Ion AmpliSeq Research Articles

12505 Prevalence Of Mody Diabetes Polymorphism Of Thai Urban Families By Targeted Next Generation Sequencing Of Diabetes Polymorphism

Abstract Disclosure: S. Niramitmahapanya: None. Prevalence of MODY diabetes polymorphism of Thai Urban families by targeted next-generation sequencing in diabetes polymorphismObjective: To study the prevalence between single nucleotide polymorphism of urban diabetes (UD) and non-diabetes (noD) in Thai population. Method: Cross-sectional study was collected 240 participant samples; 60 from UD, 60 from RnoD, 60 from non-relative, non-diabetes (noRnoD) and 60 diabetes, non-relatives (noRD). And generated diabetes variant frequency in Thai population by targeted next-generation sequencing. Result; Among group of UD and RnoD found that age, fasting blood glucose (FBG), HbA1c and LDL level were statistic significant all parameter by p<0.001. Age in UD was 64.15±10.74 vs. 43.43±12.46 in RnoD, FBs in UD was 129.39±44.91 vs. 99.74±39.75 in RnoD, A1c in UD was 8.26±4.76 vs. 6.17±1.35 in RnoD and LDL level in UD was 98.28±38.52 vs. 129.35±39.75 in RnoD.In subgroup analysis of diabetes variant, we designed Ion AmpliSeq On-Demand Panels (25 genes related diabetes) for targeted sequencing. We didn’t found statistic significant between UD and RnoD group by p-value =0.998 (23 genes; ABCC8, FGA, SELE, PAX4, HNF1A, BLK, LOC10028732, NEUROD1, KCNJ11, MMP2, APPL1, LPL, CAT, CEL, HNF1A, HNF1B, ICAM1, INSR, SOD, GCK, INS and UCP3). We identified 12 mutations of MODY in UD, the most polymorphism was HNF1a and ABCC8 that found 16.4%, PAX4 found 13.1%, APPL1 found 11.5%, KCNJ11 and NEUROD1 found 8.2%, BLK 6.6% and other less polymorphism that found <5% (HNF1B, HNF4a, CEL, GCK, INS). Conclusion; From the study, we analyzed genetic data from 12 genes that involved pathogenesis for diabetes polymorphism between UD and noD groups. We focus on diabetes variant frequency. We found that 5 pathogenic polymorphism such as HNF1a, GCK, CEL, INS and APPL1. Further study needs to evaluate correlation diabetes polymorphism and their treatment protocols. Keywords; Thai urban diabetes, first degree relatives and MODY diabetes polymorphism Presentation: 6/1/2024

Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue

BackgroundNext-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients.Methods and resultsGenomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage.ConclusionsWith the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

Unusual PEComa With PRCC :: TFE3 Fusion Mimicking Sinonasal Tract Melanoma.

We report a nasal cavity unusual perivascular epithelioid cell tumor (PEComa) mimicking mucosal melanoma. Immunohistochemistry was performed using BenchMark Ultra and panel of antibodies. The Ion Torrent platform and Ion AmpliSeq cancer hotspot panel were utilized for DNA genotyping. Target-specific RNA libraries for the detection of fusion transcripts were constructed using Archer Universal RNA Reagent Kit v2 and Archer FusionPlex Solid Tumor panel and sequenced on the MiSeqDx instrument. The tumor, diagnosed in 46-year-old female, was composed of spindle cells, and lacked pigmentation. Immunohistochemically, it showed a patchy HMB-45 positivity. Other melanocytic markers (S100 protein, Melan-A, SOX10) were negative. The tumor cells were weakly positive for KIT (CD117) while negative for smooth muscle actin, pancytokeratin cocktail (AE1/AE3), and synaptophysin. Diagnosis of primary sinonasal tract mucosal melanoma was favored. Additional molecular studies detected PRCC :: TFE3 fusion as the sole genetic change, and suggested the diagnosis of unusual PEComa. Previously, TFE3 fusions were reported in a subset of PEComas but not in melanomas, while PRCC involvement has only been documented once in an ocular PEComa. Immunohistochemistry revealed strong nuclear TFE3 expression concordant with the molecular findings. This report emphasis the importance of molecular testing in the differential diagnosis between PEComa and melanoma, especially when the tumor arises in a site typical of melanoma but showing an unusual morphology and immunophenotype. The detection of TFE3 fusion transcripts suggested the diagnosis of SNT PEComa, although it cannot be excluded that this and similar tumors represent a distinct diagnostic category.

