Targeted Next-generation Sequencing Panel Research Articles

Performance of a Rapid Digital PCR Test for the Detection of Non-Small Cell Lung Cancer (NSCLC) Variants.

Next-generation sequencing is widely used for comprehensive molecular profiling for many cancers including lung cancer. However, the complex workflows and long turnaround times limit its access and utility. ChromaCode's High Definition PCR Non-Small Cell Lung Cancer Panel (HDPCR™ NSCLC Panel) is a low-cost, rapid turnaround, digital polymerase chain reaction assay that is designed to detect variants in nine NSCLC genes listed in National Comprehensive Cancer Network guidelines. This assay uses TaqMan® probe limiting chemistry and proprietary analysis software to enable multi-target detection within a single-color channel. We compared the performance of the HDPCR™ NSCLC Panel against an in-house, laboratory-developed, targeted next-generation sequencing panel used in the Molecular Diagnostics Laboratory at the University of Minnesota Medical Center to detect biomarkers for NSCLC. The overall accuracy of the HDPCR panel was 99.48% (95% confidence interval 99.01-99.76) with a sensitivity of 95.35% (95% confidence interval 88.52-98.72) and a specificity of 99.69% (95% confidence interval 99.29-99.90). The HDPCR wet lab workflow was 4 h, and the time to generate variant calls from raw data using the ChromaCode Cloud was 2 minutes. We demonstrated that the HDPCR™ NSCLC Panel provides timely, comprehensive, and sensitive mutation detection in NSCLC samples with results in less than 24 h.

Effectiveness of whole exome sequencing analyses in the molecular diagnosis of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of phenotypically and genetically heterogeneous connective tissue disorders that share similar skeletal anomalies causing bone fragility and deformation. This study aimed to investigate the molecular genetic etiology and to determine the relationship between genotype and phenotype in OI patients with whole exome sequencing (WES). Multiplex-Ligation dependent Probe Amplification (MLPA) analysis of COL1A1 and COL1A2 and WES were performed on cases between the ages of 0 and 18 whose genetic etiology could not be determined before using a targeted next-generation sequencing panel, including 13 genes (COL1A1, COL1A2, IFITM5, SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, SP7, BMP1, MBTPS2, PLOD2) responsible for OI. Twelve patients (female/male: 4/8) from 10 different families were included in the study. In 6 (50 %) families, consanguineous marriage was noted. The clinical typing based on Sillence classification; 3 (25 %) patients were considered to be type I, 7 (58.3 %) type III, and 2 (16.7 %) type IV. Deletion/duplication wasn't detected in the COL1A1 and COL1A2 genes in the MLPA analysis of the patients. Twelve patients were molecularly analyzed by WES, and in 6 (50 %) of them, a disease-causing variant in three different genes (FKBP10, P3H1, and WNT1) was identified. Two (33.3 %) detected variants in all genes have not been previously reported in the literature and were considered deleterious based on prediction tools. In 6 cases, no variants were detected in disease-causing genes. This study demonstrates rare OI types' clinical and molecular features; genetic etiology was determined in 6 (50 %) 12 patients with the WES analysis. In addition, two variants in OI genes have been identified, contributing to the literature.

Multiomic analysis in ovarian clear cell carcinoma to gain insight into biology and therapeutic opportunities.

