Next-generation Sequencing Gene Panel Research Articles

Discovery and Validation of Survival-Specific Genes in Papillary Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

Papillary renal cell carcinoma (PRCC), the second most common kidney cancer, is morphologically, genetically, and molecularly heterogeneous with diverse clinical manifestations. Genetic variations of PRCC and their association with survival are not yet well-understood. This study aimed to identify and validate survival-specific genes in PRCC and explore their clinical utility. Using machine learning, 293 patients from the Cancer Genome Atlas-Kidney Renal Papillary Cell Carcinoma (TCGA-KIRP) database were analyzed to derive genes associated with survival. To validate these genes, DNAs were extracted from the tissues of 60 Korean PRCC patients. Next generation sequencing was conducted using a customized PRCC gene panel of 202 genes, including 171 survival-specific genes. Kaplan-Meier and Log-rank tests were used for survival analysis. Fisher's exact test was performed to assess the clinical utility of variant genes. A total of 40 survival-specific genes were identified in the TCGA-KIRP database through machine learning and statistical analysis. Of them, 10 (BAP1, BRAF, CFDP1, EGFR, ITM2B, JAK1, NODAL, PCSK2, SPATA13, and SYT5) were validated in the Korean-KIRP database. Among these survival gene signatures, three genes (BAP1, PCSK2, and SPATA13) showed survival specificity in both overall survival (OS) (p = 0.00004, p = 1.38 × 10-7, and p = 0.026, respectively) and disease-free survival (DFS) (p = 0.00002, p = 1.21 × 10-7, and p = 0.036, respectively). Notably, the PCSK2 mutation demonstrated survival specificity uniquely in both the TCGA-KIRP (OS: p = 0.010 and DFS: p = 0.301) and Korean-KIRP (OS: p = 1.38 × 10-7 and DFS: p = 1.21 × 10-7) databases. We discovered and verified genes specific for the survival of PRCC patients in the TCGA-KIRP and Korean-KIRP databases. The survival gene signature, including PCSK2 commonly obtained from the 40 gene signature of TCGA and the 10 gene signature of the Korean database, is expected to provide insight into predicting the survival of PRCC patients and developing new treatment.

Pediatric neuropathology practice in a low- and middle-income country: capacity building through institutional twinning.

Accurate and precise diagnosis is central to treating central nervous system (CNS) tumors, yet tissue diagnosis is often a neglected focus in low- and middle-income countries (LMICs). Since 2016, the WHO classification of CNS tumors has increasingly incorporated molecular biomarkers into the diagnosis of CNS tumors. While this shift to precision diagnostics promises a high degree of diagnostic accuracy and prognostic precision, it has also resulted in increasing divergence in diagnostic and management practices between LMICs and high-income countries (HICs). Pathologists and laboratory professionals in LMICs lack the proper training and tools to join the molecular diagnostic revolution. We describe the impact of a 7-year long twinning program between Canada and Pakistan on pathology services. During the study period, 141 challenging cases of pediatric CNS tumors initially diagnosed at Aga Khan University Hospital (AKUH), Karachi, were sent to the Hospital for Sick Children in Toronto, Canada (SickKids), for a second opinion. Each case received histologic review and often immunohistochemical staining and relevant molecular testing. A monthly multidisciplinary online tumor board (MDTB) was conducted to discuss the results with pathologists from both institutions in attendance. Diagnostic discordance was seen in 30 cases. Expert review provided subclassification for 53 cases most notably for diffuse gliomas and medulloblastoma. Poorly differentiated tumors benefited the most from second review, mainly because of the resolving power of specialized immunohistochemical stains, NanoString, and targeted gene panel next-generation sequencing. Collaboration with expert neuropathologists led to validation of over half a dozen immunostains at AKUH facilitating diagnosis of CNS tumors. LMIC-HIC Institutional twinning provides much-needed training and mentorship to pathologists and can help in infrastructure development by adopting and validating new immunohistochemical stains. Persistent unresolved cases indicate that molecular techniques are indispensable in for diagnosis in a minority of cases. The development of affordable alternative molecular techniques may help with these histologically unresolved cases.