P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis

Abstract Study question Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes? Summary answer PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need. What is known already Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative. Study design, size, duration In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study. Participants/materials, setting, methods The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol. Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis. Main results and the role of chance The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo. In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%). Limitations, reasons for caution The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases. Wider implications of the findings A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy. Trial registration number Not applicable

Molecular profiling of metastatic breast cancer and target-based therapeutic matching in an Asian tertiary phase I oncology unit.

Molecular profiling of metastatic breast cancer (MBC) through the widespread use of next-generation sequencing (NGS) has highlighted actionable mutations and driven trials of targeted therapy matched to tumour molecular profiles, with improved outcomes reported using such an approach. Here, we review NGS results and treatment outcomes for a cohort of Asian MBC patients in the phase I unit of a tertiary centre. Patients with MBC referred to a phase I unit underwent NGS via Ion AmpliSeq Cancer Hotspot v2 (ACH v2, 2014-2017) prior to institutional change to FoundationOne CDx (FM1; 2017-2022). Patients were counselled on findings and enrolled on matched therapeutic trials, where available. Outcomes for all subsequent treatment events were recorded to data cut-off on January 31, 2022. A total of 215 patients were enrolled with successful NGS in 158 patients. The PI3K/AKT/PTEN pathway was the most altered with one or more of the pathway member genes PIK3/AKT/PTEN affected in 62% (98/158) patients and 43% of tumours harbouring a PIK3CA alteration. Tumour mutational burden (TMB) was reported in 96/109 FM1 sequenced patients, with a mean TMB of 5.04 mt/Mb and 13% (12/96) with TMB ≥ 10 mt/Mb. Treatment outcomes were evaluable in 105/158 patients, with a pooled total of 216 treatment events recorded. Matched treatment was administered in 47/216 (22%) events and associated with prolonged median progression-free survival (PFS) of 21.0 weeks [95% confidence interval (CI) 11.7, 26.0 weeks] versus 12.1 weeks (95% CI 10.0, 15.4 weeks) in unmatched, with hazard ratio (HR) for progression or death of 0.63 (95% CI 0.41, 0.97; p = 0.034). In the subgroup of PIK3/AKT/PTEN-altered MBC, the HR for progression or death was 0.57 (95% CI 0.35, 0.92; p = 0.02), favouring matched treatment. Per-patient overall survival (OS) analysis (n = 105) showed improved survival for patients receiving matched treatment versus unmatched, with median OS (mOS) of 30.1 versus 11.8 months, HR = 0.45 (95% CI 0.24, 0.84; p = 0.013). Objective response rate (ORR) in the overall population was similar in matched and unmatched treatment events (23.7% versus 17.2%, odds ratio of response 1.14 95% CI 0.50, 2.62; p = 0.75). Broad-panel NGS in MBC is feasible, allowing therapeutic matching, which was associated with improvements in PFS and OS.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Genome-wide characterization of the mutational landscape of proliferative verrucous leukoplakia