e17540 Background: Ovarian clear cell carcinoma (OCCC) is a rare subtype with distinct histologic and molecular features. The prognosis of advanced/recurrent OCCC treated with standard of care surgery and platinum-based chemotherapy remains poor due to inherent chemoresistance. Therefore, there is an urgent need to identify novel biological insights and personalized treatment approaches to improve outcomes. Methods: Participants were referred to the SMMART Program at the OHSU Knight Cancer Institute. Archival or fresh tissue was obtained from metastatic sites and processed through a multiomics platform that included immunohistochemistry (Ki67, HER2, CD4, CD8, PD-L1), fluorescent in-situ hybridization, targeted next-generation sequencing panel (225 genes), whole transcriptomic sequencing, chromosomal microarray, and multiplexed analysis of 67 key cancer proteins and phosphoproteins on the Nanostring GeoMx Digital Spatial Profiler. Results: 5 participants were enrolled with an average age of 62 years (range 52-72). Therapeutically relevant findings included detectable HER2 expression by IHC (2+, N=2; 3+, N=2), HER2 amplification by FISH (N=2), and high HER2 and phospho-HER2 protein expression compared to a breast cancer cohort (phospho-HER2 supports active HER2 signaling in the tumor cells). Additional findings include TMB <10 muts/Mb (N=5), microsatellite stable (N=5), Ki67 40-60% (N=4), mild to moderate CD4 and/or CD8 infiltration (N=4), PD-L1 >1% (N=3), low homologous recombination deficiency score (N=4), chromosomal losses including ATM, SMAD4, RB1, and TP53, and single nucleotide variations in ARID1A (N=4). Two participants with HER2 expression were treated with off-label trastuzumab deruxtecan for 6 or 7 cycles yielding significant clinical benefit. Participant A (HER2 = 3+, FISH negative) had an exceptional response while Participant B (HER2 = 2+, FISH amplified) had stable disease. The toxicity profile was consistent with literature. Conclusions: Our OCCC cohort revealed elevated HER2 expression with or without genomic amplification in 4/5 cases. Two participants experienced clinical benefit when treated with trastuzumab deruxtecan, supporting further exploration of HER2 antibody drug conjugates in this aggressive disease. More study is needed to determine the frequency of HER2 overexpression and amplification in OCCC, the importance of HER2 on OCCC pathophysiology, and the therapeutic benefit of targeting HER2 in this population.

Comparative analysis of actionable gene reporting in targeted panels versus comprehensive NGS testing for solid tumor samples.

11185 Background: The landscape of actionable genes significantly influences clinical decisions in cancer diagnosis, prognosis, and treatment planning. In a clinical context, the selection of NGS panels hinges on striking a balance among informative data, insurance coverage, and the preferences of patients and healthcare providers. Molecular pathology reference laboratories commonly employ large-scale next-generation sequencing (NGS) testing, curating smaller, disease-specific targeted panels. This study investigates the prevalence of unreported actionable genes in real-world clinical samples subjected to disease-specific panels. Methods: We analyzed SNV/InDel DNA variants in 795 clinical solid tumor samples using the Illumina TSO 500 platform for comprehensive NGS panel testing of 517 genes. Comprehensive NGS panel-tested samples were intersected with disease-specific targeted NGS panels for breast (54 genes), brain (62 genes), colorectal (36 genes), and lung (44 genes). An artificial filter was applied to align targeted panels with patient disease. Variants were classified based on pathogenicity, and benign variants were filtered out. Detected variants were assessed for actionable mutations as defined by the FDA recognized OncoKB actionable gene database criteria. Additionally, 1484 lung patient samples tested with a lung-specific targeted panel were de-identified and unmasked to the comprehensive NGS tumor profiling to investigate the prevalence of actionable alteration beyond those covered by the disease-specific panel. Results: The study revealed that 3.2% of breast, 0% of brain, 66.2% of colorectal, and 2.7% of lung samples had additional actionable mutations undisclosed by the targeted panels. The number of actionable genes beyond those included in the targeted panels were 14 for breast, 2 for brain, 25 for lung, and 41 for colorectal cancer. This underscores the importance of comprehensive testing to fully capture each patient tumor’s actionable mutation profile. Conclusions: Our findings detect masked actionable mutations in down-sampled targeted panels and highlight the need for comprehensive testing to accurately identify actionable mutations in various cancer types. Reporting actionable mutations can significantly influence clinical decision-making, emphasizing the importance of NGS test selection in routine molecular diagnostics as well as the impact of integrating comprehensive NGS testing.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2—8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Clinical actionability of BRCA2 alterations in uterine leiomyosarcoma: a molecular tumor board case report and a cBioPortal comprehensive analysis.