Genetic determinants of global developmental delay and intellectual disability in Ukrainian children

BackgroundGlobal developmental delay or intellectual disability usually accompanies various genetic disorders as a part of the syndrome, which may include seizures, autism spectrum disorder and multiple congenital abnormalities. Next-generation sequencing (NGS) techniques have improved the identification of pathogenic variants and genes related to developmental delay. This study aimed to evaluate the yield of whole exome sequencing (WES) and neurodevelopmental disorder gene panel sequencing in a pediatric cohort from Ukraine. Additionally, the study computationally predicted the effect of variants of uncertain significance (VUS) based on recently published genetic data from the country’s healthy population.MethodsThe study retrospectively analyzed WES or gene panel sequencing findings of 417 children with global developmental delay, intellectual disability, and/or other symptoms. Variants of uncertain significance were annotated using CADD-Phred and SIFT prediction scores, and their frequency in the healthy population of Ukraine was estimated.ResultsA definitive molecular diagnosis was established in 66 (15.8%) of the individuals. WES diagnosed 22 out of 37 cases (59.4%), while the neurodevelopmental gene panel identified 44 definitive diagnoses among the 380 tested patients (12.1%). Non-diagnostic findings (VUS and carrier) were reported in 350 (83.2%) individuals. The most frequently diagnosed conditions were developmental and epileptic encephalopathies associated with severe epilepsy and GDD/ID (associated genes ARX, CDKL5, STXBP1, KCNQ2, SCN2A, KCNT1, KCNA2). Additionally, we annotated 221 VUS classified as potentially damaging, AD or X-linked, potentially increasing the diagnostic yield by 30%, but 18 of these variants were present in the healthy population of Ukraine.ConclusionsThis is the first comprehensive study on genetic causes of GDD/ID conducted in Ukraine. This study provides the first comprehensive investigation of the genetic causes of GDD/ID in Ukraine. It presents a substantial dataset of diagnosed genetic conditions associated with GDD/ID. The results support the utilization of NGS gene panels and WES as first-line diagnostic tools for GDD/ID cases, particularly in resource-limited settings. A comprehensive approach to resolving VUS, including computational effect prediction, population frequency analysis, and phenotype assessment, can aid in further reclassification of deleterious VUS and guide further testing in families.

Abstract 5038: Validation of a comprehensive next-generation sequencing liquid biopsy assay for clinical diagnostics and clinical trial applications

Abstract Liquid biopsy next generation sequencing (NGS) provides a powerful non-invasive approach to detect solid tumor derived somatic variants in a massively parallel manner. Liquid biopsy NGS has rapidly become an invaluable choice of testing technologies for clinical utility where invasive tissue biopsy is inaccessible and/or inadequate for molecular characterization. We present here a targeted NGS gene panel assay designed and validated specifically for liquid biopsy characterization. The assay was designed to test cell free DNA and cell free RNA from blood plasma for detection of single nucleotide variants (SNV), insertion/deletion (Indel), copy number variants (CNV), and gene fusions present in 52 most commonly mutated genes in cancers. The validation was performed to assess assay accuracy, specificity, sensitivity, repeatability, and reproducibility, with including pre-characterized reference controls, retrospective plasma samples from cancer patients, and prospectively-collected clinic blood specimen. Triplicates were included and testing was performed at different times and by different operators to assess the assay precision. Over 100 clinic blood specimens were collected in Streck cfDNA tubes from multiple trial centers, including treatment naïve phase III-IV patients with solid tumor (lung cancer, breast cancer, brain cancer, prostate cancer, kidney cancer, melanoma, etc) as well as heathy donors, processed within 7 days for plasma separation and total nucleotide acid (TNA) extraction, and assessed by the NGS assay for mutation detection. The identified mutations and wild-types were verified with orthogonal ddPCR. Assay sensitivity as low as 0.1% for SNV and Indel, 1.3 fold change for CNV, and 10 copies for gene fusion were observed, with near-perfect assay specificity (>99.99%) at these sensitivity levels. In conclusion, a targeted NGS liquid biopsy assay was analytically validated under medical oversight, with a demonstrated rigor of the test by its high sensitivity/specificity and robust reproducibility. The validated assay has been utilized for clinical diagnostics and to support research and development efforts in clinical trials. Citation Format: Xin-Xing Tan, Tricia Peters, Meredith Berry, Kimberly Barber, Kristyn Jeter, Brigitte Lovell, Paula Elizondo, Yanglong Mou, Brad Thomas, Heng Xie, Jiannan Guo. Validation of a comprehensive next-generation sequencing liquid biopsy assay for clinical diagnostics and clinical trial applications [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 5038.

Novel MIP gene mutation causes autosomal-dominant congenital cataract.