ObjectiveProliferative verrucous leukoplakia (PVL) is a rare but highly aggressive variant of oral leukoplakia that almost inevitably progresses to oral squamous cell carcinoma (OSCC). The aims of this study were to perform whole exome sequencing of a cohort of patients diagnosed with PVL and identify potential mutational profiles and pathways in this disorder. Study DesignA total of 12 oral cavity mucosal biopsies from 6 patients with oral lesions clinically compatible with PVL were used. Of these, 9 were diagnosed as dysplasia, 1 OSCC and 2 hyperkeratosis/hyperplasia. Exome sequencing used the Ion AmpliSeq Exome platform. Ion Reporter software was used for variant calling, annotation and filtering. Analysis and visualization of somatic mutations was carried out using the MAFtools R package. ResultsFollowing exome sequencing and mutational profiling we analyzed the profiles for cancer associated genes and signatures. Genes previously associated with OSCC including HYDIN, MUC16, MAML3, CDKN2A, FAT1 and CASP8 were mutated in multiple samples. Several DNA damage repair genes including PARP1 were mutated in PVL samples. NOTCH and Hippo pathways were the most frequently impacted by mutation. ConclusionThis genome wide characterization of premalignant PVL identifies both known and potentially novel oncogenic mechanisms in this disorder.

Variants in Candidate Genes for Phenotype Heterogeneity in Patients with the 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11.2DS) is a microdeletion syndrome with a broad and heterogeneous phenotype, even though most of the deletions present similar sizes, involving ∼3 Mb of DNA. In a relatively large population of a Brazilian 22q11.2DS cohort (60 patients), we investigated genetic variants that could act as genetic modifiers and contribute to the phenotypic heterogeneity, using a targeted NGS (Next Generation Sequencing) with a specific Ion AmpliSeq panel to sequence nine candidate genes (CRKL, MAPK1, HIRA, TANGO2, PI4KA, HDAC1, ZDHHC8, ZFPM2, and JAM3), mapped in and outside the 22q11.2 hemizygous deleted region. In silico prediction was performed, and the whole-genome sequencing annotation analysis package (WGSA) was used to predict the possible pathogenic effect of single nucleotide variants (SNVs). For the in silico prediction of the indels, we used the genomic variants filtered by a deep learning model in NGS (GARFIELD-NGS). We identified six variants, 4 SNVs and 2 indels, in MAPK1, JAM3, and ZFPM2 genes with possibly synergistic deleterious effects in the context of the 22q11.2 deletion. Our results provide the opportunity for the discovery of the co-occurrence of genetic variants with 22q11.2 deletions, which may influence the patients´ phenotype.

Abstract 3489: MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs

Abstract Introduction: DNA methylation plays a crucial role in development and progression of cancer. Identification of DNA methylation profiles in cancer is indispensable for early detection. To address this research need, Ion AmpliSeqTM offers targeted next-generation sequencing (NGS) methylation community amplicon panels for DNA methylation profiling in cancer-associated genes. However, the process of designing custom methylation amplicon panels using the Ion AmpliSeq primer design pipeline is intricate, labor-intensive, and time-consuming. Here, we developed a one-step, fully automated, faster, and reproducible nextflow-based primer design pipeline (MetFlow) for development of custom Ion AmpliSeqTM methylation panels. MetFlow streamlines the development of custom methylation panels for cancer-related genes, and thus significantly reduces the time and effort traditionally required for amplicon design. Methods: The fully automated MetFlow pipeline is implemented using Nextflow. MetFlow necessitate an input configuration file for the execution of methylation Ion AmpliSeqTM designs. The configuration file specifies the essential parameters, including target CpG input, reference genome, pool number, and amplicon lengths, which are required for designing the amplicons. The MetFlow pipeline designs candidate amplicons for CpG targets on both Watson and Crick strands, facilitating the subsequent creation of files required for the generation of a methylation panel. Results: The fully automated MetFlow pipeline has been used for designing the customized Ion AmpliSeqTM methylation amplicon panel(s) for cancer and age-related genes. The amplicons lengths in these AmpliSeqTM methylation panels were ranged from 125 bp to 175 bp for formalin-fixed, paraffin-embedded (FFPE) research samples. These customized methylation panels were run on the Ion GeneStudioTM S5 sequencer followed by performance analysis using Ion TorrentTM Suite. The published data for these custom amplicon panels suggest that the Ion AmpliSeqTM has sufficient read depth and mapping efficiency, higher sensitivity and accuracy for methylation measurement, and strong correlation (r=0.993) for methylation profiling among sample replicates. Conclusion: We present MetFlow, an advanced automated primer design pipeline for designing the custom Ion AmpliSeqTM methylation amplicon panels tailored to targeted NGS applications in cancer research. The custom designed Ion AmpliSeqTM methylation panels exhibited sufficient read depth, higher sensitivity and accuracy, and reproducibility. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Renesh Bedre, Drishti Kaul, Anupma Sharma, Andrew Hatch, Loni Pickle, Na Li, Fiona Hyland. MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3489.

