseline characteristics are noted.30 distinct CH mutations are noted in these 22 patients. Most patients have a single gene mutation (77%). Gene mutations in DNMT3A are the most common (46%), followed by TET2 (13%), and PPM1D (10%) (see figure). Most mutations are missense (57%), followed by nonsense (30%) and insertion/deletion mutations (13%). One patient has a JAK2 V617F mutation without known history of myeloproliferative disease. 8 CH patients with serial samples collected over a median duration of 136 days (range 91 - 490 days) showed no significant change in CH clone size over time.Patients with CH have no differences in rates of recurrent VTE or arterial thrombosis compared to those without CH. Patients with CH have a trend towards increased rate of combined major bleeding and CRNMB events (23% vs 10%, p=0.14). Patients with CH also had a trend towards increased mean corpuscular volume (MCV) (mean 93.5 vs 90.7, p=0.13), although no other cytopenias or differences in complete blood count parameters were noted.ConclusionThe CH rate in patients with VTE was not found to be much higher than what might be expected by age alone. We found no change in the CH rate after excluding cancer patients. A larger VTE population with age-matched controls will better determine the CH rate in patients with VTE. CH can be a consideration in designing future clinical VTE studies that assess bleeding risk. [Display omitted] DisclosuresByrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Wang: Servier: Membership on an entity's Board of Directors or advisory committees; Leo Pharma: Research Funding.

Characteristics and Philogenetic Analysis of &lt;I&gt;Yersinia pseudotuberculosis&lt;/I&gt; Strains from the Sarydzhaz High-Mountain Focus in the Tien-Shan

The aim of the study was to characterize and carry out molecular-genetic identification of Yersinia pseudotuberculosis strains isolated in the Sarydzhaz high-mountain focus of the Tien-Shan.Materials and methods. The Y. pestis and Y. pseudotuberculosis cultures were differentiated according to their sensitivity to diagnostic bacteriophages, motility, urea fermentation, and pesticin production. Whole genome sequencing was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package, 5.12 and Newbler gsAssembler 2.6 were used for data processing and de novo assembly of raw read sequences. The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 software.Results and discussion. Strains of Y. pseudotuberculosis were isolated from museum mixed with Y. pestis cultures obtained from marmots in the Sarydzhaz high-mountain focus. According to whole genome SNP analysis, they belong to the phylogenetic group O:3 serovar, which is widespread throughout the world. Y. pseudotuberculosis is often present in mixed with Y. pestis cultures, isolated from rodents, which should be taken into account when carrying out laboratory diagnostics of plague in natural foci.

Molecular profiling by NGS upon progression disease in advanced stage NSCLC patients.

e21196 Background: Non-small cells lung cancer (NSCLC) patients treated with Tyrosine Kinase Inhibitors (TKI) at first-line ultimately develop disease progression after 10 to 14 months. Mechanisms underlying TKI-resistance are not complete understood. Tumor re-biopsy is often unfeasible for NSCLC patients and liquid biopsy is a suitable alternative in identify mechanism by which tumors progress. Methods: 113 plasma samples were collected from advanced stage EGFR positive NSCLC patients upon disease progression according to RECIST V1.1. cfDNA NGS profiling was carried out using the Oncomine™ Pan-Cancer Cell-Free Assay and sequenced on an Ion GeneStudio S5 Plus System. Variant calling and annotation were performed with Torrent Suite and Ion Reporter (v5.16) software, respectively. In addition, we developed a bioinformatic pipeline, using RStudio for variant filtering. Results: Overall, 344 somatic variants were detected with an average number of variants per patient of 2.32 and a median Mutant Allele Frequency (MAF) of 0.83%. Most mutated genes were EGFR (63.72%), TP53 (60.18%), APC (16.81%), MAP2K1 (11.50%), PIK3CA (10.62%), FGFR3 (7.08%), KRAS (7.08%) and BRAF (6.19%). As expected, SNPs were the most frequent mutation type (72.67%), following by INDELs (24.41%) and CNVs (2.91%). We found high co-occurrence between MYC-GNA11, MYC-CCND2, HRAS-AR and EGFR-TP53 genes using Jaccard’s score (0.5, 0.5, 0.5 and 0.46, respectively). Within EGFR-mutated patients, 40% had T790M resistance mutation. Finally, we found KRAS mutations and missense PIK3CA mutations were mutually exclusive. Conclusions: Molecular profiling of liquid biopsies collected upon disease progression using NGS help to identify molecular mechanisms underlying treatment failure.