To identify disease-causative mutations in families with congenital cataract. Two Chinese families with autosomal-dominant congenital cataract (ADCC) were recruited and underwent comprehensive eye examinations. Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family. Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis. The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis. Major intrinsic protein (MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos. Zebrafish embryonic lens phenotypes were screened using confocal microscopy. A novel heterozygous mutation (c.85G>A; p.G29R) in the MIP gene was identified in the proband of one family. A known heterozygous mutation (c.97C>T; p.R33C; rs864309693) in MIP was found in the proband of another family. In-silico prediction indicated that the novel mutation might affect the MIP protein function. Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP. Two missense mutations in the MIP gene in Chinese cataract families are identified, and one of which is novel. These findings expand the genetic spectrum of MIP mutations associated with cataracts. The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

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.

Large-Scale Screening: Phenotypic and Mutational Spectrum in Isolated and Combined Dystonia Genes.

Pathogenic variants in several genes have been linked to genetic forms of isolated or combined dystonia. The phenotypic and genetic spectrum and the frequency of pathogenic variants in these genes have not yet been fully elucidated, neither in patients with dystonia nor with other, sometimes co-occurring movement disorders such as Parkinson's disease (PD). To screen >2000 patients with dystonia or PD for rare variants in known dystonia-causing genes. We screened 1207 dystonia patients from Germany (DysTract consortium), Spain, and South Korea, and 1036 PD patients from Germany for pathogenic variants using a next-generation sequencing gene panel. The impact on DNA methylation of KMT2B variants was evaluated by analyzing the gene's characteristic episignature. We identified 171 carriers (109 with dystonia [9.0%]; 62 with PD [6.0%]) of 131 rare variants (minor allele frequency <0.005). A total of 52 patients (48 dystonia [4.0%]; four PD [0.4%, all with GCH1 variants]) carried 33 different (likely) pathogenic variants, of which 17 were not previously reported. Pathogenic biallelic variants in PRKRA were not found. Episignature analysis of 48 KMT2B variants revealed that only two of these should be considered (likely) pathogenic. This study confirms pathogenic variants in GCH1, GNAL, KMT2B, SGCE, THAP1, and TOR1A as relevant causes in dystonia and expands the mutational spectrum. Of note, likely pathogenic variants only in GCH1 were also found among PD patients. For DYT-KMT2B, the recently described episignature served as a reliable readout to determine the functional effect of newly identified variants. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Evaluation of T-cell clonality by anti-TRBC1 antibody-based flow cytometry and correlation with T-cell receptor sequencing.

Flow cytometry (FC) incorporating the T-cell receptor β constant chain-1 (TRBC1) has been recently proposed as a new standard in T-cell clonality assessment. While early studies demonstrated high sensitivity in samples with conspicuous tumour burden, performance in real-world samples, including those with low tumour burden and correlation with molecular methods has been limited. We evaluated TRBC1-FC performance and correlated the results with high-throughput TRB sequencing and a targeted next-generation sequencing gene panel. Our cohort consisted of 90 evaluable samples from 57 patients. TRBC1-FC confirmed T-cell clonality in 37 out of 38 samples (97%) that were involved in a mature T-cell neoplasm (MTCN). T-cell clonality was also identified in nine samples from patients lacking a current or prior diagnosis of MTCN, consistent with the emerging entity T-cell clonality of uncertain significance. TRBC-FC was polyclonal in all samples and negative for disease involvement by standard pathology assessment. However, correlation with TRB sequencing in 17 of these samples identified two cases that harboured the known clonal sequence from index testing, indicating the presence of measurable residual disease not otherwise detected. Our study provides real-world correlative validation of TRBC1-FC, highlighting the strengths and limitations pertinent to its increasing implementation by general diagnostic laboratories.

Genomic analysis across 53 canine cancer types reveals novel mutations and high clinical actionability potential.