Abstract 3659: Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer

Abstract Biliary tract cancer (BTC) is a rare type of abdominal cancer and is more frequent in Asian countries than in Western. The 5-year survival rate for BTC is very low 7 to 20% and only 20% of BTC patients could undergo curative-intent surgery. BTC diagnosis based on biopsy and cytology are low in sensitivity that prompted the necessity to identify alternative biopsy. Liquid biopsy, specifically circulating tumor DNA (ctDNA) is known for the accessibility and low invasiveness received great interest for their potential uses in various clinical applications for patients with insufficient tumor tissue, including mutation profiling, treatment monitoring, and cancer detection. However, the panel available in the market up-to-date focused mainly targeted, actionable alterations and thus not suitable for minimal residual disease (MRD) monitoring. In this study, we aimed to develop a customized ctDNA panel for MRD monitoring in BTC. In this study, 124 BTC patients undergone curative resections were enrolled at Hokkaido University and Sapporo Medical University from May 2019 to April 22. All enrolled patients have BTC diagnosis confirmed by pathological diagnosis after surgery, and without other malignant tumors. Genomic profiles of FFPE tissue were conducted using Ampliseq CCP. 13 patients were excluded for high fragmentation. Referring to the genomic profiles of 111 patients in our study and 1924 patients in TCGA established a customized panel using Ion Ampliseq HD technology. The Ampliseq HD panel claims to detect variants at 0.1% LOD for ctDNA detection. It also integrates molecular barcodes to reduce NGS-related errors. The designed panel was 258 Amplicons and 20 genes including eleven therapeutically targetable genes. Clinical characteristics of ctDNA detection were also assessed. Results:The panel validation: concordance of the mutation allele frequency (MAF) between the NGS standard control and Ampliseq HD panel showed a coefficient of determination (R2) of 0.96. Also, concordance of MAF Ampliseq CCP and Ampliseq HD panel on FFPE tumor profiling showed R2 of 0.78. When assessing 70% of BTC patients were covered to have at least one tumor-derived Ampliseq HD panel. When assessing using plasma cell-free DNA, 55% of BTC patients were detected to have at least one tumor-derived ctDNA. Distribution of detected altered top4 genes using Ampliseq HD panels shows TP53 35%, KRAS 10%, SMAD4 5%, ARID1A 2.5%. Clinical characteristics may affected the concordance of plasma and FFPE tumor tissue; stage (P=0.035), size of tumors (P=0.0088), subtype (P=0.013), LN metastasis (P=0.11). Conclusions: A highly sensitive ctDNA customized panel for BTC was established. We also found that clinical characteristics of the BTC affects the detection of ctDNA from blood liquid biopsy. Future study: MRD evaluation using this customized panel. Citation Format: Yoshihito Shinohara, Siew-Kee Low, Toru Nakamura, Yasutoshi Kimura, Takeshi Murakami, Kazuma Kiyotani, Satoshi Hirano. Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3659.