Uterine leiomyosarcoma (uLMS) represents one of the most common sarcoma histotypes, demonstrating an overall dismal prognosis. Previous studies reported uLMS to carry recurrent somatic BRCA2 homozygous deletions, related to significant clinical benefits from the use of PARP inhibitors. To investigate the prevalence in uLMS of genomic alterations (alt) in BRCA2 and other homologous recombination (HR) and DNA damage response (DDR) genes, cBioPortal was accessed and data were retrieved from studies including pan-sarcoma histologies. HR-/DDR-genes included BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, NBN, PALB2, RAD51C, RAD51D, RAD50, and ATR. Only oncogenic/likely oncogenic alterations were included according to OncoKB. We reported a clinical case of a patient affected by a highly pretreated uLMS discussed at the European Institute of Oncology Molecular Tumor Board. A targeted next-generation sequencing panel demonstrated a somatic BRCA2 homozygous deletion (homDel). Upon access to Niraparib, a remarkable response of 15 months was observed before experiencing disease progression. In the genomic query, among 2393 cases, uLMS (n = 193) displayed 9 of all 31 BRCA2alt observed, representing the only sarcoma histotype showing an enrichment in BRCA2alt (4.66%; q < 0.001). All of 9 BRCA2alt were represented by homDel, which related to a high fraction of genome altered. uLMS displays a significant frequency of somatic BRCA2alt homDel. Considering their dismal prognosis, further investigation is warranted to test the use of PARPi in uLMS, and particularly in the setting of BRCA1/2 alterations.

Increased prevalence of germline pathogenic CHEK2 variants in individuals with pituitary adenomas.

CHEK2 is a cell cycle checkpoint regulator gene with a long-established role as a clinically relevant, moderate risk breast cancer predisposition gene, with greater risk ascribed to truncating variants than missense variants. We assessed 165 individuals with pituitary adenomas for CHEK2 variants. The study consisted of a primary cohort of 29 individuals who underwent germline and tumour whole exome sequencing, and a second, independent cohort of 136 individuals who had a targeted next-generation sequencing panel performed on both germline and tumour DNA (n=52) or germline DNA alone (n=84). We identified rare, coding, non-synonymous germline CHEK2 variants amongst 3/29 (10.3%) patients in our primary cohort and 5/165 (3.0%) patients overall, with affected patients having a range of hormone secretion types (prolactinoma, thyrotrophinoma, somatotrophinoma and non-functioning pituitary adenoma). No somatic variants were identified. Two variants were definitive null variants (c.1100delC, c.444+1G>A), classified as pathogenic. Two variants were missense variants (p.Asn186His, p.Thr476Met), classified as likely pathogenic. Even when considering the null variants only, the rate of CHEK2 variants was higher in our cohort compared to national control data (1.8% vs. 0.5%, P=0.049). This is the first study to suggest a role for the breast cancer predisposition gene, CHEK2, in pituitary tumorigenesis, with pathogenic/likely pathogenic variants found in 3% of patients with pituitary adenomas. As pituitary adenomas are relatively common and typically lack classical autosomal dominant family histories, risk alleles - such as these variants found in CHEK2 - might be a significant contributor to pituitary adenoma risk in the general population.

Beyond the phenotype: Exploring inherited retinal diseases with targeted next-generation sequencing in a Turkish cohort.

This research aims to compile recent clinical and genetic data from Turkish patients with inherited retinal disorders and evaluate the effectiveness of targeted Next-generation sequencing panels. The study included Turkish individuals with hereditary retinal diseases who visited the Medical Genetic Department of Erciyes University between 2019 and 2022. One proband per family was selected based on eligibility. We used Hereditary Disorder Solution (HDS) by Sophia Genetics and performed next-generation sequencing (NGS) with Illumina NextSeq-500. Bioinformatics analysis using Sophia DDM® SaaS algorithms and ACMG guidelines classified genomic changes. The study involved 354 probands. Disease-causing variants were found in 58.1% of patients, with ABCA4, USH2A, RDH12, and EYS being the most frequently implicated genes. Forty-eight novel variants were detected. This study enhances the knowledge of clinical diagnoses, symptom onset, inheritance patterns, and genetic details for Turkish individuals with hereditary retinal disease. It contributes to broader health strategies by enabling comparisons with other studies.

Clinical Validation of a Targeted Next-Generation Sequencing Panel for Lymphoid Malignancies

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin.

Novel compound heterozygous variants in LTBP2 associated with relative anterior microphthalmos.