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαβ+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors’ laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

Comparative Genetic Analysis of Yersinia pestis Strains Isolated on the Ukok Plateau and other Territories of the Altai Mountains

The aim of the study was molecular-genetic identification and analysis of the phylogenetic relationship of Yersinia pestis strains isolated on the Ukok Plateau in 2020, in order to establish the current boundaries of the natural mega focus of plague in the Altai Mountains in Russia and Mongolia.Materials and methods. 37 strains of Y. pestis of the main subspecies isolated in the Tuva mountain and Gorno-Altai high-mountain plague foci and adjacent territories of Mongolia in 1971–2020 were studied. The whole genome sequencing of the strains was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package 5.12 and Newbler gsAssembler 2.6 were used to process the data and assemble de novo the sequences of raw reads. The average size of the collected genome was 4.55 million base pairs. Core SNPs were detected by aligning the contigs of Y. pestis strains on the CO92 genome using the Snippy 4.6 program, then 28 SNP homoplasies were removed. The resulting set of SNPs contained only the core region of the genome (955 SNPs). The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 program.Results and discussion. The current population structure of Y. pestis of the main subspecies, antique biovar, phylogenetic line 4.ANT, endemic to the foci of the Altai Mountains in Russia and Mongolia has been determined. The presence of 4.ANT-21 clone, which became widespread in the territory of these natural foci of plague at the begining of the XXI century, was revealed. It is shown that three strains isolated on the Ukok Plateau in 2020 belong to clone 4.ANT-21. According to phylogenetic analysis, evidence of 4.ANT circulation on the Ukok Plateau before 2018 was obtained. The lesson that has been learned is that it is necessary to study the territories of Mongolia, Kazakhstan and China bordering the Ukok Plateau in order to establish the current boundaries of the 4.ANT mega focus.

Identification of novel somatic cell-free DNA variants by next-generation sequencing in breast cancer patients

Objectives: Breast cancer is a heterogeneous disease affecting women worldwide and is one of the leading causes of mortality in India. Sampling bias due to tumor heterogeneity and invasive nature of biopsies necessitate noninvasive methods for comprehensive tumor profiling. Circulating cell-free DNA presents a complete mutation profile of the tumor, enabling the non-invasive monitoring of disease in real-time. This study aimed to identify tumor-specific variants in cfDNA with potential applications in the liquid-biopsy based testing of breast cancer. Material and Methods: Next-generation sequencing was performed for cell-free DNA, lymphocyte DNA, and tumor DNA from 21 breast cancer patients. Variant calling was performed using Torrent Suite Server v.5.0 and somatic variants were annotated using web-based tools. Pathogenic variants detected in cell-free DNA and tumor DNA of three patients were validated by Sanger sequencing. Results: Fifty-nine somatic variants were detected in the cell-free DNA of 10 breast cancer patients. Hotspot variants were detected in PIK3CA, TP53, and KRAS genes. In addition, previously unreported missense variants in ABL1 and PIK3CA genes were predicted to be pathogenic and potential driver mutations. Several frameshift indels were detected in two triple negative breast cancer patients. Conclusion: Sequencing of cell-free DNA from breast cancer patients identified somatic variants including several potentially pathogenic variants which have not been reported previously. These variants may have potential applications as non-invasive biomarkers for breast cancer.