A genomic understanding of the oncogenic processes and individual variability of human cancer has steadily fueled improvement in patient outcomes over the past 20 years. Mutations within tumour tissues are routinely assessed through clinical genomic diagnostic assays by academic and commercial laboratories to facilitate diagnosis, prognosis and effective treatment stratification. The application of genomics has unveiled a wealth of mutation-based biomarkers in canine cancers, suggesting that the transformative principles that have revolutionized human cancer medicine can be brought to bear in veterinary oncology. To advance clinical genomics and genomics-guided medicine in canine oncology, we have developed and validated a canine cancer next-generation sequencing gene panel for the identification of multiple mutation types in clinical specimens. With this panel, we examined the genomic landscapes of 828 tumours from 813 dogs, spanning 53 cancer types. We identified 7856 alterations, encompassing copy number variants, single nucleotide variants, indels and internal tandem duplications. Additionally, we evaluated the clinical utility of these alterations by incorporating a biomarker framework from comprehensive curation of primary canine literature and inferences from human cancer genomic biomarker literature and clinical diagnostics. Remarkably, nearly 90% of the cases exhibited mutations with diagnostic, prognostic or therapeutic implications. Our work represents a thorough assessment of genomic landscapes in a large cohort of canine cancers, the first of its kind for its comprehensive inclusion of multiple mutation types and structured annotation of biomarkers, demonstrating the clinical potential of leveraging mutation-based biomarkers in veterinary oncology.

Feasibility and Relevance of Incorporating Genetic Testing By Next-Generation Sequencing for Acute Myeloid Leukemia Patients Treated in Low- and Middle-Income Countries

Background: The outcome of the treatment for acute myeloid leukemia (AML) in low- and middle-income countries (LMIC) is significantly worse than in high-income ones due to several factors, including higher non-relapse mortality from infections and limited access to hematopoietic stem-cell transplantation (HSCT). In this resource-constrained setting, AML-risk stratification is based exclusively on cytogenetics, which has longer turn-around time and higher failure rates compared to next-generation sequencing (NGS). Here, we report the benefit of adding a simplified NGS gene panel to improve risk stratification for 279 adult Brazilian patients with AML (male=146, female=136; mean age=50 years; range 18-65) included in International Consortium of Acute Leukemia (ICAL) 2015 Study. Material &amp; Methods: Samples from 10 ICAL centers were shipped to the National Central Lab at the University of São Paulo at Ribeirão Preto Medical School, Brazil (located zero to 1420 kilometers from centers). All patient samples were assessed by conventional cytogenetics and molecular tests by RT-PCR for RUNX1::RUNX1T1 and CBFB::MYH11 , PCR and fragment analysis for NPM1 and FLT3-ITD allelic ratio, and Skyline AML profiler assay for CEBPA double mutant. We retrospectively searched for mutations in the ASXL1, CEBPA, FLT3, IDH1, IDH2, NPM1, RUNX1, TP53 genes using NGS in 53 out of the 279 samples. The tests were performed in the Illumina iSeq100 Sequencing System using reagents and methodology developed under the supervision of the Erasmus MC Cancer Institute (Valk Laboratory) as part of the activities of the ICAL Laboratory Subcommittee. Training, including bioinformatics analysis, was provided virtually and through a short-term visiting program (ASH Visitor Training Program) to Erasmus MC. Datasets with genetic testing results obtained locally were sent and reanalyzed at Erasmus MC and discussed in bi-monthly meetings. Risk stratification was determined either by cytogenetics only or by cytogenetic and molecular analysis according to the ELN 2017 proposal. Overall survival (OS) was defined from date of diagnosis to last follow up or death and compared by Kaplan-Meier and Log-Rank test followed by Benjamini-Hochberg p-value adjustment method, using R 4.3.1. Results: conventional cytogenetics was informative in 68% (190/279) of the cases, and based on those results 32 (16.8%), 116 (61%) and 42 (22.1%) patients were stratified into favorable, intermediate and adverse categories, respectively. However, no significant difference in the OS was detected according to cytogenetic risk. When genetic testing by NGS was added, 54 (27.3%), 98 (49.5%) and 46 (23.2%) patients were assigned into favorable, intermediate and adverse categories, respectively. Twenty-six (48%) patients had risk re-classified as a consequence of the NGS results. Moreover, two out of four patients whose risk stratification could not be assigned due to insufficient metaphasis were classified based on NGS results. Of note, the median OS of the favorable group was significantly longer (not reached) than that of intermediate and of the adverse groups (6 months), ( p&amp;lt;0.0001), but no difference was detected between the latter two. We also assessed whether genetic testing by NGS would change the risk category of AML patients previously classified based on conventional cytogenetics in association with the evaluation of gene rearrangements, NPM1, FLT3 and CEBPA mutational analysis by methods other than NGS. NGS results changed the category of 3 out of 54 patients. Our results indicate the feasibility of developing a simplified platform of genetic testing for AML based on NGS in LMIC, which was able to improve the identification of favorable-risk patients and may help to better allocate resources.