Relative anterior microphthalmos (RAM) is a rare congenital defect associated with severe vision impairment that is primarily caused by genetic alterations. The purpose of this study was to identify the causative genetic variants in two Chinese families with RAM with an autosomal recessive inheritance pattern. DNA samples were obtained from two probands and their family members. Targeted next-generation sequencing (NGS) was used to screen 425 genes associated with inherited eye diseases to identify possible disease-causing variants in the two patients. Sanger sequencing was subsequently used to validate the results in both families. The targeted NGS panel identified potentially causative novel variants of the latent transforming growth factor beta binding protein 2 (LTBP2) gene in the two RAM families: a missense variant (c.2771C > T; p.Ala924Val) and an intronic variant (c.4582 + 9A > G) in Family A and a different missense variant (c.5239C > A; p.Arg1747Ser) and a synonymous variant (c.951G > A; p.Pro317Pro) in Family B. These four novel variants all cosegregated with the disease phenotype. To our knowledge, this is the first study to report novel LTBP2 gene variants related to RAM. Considering the importance of LTBP2 in ocular development, we provide initial insights into the potential pathogenic mechanisms of LTBP2 in RAM.

Abstract 5058: Genomic alterations predictive of outcome in early staged cervical cancer: A translational investigation from the SENTICOL III trial

Abstract Background: The risk of relapse in patients with early-stage cervical cancer (ESCC) ranges from 4-14% (LACC study). Risk-factors for recurrence are mainly derived from histopathological factors. Senticol III is a large multicenter international randomized prospective study aims to validate sentinel lymph node biopsy in the surgical management of ESCC. Our objective is to characterize prognostic and actionable biomarkers in ESCC. Methods: FFPE-fixed tumoral slides of the first 150 randomized patients were analyzed based on SEDLIS criteria. We characterized TIL’s infiltration, PD-L1 expression and HPV status. Sequencing of tumor DNA was carried out using an in-house targeted next-generation sequencing panel (571 genes). Relapse-free survival (RFS) was defined as the time between randomization and relapse of any type. We performed Fisher’s exact test and Student’s t-test to compare distributions of categorical variables and Log Rank test. Results: The sequencing quality control criteria were met for 135 samples. PIK3CA was the most commonly altered gene (17%). Loss-of-function variants in KMT2-family genes (KTM2C 9%, KMT2D 6%, KMT2A 1%) were found in 16% of samples. The loss-of-function variant of ARID1A was mostly non-sense mutation (6%). Activations of ERBB2 (5%) were 3 focal amplifications and 4 hotspot missense mutations. Two samples were found to have microsatellite instability due to an alteration of MLH1. The Pik3/AKT/mTOR (26%) and the chromatin remodeling (23%) pathways were the most altered pathways. The median follow-up period was 20 months. There were 6 relapses, of which 80% were considered as low risk based on SEDLIS criteria. Recurrences were local and occurred 9-23 months following surgery. Multiparity was the only clinical feature associated with RFS (p &amp;lt; 0,001). Tumor size, histological subtypes, PD-L1 status, number of TIL’s, presence or absence of lymphovascular emboli and deep stromal invasion were not associated with RFS in our cohort.KMT2C (p=0,018) and ARID1A (p &amp;lt; 0,001) pathogen variants were significantly associated with RFS. The pathway chromatin remodeling (p=0,063) showed a trend toward association with RFS. We found 32% of genomic alterations actionable for a target therapy including mutations of PIK3CA, ERBB2, ARID1A, PTEN, BRCA2, KRAS, BRAF, CDK12, FGFR2, NF1, AKT1, BRCA1, TP53 and MSI-high. Conclusion: Our prospective, randomized and multicentric study suggests a genomic profile from a homogeneous cohort of 150 patients. We identified genes involved in chromatin remodeling (ARID1A and KMT2C) as prognostic biomarkers of clinical interest and suggesting the relevance of epidrugs in ESCC. Theranostic annotations of variants identify a matched to target therapy for 32% of alterations. Those targets represent multiple tracks for personalized therapy in ESCC. Citation Format: Maryame El Gani, Sabrina Ibadioune, Zakhia El Beaino, Abderraouf Hamza, Sophie Vacher, Emmanuelle Jeannot, Julien Masliah-Planchon, Anne Salomon, Alexandre Degnieau, Anne-Sophie Bats, Martin Koskas, Virginie Fourchotte, Estelle Wafo, Nicolas Bourdel, Raffaele Fauvet, Marie Plante, Patrice Mathevet, Maud Kamal, Fabrice Lecuru, Ivan Bièche. Genomic alterations predictive of outcome in early staged cervical cancer: A translational investigation from the SENTICOL III 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 5058.