BIOM-08. TARGETSELECTORTM CEREBROSPINAL FLUID (CSF) CIRCULATING TUMOR CELLS AND BIOMARKER ANALYSIS: IMPROVING SENSITIVITY AND TARGETED TREATMENT OPTIONS IN BREAST AND NSCLC CANCER PATIENTS WITH CNS INVOLVEMENT

Abstract BACKGROUND Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal disease (LMD). Here we report the analytical and clinical validation of Target Selector™ CSF assays. METHODS CSF was collected prospectively from pts with a prior solid tumor diagnosis and confirmed or clinical/ radiological suspicion of BM or LMD. CTCs were captured in microfluidic channel and classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) NSCLC and breast multi-gene testing. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software. RESULTS The Target Selector™ CTC platform assays performed on clinical samples (n = 89) resulted in clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0% at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%. CONCLUSIONS Target Selector™ is a viable and sensitive platform for CTC detection and molecular analysis of CSF samples from patients with NSCLC or breast cancer with CNS metastases compared to the current standard of care (CSF cytology) and when imaging findings are equivocal. Identifying CTCs and actionable biomarkers can help to confirm CNS involvement when clinically suspected, guide targeted therapy selection and potentially monitor treatment response.

Identifying Genomic Alterations in Patients With Stage IV Breast Cancer Using MammaSeq: An International Collaborative Study

BackgroundIdentification of genomic alterations present in cancer patients may aid in cancer diagnosis, prognosis and therapeutic target discovery. In this study, we aimed to identify clinically actionable variants present in stage IV breast cancer (BC) samples. Materials and MethodsDNA was extracted from formalin-fixed paraffin-embedded samples of BC (n = 41). DNA was sequenced using MammaSeq, a BC-specific next-generation sequencing panel targeting 79 genes and 1369 mutations. Ion Torrent Suite 4.0 was used to make variant calls on the raw data, and the resulting single nucleotide variants were annotated using the CRAVAT toolkit. Single nucleotide variations (SNVs) were filtered to remove common polymorphisms and germline variants. CNVkit was employed to identify copy number variations (CNVs). The Precision Medicine Knowledgebase (PMKB) and OncoKB Precision Oncology Database were used to associate clinical significance with the identified variants. ResultsA total of 41 samples from Turkish patients with BC were sequenced (read depth of 94-13,340; median of 1529). These patients were diagnosed with various BC subtypes including invasive ductal carcinoma, invasive lobular carcinoma, apocrine BC, and micropapillary BC. In total, 59 different alterations (49 SNVs and 10 CNVs) were identified. From these, 8 alterations (3 CNVs – ERBB2, FGFR1, and AR copy number gains and 5 SNVs – IDH1.R132H, TP53.E204∗, PI3KCA.E545K, PI3KCA.H1047R, and PI3KCA.R88Q) were identified to have some clinical significance by PMKB and OncoKB. Moreover, the top 5 genes with the most SNVs included PIK3CA, TP53, MAP3K1, ATM, and NCOR1. Additionally, copy number gains and losses were found in ERBB2, GRB7, IGFR1, AR, FGFR1, MYC, and IKBKB, and BRCA2, RUNX1, and RB1, respectively. ConclusionWe identified 59 unique alterations in 38 genes in 41 stage IV BC tissue samples using MammaSeqTM. Eight of these alterations were found to have some clinical significance by OncoKB and PKMB. This study highlights the potential use of cancer specific next-generation sequencing panels in clinic to get better insight into the patient-specific genomic alterations.

Study of Ras Mutations' Prognostic Value in Metastatic Colorectal Cancer: STORIA Analysis.