Familial Alzheimer’s disease associated with heterozygous NPC1 mutation

IntroductionNPC1 mutations are responsible for Niemann-Pick disease type C (NPC), a rare autosomal recessive neurodegenerative disease. Patients harbouring heterozygous NPC1 mutations may rarely show parkinsonism or dementia. Here, we describe...

Recombinant Human Erythropoietin Combined with All-Trans Retinoic Acid and Testosterone Undecanoate in the Treatment of Anemia in Patients with Lower-Risk Myelodysplastic Syndromes: Updated Results of a Multicenter, Single-Arm, Prospective Trial

[Background] More than 90% of low-risk MDS face refractory anemia and blood transfusion dependence for a long time. At present, recombinant human erythropoietin (EPO) is the main treatment, but the effective rate of single agent is 30%-40%, which seriously affects the quality of life of patients. Previous studies had shown that testosterone undecanoate could promote erythroid hematopoiesis and all-trans retinoic acid (ATRA) had the function of promoting blood cell differentiation in vitro. We previously reported that EPO combined with ATRA and testosterone undecanoate in the treatment of patients with low-risk MDS and demonstrated promising efficacy and safety in a prospective trial (Chen et al, ASH 2022). Here we present an updated analysis of efficacy and safety results in the ongoing study. [Methods] In this multicenter, single-arm trial, patients with newly diagnosed lower-risk MDS based on the 2016 WHO classification and the Revised International Prognostic Scoring System (IPSS-R) from 3 different tertiary medical centers in China were included. Patients were received EPO (10000IU/day) plus ATRA (25mg/m 2/day) and testosterone undecanoate (80mg twice daily) for 3 months. The primary endpoint was overall hemoglobin response rate, defined as an increase in hemoglobin levels of ≥ 1.5g/dL from baseline in 2 or more consecutive assessments (2 weeks apart) and no red blood cell transfusions during this period. Response was assessed after completion of 3months. The data cut-off date was July 25, 2023. [Results] As of July 2023, a total of 43 patients were enrolled, with a median age of 65 years, 24 males and 19 females, with median hemoglobin of 63 g/L, EPO concentration &amp;gt; 500 IU/L in 18 patients, and the overall hemoglobin response rate of 65.1% (28/43). The hemoglobin response rates were 61.1% (11/18) in patients with EPO concentrations greater than &amp;gt; 500 IU/L and 68.0% (17/25) in patients with EPO concentrations ≤ 500, which were not statistically different. Among them, 112 known or putative mutational gene targets in hematologic malignancy were examined for mutations in 32 patients using a custom targeted next-generation sequencing (NGS) gene panel. The most frequent mutations were SF3B1 (15/32, 46.8%) and ASXL1 (8/32, 25.0%). The hemoglobin response rate was 80.0% (12/15) in patients with SF3B1 mutation and 37.5% (3/8) in patients with ASXL1 mutation, respectively. Among 43 patients evaluable for safety analysis, the most common non-hematological adverse events were dry skin (17/43, 39.5%) and fatigue (12/43, 27.9%). No patients had disease progression during the treatment period. [Conclusion]: This study showed that EPO combined with tretinoin and testosterone undecanoate regimen can effectively improve the anemia of lower risk MDS in approximately 65.1% of patients and is not affected by EPO concentration. It has the better efficacy in patients with SF3B1 mutation, but has worse efficacy in patients with ASXL1 mutation. It is worthy of further randomized controlled studies to verify the efficacy.

Novel Biomarkers for Assessing Clinical Severity in Hereditary Spherocytosis - Application of Routine and Advanced Diagnostic Tests