Comprehensive genomic and clinical analyses identify APOBEC mutational signatures as a brain metastasis risk factor in lung adenocarcinoma patients

BackgroundLung adenocarcinoma is the most common source of brain metastasis (BM), resulting in significant morbidity and mortality. We aimed to identify patients with high BM risk who possibly benefit from brain-penetrant drugs, prophylactic cranial irradiation, or close brain magnetic resonance imaging surveillance. MethodsMetastatic lung adenocarcinoma patients with extracranial tumor samples profiled by a next-generation sequencing panel targeting 425 tumor-related genes were retrospectively enrolled between February 2008 and July 2021. We compared BM and non-BM patients’ genomic and clinical features and studied their associations with BM risk. Two external cohorts were used for result validation and molecular mechanisms investigation, respectively. ResultsWe included 174 eligible patients, including 90 having developed BM by the end of follow-up. Age≤60, EGFR activating mutations, and high-level apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) mutational signatures were associated with elevated BM risk. Similar findings in BM-free survival were obtained by fitting Fine-Gray subdistribution hazard models addressing competing risks. Increased BM risk related to APOBEC mutational signatures was validated in an external cohort (N = 440). RNA sequencing data analyses performed in another external cohort (N = 230) revealed that expressions of metastasis-related pathways such as transforming growth factor (TGF)β and epithelial-mesenchymal transition (EMT) were upregulated in the patients with high-level APOBEC mutational signatures. ConclusionAPOBEC mutational signatures related to upregulated TGFβ and EMT, could serve as an independent risk factor for BM and BM-free survival in metastatic lung adenocarcinoma patients. Further investigations are warranted to tailor personalized treatments to improve the susceptible patient's outcomes.

The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n=147).

Genetic profiling of rat gliomas and cardiac schwannomas from life-time radiofrequency radiation exposure study using a targeted next-generation sequencing gene panel.

The cancer hazard associated with lifetime exposure to radiofrequency radiation (RFR) was examined in Sprague Dawley (SD) rats at the Ramazzini Institute (RI), Italy. There were increased incidences of gliomas and cardiac schwannomas. The translational relevance of these rare rat tumors for human disease is poorly understood. We examined the genetic alterations in RFR-derived rat tumors through molecular characterization of important cancer genes relevant for human gliomagenesis. A targeted next-generation sequencing (NGS) panel was designed for rats based on the top 23 orthologous human glioma-related genes. Single-nucleotide variants (SNVs) and small insertion and deletions (indels) were characterized in the rat gliomas and cardiac schwannomas. Translational relevance of these genetic alterations in rat tumors to human disease was determined through comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) database. These data suggest that rat gliomas resulting from life-time exposure to RFR histologically resemble low grade human gliomas but surprisingly no mutations were detected in rat gliomas that had homology to the human IDH1 p.R132 or IDH2 p.R172 suggesting that rat gliomas are primarily wild-type for IDH hotspot mutations implicated in human gliomas. The rat gliomas appear to share some genetic alterations with IDH1 wildtype human gliomas and rat cardiac schwannomas also harbor mutations in some of the queried cancer genes. These data demonstrate that targeted NGS panels based on tumor specific orthologous human cancer driver genes are an important tool to examine the translational relevance of rodent tumors resulting from chronic/life-time rodent bioassays.

Validation of targeted next-generation sequencing panels in a cohort of Polish patients with epilepsy: assessing variable performance across clinical endophenotypes and uncovering novel genetic variants.