Background: Colorectal cancer (CRC) is the second most common cause of cancer-specific death in both sexes in Western countries. KRAS mutations occur in about 50% of metastatic CRCs (mCRCs). The prognostic value of specific KRAS mutations still remains unexplored and unclear. Methods: Two hundred and forty KRAS wild-type and 206 KRAS/NRAS mutant consecutive unresectable mCRC patients with PS Eastern Cooperative Oncology Group (ECOG) 0 or 1, aged < 80 years, and with a life expectancy >3 months entered into this study. DNA was extracted from paraffin-embedded formalin-fixed tumour tissues, and it was sequenced with the Oncomine Solid Tumour DNA kit (Thermo Fisher Scientific, Waltham, MA, USA). Data were analysed using the Torrent Suite Software v5.0 (Thermo Fisher Scientific). The primary outcome was the analysis of the prognostic role of different KRAS mutations in terms of overall survival (OS). Results: There were no significant differences among the most prevalent mutations (p.G12D, p.G12V, p.G13D, p.G12A, p.G12C, and p.G12S) in terms of age (<65 vs. ≥65 years), gender (male vs. female), grading (G1/G2 vs. G3), side of primary tumour (left vs. right), pT, and pN. At the median follow-up of 25.6 months, there were 77 deaths in KRAS-mutated patients and 94 in wild-type patients. Three homogeneous prognostic groups were identified: wild-type patients (group A, median survival: 27.5 months), p.G13D/p.G12A/p.G12V/p.G12D mutants (group B, median survival: 17.3 months), and p.G12C/p.G12S mutants (group C, median survival: 5.0 months, p < 0.0001 according to Log Rank test). Upon multivariate analysis, metastatic involvement and p.G12C/p.G12S KRAS mutation group C (vs. other mutations) emerged as independent prognostic variables for survival. Conclusions: We show that mutant KRAS is a negative prognostic factor and that p.G12C/p.G12S variants present the worst clinical courses. This information suggests a clear difference among KRAS mutations, and it will be useful to test potentiated and/or innovative therapeutic strategies in p.G12C/p.G12S metastatic CRC patients.

Abstract B091: Molecular alterations in black and Hispanic women with early breast cancer

Abstract Background: Black race is associated with inferior outcomes in early breast cancer, including hormone-receptor (HR) positive and HER2-negative disease, even when receiving comparable adjuvant systemic chemotherapy and endocrine therapy (Sparano et al. JNCI 2012; Albain et al. AACR SABCS 2018, abstract GS4-07). Hispanic ethnicity is associated with lower breast cancer incidence but higher mortality rates than non-Hispanic whites. Molecular differences in breast cancer could account for these disparities. A prior report based on Whole Exome Sequencing (WES) from the Cancer Genome Atlas (TCGA) in 105 African-American women showed higher rates of TP53 mutations and lower frequency of PIK3CA mutations (Keenan et al. JCO 2017), whereas data in Hispanics is limited. We use a custom sequencing panel called the Einstein Comprehensive Cancer Panel (ECCP) of ~150 genes commonly mutated in malignancies (Miller et al. Oncotarget 2017) in a cohort of 20 non-Hispanic black (NHB) and 24 Hispanic (H) women with early breast cancer. Methods: Ion AmpliSeq targeted libraries were prepared from genomic DNA isolated from breast tumor and adjacent non-tumor tissues and sequenced on the Ion Proton platform. Sequencing reads were analyzed using the Ion Torrent Suite’s Torrent Variant Caller followed by the Ion Reporter Software for variant calling and annotation. Results: Samples for 20 NHB and 24 H women were analyzed. The characteristics of the NHB/H cohorts were similar in age (median 55/59 years), stage (61%/73% with Stage II-III), and HR+/HER2-negative subtype (70%/77%). In NHB women, the most frequent pathogenic mutations were in TP53 (20%), PIK3CA (15%), BRCA1 (10%), CDH1 (10%), and KM2TC (10%). Of the NHB patients who recurred, 33% had a pathogenic mutation in TP53 and 16% in KRAS. Of NHB women with HR+ HER2- disease, the most common pathogenic mutations were in PIK3CA (21%) followed by CDH1, TP53, KM2TC, and BRCA1 (all 14%). In H women, the most frequent pathogenic mutations were in PIK3CA (50%) and TP53 (21%) followed by CDH1, FAT4, ATM, and KM2TC (all 8%). Of NHB women with HR+ HER2- disease, the most common pathogenic mutations were in PIK3CA (55%), TP53 (11%), and FAT4 (11%). Of the H patients who recurred, one had a deleterious mutation in TP53, BRCA1, and PIK3CA while the other had a deleterious mutation in GATA3. Conclusions: We found that the NHB and H patients had different molecular profiles despite similar clinical presentations with regard to age, stage, and subtype. The most notable finding was the very high rate of PIK3CA/PIK3R1 (50%) in breast cancers occurring in Hispanic women, which has implications for screening H women with metastatic breast cancer for PIK3CA-directed therapies such as alpelisib. The cohort has variants of unknown significance with mutations in genes that are not described in the literature as well as some hypermutated patients. This study indicates the importance of expanding molecular testing in diverse populations. Citation Format: Preethi Prasad, Cristina Montagna, Jesus D. Anampa Mesias, Maja Oktay, Joseph A. Sparano. Molecular alterations in black and Hispanic women with early breast cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B091.