Abstract 6684: Impact of fecal microbiome on the efficacy of neoadjuvant chemo-immunotherapy and colitis adverse events: Findings from the NADIM II Trial

Abstract Introduction: The balance and composition of the gut microbiome play a pivotal role in modulating the host immune system, and they have been associated with survival outcomes in patients undergoing immunotherapy-based treatments and immune-related adverse events. In this study, our objective was to analyze the fecal microbiota of non-small-cell lung cancer (NSCLC) patients with locally advanced disease undergoing neoadjuvant chemo-immunotherapy (IO-CT). Methods and patients: Baseline stool samples were collected from 86 resectable stage IIIA/B NSCLC patients enrolled in the NADIM II clinical trial (NCT03838159). The patients were randomly assigned to receive either neoadjuvant nivolumab plus chemotherapy (CT) or CT alone. DNA extraction was performed using the QIAamp PowerFecal DNA Kit (Qiagen), and the samples were analyzed using the Ion AmpliSeq Microbiome Health Research assay (ThermoFisher). The NGS analysis utilized the AmpliSeq Microbiome Health w1.3 workflow. Alpha diversity was assessed using the Simpson and Chao1 Index. Results: Among the 86 collected stool samples, 77 were deemed valid for analysis. Bacteroides were the most common genus with a relative percentage (RP) of 18.9% ± 14.8 followed by Faecalibacterium (13.4% ± 7.8). The presence of bacteria from the genera Bifidobacterium, and Faecalibacterium was associated with improved progression-free survival (PFS) [HR = 0.4 (0.16-0.98), and HR = 0.08 (0.01-0.72), respectively] and overall survival (OS) [HR = 0.19 (0.06-0.62)], and HR = 0.04 [0.01-0.43], respectively), in the experimental arm. The detection of Faecalibacterium prausnitzii (validated by qPCR) was associated with improved PFS [HR = 0.17 (0.04-0.76)] and OS [HR = 0.05 (0.01-0.29)] in the experimental. On the other hand, detection of bacteria from the genera Synergistes, Lactobacillus, and Anaerotruncus was associated to higher risk of disease progression [HR = 9.9 (1.2-85), HR = 3.7 (1.5-9.1), and HR = 12 (1.4-110)] and death [HR = 51 (3.2-820), HR = 6.7 (2-23), and HR = 25 (2.3-280)] in this set of patients.In the entire cohort, patients whose samples were in the highest tertile of alpha diversity, had significantly prolonged PFS compared to those with intermediate or low diversity [HR = 0.39 (0.16-0.94)]. Finally, 25 colitis adverse events (CAE) occurred in 18 patients, including two Grade 3 (2.3%) and four Grade 2 (4.6%) events. A higher RP of Bacteroides were found in patients who did not suffer colitis events (P = 0.016), whereas higher RP of Coriobacterium were detected in patients who suffered CAE (P = 0.023). Conclusions: Our findings suggest that composition of the gut microbiome may play a crucial role in the effectiveness of neoadjuvant IO-CT in NSCLC patients. Specifically, the presence of F. prausnitzii may enhance the antitumor activity of these treatments. Citation Format: Roberto Serna-Blasco, Alejandro Rodriguez Festa, Sandra Sanz-Moreno, Lucía Robado de Lope, Pilar Mediavilla Medel, Ernest Nadal, Jose Luis González Larriba, Alex Martínez-Martí, Reyes Bernabé, Joaquim Bosch-Barrera, Joaquín Casal-Rubio, Virginia Calvo, Amelia Insa, Santiago Ponce, Noemí Reguart, Javier de Castro, Joaquín Mosquera, Manuel Cobo, Andrés Aguilar, Carlos Camps, Rafael López de Castro, Teresa Morán, Isidoro Barneto, Delvys Rodríguez-Abreu, Carlos Aguado de la Rosa, Ramón Palmero, Florentino Hernando-Trancho, Javier Martín-López, Alberto Cruz-Bermúdez, Bartomeu Massuti, Miguel Ángel Molina Vila, Atocha Romero, Mariano Provencio. Impact of fecal microbiome on the efficacy of neoadjuvant chemo-immunotherapy and colitis adverse events: Findings from the NADIM II Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6684.