Targeted Next-Generation Sequencing Panels (TNGSP) have become a standard in global clinical practice. Instead of questioning the necessity of next-generation sequencing in epilepsy patients, contemporary large-scale research focuses on factors such as the size of TNGSP, the comparative advantages of exome or genome-wide sequencing over TNGSP, and the impact of clinical, electrophysiological, and demographic variables on genetic test performance. This study aims to elucidate the demographic and clinical factors influencing the performance of TNGSP in 138 Polish patients with epilepsy, recognizing the pivotal role of genetic testing in guiding patient management and therapy. A retrospective analysis was conducted on patients from a genetic clinic in Poznań, Poland, who underwent commercial gene panel studies at Invitae Corporation (USA) between 2020 and 2022. Patient groups were defined based on the age of onset of the first epileptic seizures, seizure type, gender, fever dependence of seizures, presence of intellectual disability or developmental delay, abnormalities in MRI, and the presence of dysmorphic features or congenital malformations. Seizure classification followed the 2017 ILAE criteria. Among the 138 patients, 30 (21.7%) exhibited a pathogenic or likely pathogenic variant, with a distribution of 20.7% in males and 22.5% in females. Diagnostic performance correlated with the patient's age at the onset of the first seizure and the type of seizure. Predominant variants were identified in the SCN1A, PRRT2, CDKL5, DEPDC5, TSC2, and SLC2A1 genes. Additionally, 12 genes (CACNA1A, SCN2A, GRIN2A, KCNQ2, CHD2, DYNC1H1, NEXMIF, SCN1B, DDX3X, EEF1A2, NPRL3, UBE3A) exhibited single instances of damage. Notably, novel variants were discovered in DEPDC5, SCN1A, TSC2, CDKL5, NPRL3, DYNC1H1, CHD2, and DDX3X. Identified variants were present in genes previously recognized in both European and non-European populations. A thorough examination of Variants of Uncertain Significance (VUSs), specifically focusing on gene copy number changes, may unveil more extensive chromosomal aberrations. The relatively frequent occurrence of pathological variants in X chromosome-linked genes in girls warrants further investigation, challenging the prevailing notion of male predominance in X-linked epilepsy.

Abstract A010: Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors

Abstract Title: Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors Background: Fanconi anemia repair (FAR) genes have crucial involvement in determining immunogenicity of solid tumors due to their substantial involvement in DNA damage repair (DDR). Previous work has alluded to the significance of DDR pathways such as mismatch repair and homologous recombination genes in treatment guidance. Herein, we study the potential impact of FAR genes mutations on immunotherapy parameters in solid tumors. Methods: We used the MSK immunotherapy and Tumor Mutational Burden (TMB) dataset was used in our analysis, which includes mutational and clinical data for 1661 patients on immune checkpoint inhibitors (anti-PD-1/L1, anti-CTLA-4, or combinational therapy) from ten cancer types (bladder, breast, colorectal, esophagogastric, glioma, head and neck, melanoma, non- small cell lung, renal cell, and unknown primary cancer). All clinical ang genomic data was retrieved through the cBioPortal database. Patients were categorized into the FAR mutated group if they harbored a mutation in at least one of the FAR genes (FANCA, FANCC, BRCA2, BRIP1, PALB2, RAD51C, SLX4) included in the MSK-IMPACT targeted next-generation sequencing panel. The log rank test and the Wilcoxon test, Chi-squared test were used to compare overall survival (OS), continuous and categorical data between FAR mutated and wildtype groups. Results: The MSK TMB cohort included 220 (13%) patients with FAR mutations, of which melanoma comprised the highest percentage (26.4%) followed by non-small cell lung cancer (NSCLC; 19.6%). The most common altered FAR gene was BRCA2 (6%), followed by SLX4 (5%), while the least altered gene was RAD51C (0.7%). FAR mutated patients had significantly higher OS compared to the wildtype group (median OS (mOS): 29 vs 17 months, log rank p &amp;lt; 0.001). Among melanoma patients (n = 320), there was no significant difference in OS between the mutated and FAR groups, whereas the NSCLC (n = 350) and colorectal cancer (CRC, n = 110) subgroups had significantly higher OS in the FAR mutated groups (NSCLC - mOS: 19 vs 10 months, p = 0.0256, CRC – mOS: not reached vs 13 months, p &amp;l; 0.001). TMB was significantly higher in the FAR mutated group (median TMB: 22.3 vs 5.2, p &amp;lt; 0.001). Notably, the most frequent co-mutations in the FAR mutated group included CTLA4, STK19, E2F3, CD276, MYCL. Conclusion: Our analysis highlights the potential role of FAR genes mutations in predisposing solid tumor patients to ICI benefit, specifically in NSCLC and CRC patients. Insights into the potential interactions between FAR genes and other DDR pathways as well as their impact on tumor immune microenvironment parameters are needed in future research to understand the drivers of ICI benefit in this subset of patients. Citation Format: Ahmed M. Ashour, Amro M. Al-Omari, Mohammad S. Bani Amer, Zaid R. Kamal. Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A010.