P0074EXPANDING THE VARIABILITY OF THE ADPKD-GANAB CLINICAL PHENOTYPE: A NEW FAMILY OF ITALIAN ANCESTRY

Abstract Background and Aims Causative GANAB mutations have been described in only 123 families, 98 diagnosed with late-onset mild ADPKD and 35 with ADPLD. We describe a new family with mild, late-onset ADPKD due to p. R839W GANAB mutation, previously reported in an ADPLD patient requiring liver transplantation. Method Mutation analysis of PKD1, PKD2, GANAB genes was performed by targeted NGS analysis. To analyze GANAB gene we developed a custom panel of 11 kidney cystogenes (GANAB, PKDH1, TSC1, TSC2, UMOD, HNF1B, REN, OFD1, PARN, DNAJB11, SEC61A1) designed using Ion Ampliseq Designer. Sanger sequencing was performed in order to validate all the variants classified as pathogenic, likely pathogenic and VUS. Raw sequence data analysis, including base calling, demultiplexing, alignment to the hg19 human reference genome, was performed using the Torrent Suite Software version 5.5; the average depth of total coverage was set at 500X and for variant calls at minimum of 30X. Deletion and duplication analysis of PKD1 and PKD2 was performed using MLPA P351-C1 and P352-D1 probemixes, MRC-Holland. Results Diagnosis of ADPKD was made in a 45-year old man during pre-surgical screening for umbilical and inguinal hernia repair. The patient's clinical course was characterized by several complications pertaining to the ADPKD spectrum: nephrolithiasis (20 yo); umbilical/bilateral inguinal hernia repair, hypertension and mild aortic root dilation (45 yo) and AKI due to ureteral obstruction (50 yo). Abdomen CT scan showed bilateral renal cysts (TKV 565 cc), nephrolithiasis, normal-sized liver with multiple cysts, and sigmoid colon diverticulosis. Renal function was normal (SCr 0.69 mg/dL, CKD-EPI 115 ml/min). In the index case, NGS and MLPA analysis of PKD1 and PKD2 genes did not detect variants. We then use the abovementioned multigene NGS panel and identified a missense heterozygous c.2515C&amp;gt;T (p.R839W) variant in the GANAB gene. Screening was then extended to family members. No family members displayed renal function impairment. Both the 80 yo mother and the 84 yo father were found to have multiple bilateral kidney cysts (HtTKV of 239 ml and 435 ml, respectively), no liver cysts were found in either of them. Parents segregation analysis identified the GANAB variant p.R839W in the mother and in the maternal aunt. The father tested negative for all the abovementioned cystogenes. Conclusion we confirm that the renal phenotype caused by mutations in GANAB is very different from those due to mutations in PKD1 and PKD2, giving rise to a mild form of renal cystic disease, usually not progressing to ESRD. Despite the mild renal cystic burden, the index case showed a plethora of renal and extrarenal manifestations of ADPKD. The finding that patients with GANAB mutation can present with renal and liver cystic phenotype is intriguing, indicating a commonality between pathogenic background of two different inherited disorders, ADPKD and ADPLD. The missense GANAB mutation identified in our ADPKD family was first described in a pedigree reported by Porath et al. and diagnosed as ADPLD. This suggests that, beyond the effect of the shared mutation on GII subunit α, other modifier loci and environmental factors may influence the course of liver disease development and progression. Our study illustrates the important diagnostic role of a broader genetic testing, able to screen not only for PKD1 and PKD2 variants, but also for pathogenic variants in other cystogenes.