Gene Expression Differences Identified in Skin Samples of Mycosis Fungoides, Atopic Dermatitis, and Psoriasis

Background: Recent advances in the understanding of the molecular mechanisms underlying atopic dermatitis (AD) and psoriasis (PSO) have led to the development of multiple targeted therapies. Yet, molecular heterogeneity contributes to inconsistent clinical presentation and therapeutic response. Further, systemic treatment of presumed AD or PSO can lead to delays in both diagnosis and proper treatment of patients with a true diagnosis of mycosis fungoides (MF) – a potentially dangerous clinical mimic of AD and PSO that requires a rigorous histologic and molecular workup to diagnose. Therefore, a non-invasive method to distinguish molecular profiles of MF from AD and PSO could inform accurate diagnoses and avoid inappropriate treatment of MF. We have previously shown transcriptomic differences in AD and PSO samples obtained by a non-invasive scraping technique. However, this technique has not been used to assess differences in gene expression profiles of MF samples. Objectives: To identify gene expression differences based on diagnosis of MF, AD, or PSO and response to targeted systemic AD or PSO therapies. Methods: Lesional baseline samples were assessed from 76 patients (AD, n=24; PSO, n=48; and MF, n=4) enrolled in one of two IRB-approved studies (IDENTITY or SIGNAL-MF). Lesional samples were collected by epidermal scraping and preserved in RNA buffer. Library preparation and next generation RNA sequencing was performed using the Ion AmpliSeq Transcriptome Human Gene Expression panel on the S5 Prime sequencer (ThermoFisher). Clinical responses for AD and PSO were further assessed over 3 months using the eczema and psoriasis area and severity index (EASI and PASI, respectively). Genes were considered differentially expressed if there was a log2fold change >|1.0| and padj <.05. Results: Statistically significant differences in gene expression were observed between AD, PSO, and MF lesions. Further, genes were differentially expressed in baseline skin scrapings obtained from super-responders to dupilumab, which blocks both IL-4 and IL-13 signaling, and PSO therapies including the IL-23 inhibitor risankizumab relative to those who did not achieve ≥90% improvement. Conclusion: A non-invasive molecular test could be developed to distinguish between AD, PSO, and MF, and further identify super-responders to targeted PSO and AD therapies.

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology

A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.

Characterization and Distribution of SARS-CoV-2 Omicron Variant and its Sub-lineages in Uttarakhand using Next Generation Sequencing: A Retrospective Study

The etiological agent of coronavirus disease (COVID-19) that emerged at the end of year 2019 was first reported in Wuhan, China and was found to be SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). The massive COVID-19 waves were due to various variants. As per the reports of other study it was also found that Omicron variant spread faster than various other variant such as delta variant. Omicron has been reported from various countries and now from many states of India too. Therefore, keeping this in mind, this study was undertaken to study all the lineages of SARS-CoV-2 Omicron variant of disease COVID-19 that are circulating in the population of Uttarakhand with objective to study next generation sequencing of all the RT-PCR positive of SARS-CoV-2 and to find out all the lineages of the Omicron variant of SARS-CoV-2. This was a retrospective study conducted from 1st January 2022 to 30th September 2022. Next generation sequencing was performed on all the samples that were tested for COVID-19 by using Ion AmpliSeq kit on Ion Chef instrument. A total of 2149 samples were tested in which majority of samples belong to age group of 21-40 years. Males were affected more than females. BA.2 was found to be the predominant lineage of total of 46 lineages that were identified. Their mutations were also studied. We conclude that different variants of clade 21L, 22B, 22D and Omicron subvariant BA.2, BA.2.38 and BA.2.75 were the ones that were circulating amongst the population of Uttarakhand. The characteristic mutation that was found were T19I and V213G in NTD, S373P, S375F, T376A, and D405N in RBD.