Development of ion torrent-based targeted next-generation sequencing panel for identification of animal species in pet foods

Manufacturers may intentionally or unintentionally incorporate ingredients not specified on the label of canned pet foods. Including any unacknowledged ingredients in a food product is considered food fraud or misbranding. Contamination of pet foods may occur in the processing of the foods, including potential cross-contamination in packaging facilities.Of the methods available to identify meat species in food products, Sanger sequencing and several next-generation sequencing methods are available, but there are limitations including the number of targets analyzed at a time and the method specificity. In this study, we developed a targeted next-generation sequencing panel to detect meat species in canned pet foods using Ion Torrent technology.The panel contains multiple primers targeting mitochondrial genes from as many as 27 animal species, of which 7 major animal species were validated. The meat species targets could be identified from samples spiked with as low as 0.01% w/w of the contaminating meat species in a vegetarian food matrix material. Targeted NGS in the current study enriches species-specific multiple target areas in the mitochondrial genome of the target material, which gives high accuracy in the sequencing results.

The landscape of actionable genomic alterations in lung adenocarcinomas in India.

Lung adenocarcinoma (LUAD), the most prevalent form of non-small cell lung cancer (NSCLC), remains a leading cause of cancer-related death globally, including in India, with a 5-year survival rate below 10%. Despite these grim statistics, recent advances in the use of next-generation sequencing (NGS) for identifying genetic alterations and the emergence of targeted therapies have opened new possibilities for personalized treatment based on distinct molecular signatures. To understand the molecular pattern of NSCLC, a retrospective study was conducted with 53 Indian LUAD patient samples, using a targeted NGS panel of 46 cancer-relevant oncogenes to identify clinically relevant variants. Pathogenic or likely pathogenic variants were detected in 94% of the 53 cases. Non-synonymous mutations, rearrangements, copy number alterations, insertions, and deletions of functional relevance were observed in 31 out of 46 genes. The most frequently mutated genes included TP53 (52.8%) and EGFR (50.9%), followed by RET, PIK3CA and ERBB2; some patients had multiple alterations in the same gene. Gender-based enrichment analysis indicated that ALK and IDH2 alterations were more prevalent in females, while TP53 and PTEN were more common in males. No significant correlation was found between mutations and other clinicopathological attributes, such as age, stage, and subtype. A higher prevalence of EGFR, RET, PIK3CA, ERBB2 and ALK mutations were observed compared to previous LUAD genetic studies coupled with a lower frequency of KRAS mutations. Clinically actionable variants were annotated using OncoKB and categorized into the four therapeutic levels based on their clinical evidence. Seventy-nine percent of cases had at least one clinically actionable alteration. Most patients (39.6%) had the highest level of actionability (Level 1) wherein an FDA-approved drug is available specifically for the observed mutation in lung cancer patients. EGFR Exon19 in-frame deletions and EGFR L858R were the most frequent among targetable variants (20.7%). These findings emphasize the importance of a selective NGS panel in enabling personalized medicine approaches by identifying actionable molecular alterations and informing the choice of targeted therapy for more effective treatment options in Indian NSCLC patients.

The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

Right ventricle hypertrophy in fetuses diagnosed with Noonan syndrome: Can ductus venosus agenesis represent a contributing cause?

Noonan syndrome (NS) is the most common genetic syndrome in fetuses with increased nuchal translucency (NT) and normal karyotype. NS patients present typical patterns of cardiac defects, such as pulmonary stenosis, polyvalvular dysplasia and hypertrophic cardiomyopathy. This study aims to evaluate onset and trend of cardiac disease in fetuses with molecular diagnosis of NS and to search for other cardiovascular sonographic markers of NS. We performed a fetal echocardiographic follow-up of pregnancies with increased NT in the first trimester. All the fetuses with normal Karyotype and Comparative Genomic Hybridizationarray underwent a targeted next-generation sequencing panel for RASopathies. Increased NT was the only finding up to the second trimester in half of the fetuses, while in the others ductus venosus agenesis was also observed. In all of them, during the late second trimester, a severe cardiopathy arouse.