Differential gene expression in two consecutive pregnancies between same sex siblings and implications on maternal constraint

The objective of this study was to investigate how placental gene expression differs in two consecutive pregnancies in same sex siblings, and its possible association with the “maternal constraint” hypothesis. Material was gathered from the BASIC study (Biological, Affect, Stress, Imaging, and Cognition in Pregnancy and the Puerperium), a population based prospective study that was started in 2009 in Uppsala. Over 900 specimens of placenta biopsies were collected and out of these 10 women gave birth twice, to the same sex child, and were included in this study. The total RNA was isolated and prepared from frozen villous tissue from the placenta and further analyzed by use of Ion AmpliSeq Human Transcriptome Gene Expression kit. A total of 234 genes differed significantly between the first and second pregnancy placentas, when adjusting for delivery mode, maternal BMI and gestational age. Of special interest was the down-regulated group of genes in the second pregnancy. Exemplified by Pentraxin 3, SRY-Box Transcription Factor 9, and Serum Amyloid A1, which all were associated with biological processes involved in the immune system and inflammation. Further, protein–protein interaction analysis visualized them as hub genes interacting with several of the other differentially expressed genes. How these altered gene expressions affect maternal constraint during pregnancy needs further validation in lager study cohorts and also future validation in functional assays.

Genetic characterization of intramuscular myxomas.

Introduction: Intramuscular myxomas are benign tumors that are challenging to diagnose, especially on core needle biopsies. Acquired chromosomal aberrations and pathogenic variants in codon 201 or codon 227 in GNAS complex locus gene (GNAS) have been reported in these tumors. Here we present our genetic findings in a series of 22 intramuscular myxomas. Materials and methods: The tumors were investigated for the presence of acquired chromosomal aberrations using G-banding and karyotyping. Pathogenic variants in codon 201 or codon 227 of GNAS were assessed using direct cycle Sanger sequencing and Ion AmpliSeq Cancer Hotspot Panel v2 methodologies. Results: Eleven tumors carried chromosomal abnormalities. Six tumors had numerical, four had structural, and one had both numerical and structural chromosomal aberrations. Gains of chromosomes 7 and 8 were the most common abnormalities being found in five and four tumors respectively. Pathogenic variants in GNAS were detected in 19 myxomas (86%) with both methodologies. The detected pathogenic variants were p.R201H in nine cases (seven with abnormal and two with normal karyotypes), p.R201C in five cases, all with normal karyotypes, p.R201S in three cases (two with abnormal and one with normal karyotype), p.R201G in one case with a normal karyotype, and p.Q227E in one case with a normal karyotype. Conclusion: Firstly, our data indicate a possible association between chromosomal abnormalities and GNAS pathogenic variants in intramuscular myxomas. Secondly, the presence of the rare pathogenic variants R201S, p.R201G and p.Q227E in 26% (5 out of 19) of myxomas with GNAS pathogenic variants shows that methodologies designed to detect only the common "hotspot" of p.R201C and p.R201H will give false negative results. Finally, a comparison between Ion AmpliSeq Cancer Hotspot Panel v2 and direct cycle Sanger sequencing showed that direct cycle Sanger sequencing provides a quick, reliable, and relatively cheap method to detect GNAS pathogenic variants, matching even the most cutting-edge sequencing methods.

Genetic testing of Japanese patients with serrated polyposis syndrome: A multicentric study.

75 Background: Serrated polyposis syndrome (SPS) is a rare condition associated with an increased risk of colorectal cancer. Previous studies have identified germline truncating variants of the RNF43; however, patients harboring these variants comprise a small part of those with SPS, in most of whom the causative gene remains unknown. To date, no study has described the genetic features of Japanese patients with SPS. The present study aimed to identify candidate causative genes of SPS in Japanese patients. Methods: Captures for equal to or more than 55 gene regions were performed using Agilent HaloPlex or Ion AmpliSeq technologies in SPS patients enrolled in The Study Group for Establishment of Diagnosis of Hereditary Gastrointestinal Tract Cancer Syndromes Based on a Next-Generation Sequencing Technology (SGHGCS). Whole exome sequencing of tumor tissue was performed whenever a candidate gene was identified. Results: Eleven patients (four males, seven females) were enrolled. Of these, nine had a history of colorectal cancer; four had a family history of colorectal cancer; and two had a family history of polyposis. Genetic testing identified two variants of VUS, POLD1 c.670C>T (p.R224C) and BRCA2 c.4169T>G (p.L1390W). Additionally, a nonsense variant, BUB1:c.1543G>T p.Gly515Ter was deemed likely to be pathogenic. The patient with the BUB1 variant was 47-year-old female with transverse colon cancer with more than 50 serrated polyps. She was a non-smoker. The variants detected in the transverse colon cancer did not include the canonical variants in common colorectal cancer, such as APC, KRAS or TP53 and were mostly transition substitutions (C>T). Conclusions: BUB1 was identified as a novel candidate causative gene of SPS in a patient with SPS with no smoking habit. These findings will hopefully contribute to our understanding of the genetic basis of SPS.

Further Expanding the Mutational Spectrum of Gorlin Syndrome in Three Unrelated Families

Introduction: Gorlin syndrome is a rare, autosomal dominant multi-systemic disorder with a predisposition to the development of cancers such as medulloblastoma and nevoid basal cell carcinoma. Heterozygous pathogenic variants in PTCH1 are responsible for 90% of Gorlin syndrome cases. Pathogenic variants in PTCH1 cause overstimulation of the sonic hedgehog signaling pathway, which plays a role in the development of embryonic structures and tumorigenesis. Clinical major and minor diagnostic criteria for Gorlin syndrome have been determined. Odontogenic keratocyst (OKC) is the most common reason for medical admission in Gorlin syndrome. In this article, it is aimed to draw attention to the fact that patients with Gorlin syndrome are not very rare in our country and the variability in phenotypic and dysmorphic findings may be a clue for the diagnosis. Methods: Exome sequencing was performed on the Illumina NextSeq550 System platform by using the Ion Ampliseq exome RDY kit for Illumina. Sanger sequencing was performed accordingly for the other affected individuals in both families. Results: In this study, the clinical and molecular findings of 9 Gorlin syndrome patients from three unrelated families are presented. Macrocephaly, calcification of falx cerebri, palmar-plantar pits, rib anomalies, and OKC were detected in decreasing order in more than half of the patients. A novel heterozygous frameshift PTCH1 variant in family 1, a nonsense previously reported PTCH1 variant in family 2, and a novel heterozygous splice-site PTCH1 variant in family 3 were detected. Conclusion: Gorlin syndrome should be kept in mind in patients presenting with macrocephaly, palmoplantar pits, and OKC history. Careful examination of all family members is essential in the timely diagnosis of other affected individuals with minor phenotypic findings.

Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method

PurposeA new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains.MethodsT. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples.ResultsAmong type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing.ConclusionThe new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Whole-genome analysis and evolutionary characterization of cervical and oral human papillomavirus 16.

High-throughput genome-wide sequencing has revealed high genomic variability of HPV16 in different geographic regions which is the most predominant genotype in human papillomavirus (HPV)-associated malignancies. Analysis of the HPV16 by whole-genome sequence (WGS) is an advanced method for the identification of mutations in the genome. There is limited information about HPV16 diversity in Pakistan, especially at the genomic level. Till now, WGS for HPV16 has not been previously reported in Pakistan. The current study has sequenced three HPV16 viral genomes, from two cervical and one oral cavity positive sample of women presented with general gynecological problems without any evidence of precancerous or cancerous lesions using an ion ampliseq customized panel. Sequencing analysis detected 38 variations, including single-nucleotide polymorphisms (SNPs) and two Indels, across three samples with the highest number of SNPs present in E1, E2, and L2, respectively. A total of 20 non-synonymous and 11 synonymous mutations with amino acid substitutions (T1421C, G1515A, T2223C, T1389C, G1483A, and T2191C) were identified. The phylogenetic analysis revealed the genomes of HPV16 are closely associated with those reported from Thailand and the United States. These are the first HPV16 WGS from Pakistan. However, more research is needed with a large sample size from diversified areas to assess the carcinogenic consequences and impact of HPV vaccinations.