Background: Hereditary spherocytosis (HS) is caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeletal and (trans)membrane proteins. This ultimately leads to instability of the RBC membrane and subsequent membrane loss, resulting in the transformation of the biconcave RBC into a poorly deformable spherocyte, which is susceptible to premature splenic clearance. The diagnosis of HS has substantially improved over the last years and new advanced techniques have been established, including osmotic gradient ektacytometry, eosin-5'-maleimide (EMA) binding test and next-generation sequencing (NGS). However, there is little knowledge on the applicability of these diagnostic outcome parameters as biomarkers for clinical severity in HS. Aim: To explore possible correlations between established and novel diagnostic outcome parameters and clinical severity in a well-defined cohort of HS patients. Methods: This monocenter retrospective cohort study involves patients diagnosed with HS upon referral to our department between 2012 and 2023. Clinical characteristics, routine and advanced diagnostic laboratory parameters, and genotype were evaluated. Clinical severity was classified as previously described, based on parameters such as hemoglobin, reticulocyte percentage and bilirubin 1. The following laboratory tests were included: routine hematological parameters (Cel-Dyn Sapphire or Alinity hq), EMA binding test, osmotic fragility test (OFT) and osmotic gradient ektacytometry (osmoscan, Lorrca MaxSis). The latter technique was also explored for other parameters than the commonly used EI max, O hyper and O min. NGS gene panel analysis was used for molecular diagnosis. Differences between groups were determined using either Chi-squared test, unpaired t-test or Mann-Whitney U test and correlations using point-biserial, Pearson's or Spearman's test via GraphPad Prism. Results: Eighty-two non-splenectomized patients were included. Thirty-five of these (42.7%) were classified as mild, and forty-seven as moderate-severe (57.3%)). Demographic characteristics nor genotype differed significantly, albeit SPTB variants occurred more frequently in the moderate-severe group (15 vs. 4 in the mild group). Expectedly, both reticulocyte count and EI max correlated with clinical severity. RBC distribution width (RDW) correlated moderately, suggesting greater RBC heterogeneity (r=0.606, p&amp;lt;0.0001) is associated with a more severe clinical phenotype. Similarly, EMA binding test results correlated with clinical severity (r=0.390, p&amp;lt;0.001), indicating more pronounced membrane loss in clinically more severe patients. We further evaluated three novel osmoscan parameters ( Figure 1): EI O290, the elongation index (EI) at physiologic isotonicity, which correlated with clinical severity (r=-0.500, p&amp;lt;0.0001), and also with EI max (r=0.856, p&amp;lt;0.0001). The O min-width and O max-width, defined as the difference in osmolality between the intersection points of y=EI min or EI max +/- 0.05 and the osmoscan curve. Thisassesses the effect of changes in osmolality on EI min or EI max, respectively. O min-width correlated with RDW (r-0.439, p&amp;lt;0.0001), OFT results (N=30, r=0.671, p&amp;lt;0.0001) and clinical severity (r=0.390, p&amp;lt;0.001). O max-width correlated with clinical severity (r=-0.375, p&amp;lt;0.001) and strongly with percentage of hyperchromic cells (N=76, r=-0.751, p&amp;lt;0.0001), yet weakly with RDW (-0.273, p&amp;lt;0.05). All significant correlations with clinical severity are displayed in Figure 2. Conclusion: In this study, a number of routine and advanceddiagnostic laboratory parameters were found to correlate with clinical severity in HS. O min-width and O max-width are novel osmoscan-derived biomarkers that correlated with RDW, hyperchromic cells (O max-width), OFT results (O min-width) and clinical severity. EI O290, another novel osmoscan-derived biomarker, also correlated with clinical severity, although not stronger than EI max and AUC. The biomarkers identified in this study, together with patient-reported outcome measures (PROMs), could be used to revise the previously established severity classification system and thereby aid in stratifying clinical severity and subsequent clinical decision-making.

Analytical Validation of an Automated Semiconductor-Based Next-Generation Sequencing Assay for Detection of DNA and RNA Alterations in Myeloid Neoplasms

Myeloid neoplasms are heterogeneous tumors derived from early hematopoietic progenitors. Most international guidelines, including the European LeukemiaNet 2022 update, recommend testing a comprehensive set of genes, most within a 3- to 5-day period for optimal treatment decisions. Next-generation sequencing gene panels are essential for identifying genetic alterations, risk stratification, and determining targeted therapies for myeloid malignancies. This study describes the analytical validation of the Oncomine Myeloid Assay GX v2 (Myeloid GX v2) in combination with the Ion Torrent Genexus System using commercial controls, 16 variant-negative samples, and 130 clinical samples of myeloid neoplasms. The Myeloid GX v2 panel detected single nucleotide variants (SNVs), insertions/deletions (indels) (allele frequency >5%), and gene fusions (minimum 11 fusion copies/μL) in synthetic controls with a sensitivity of 100%. Specificity for detection of SNVs, indels, or fusions in 16 variant-negative samples was 100%. Sensitivity for detection of SNVs, indels, and gene fusions in 130 clinical samples was 99%, 97%, and 100%, respectively. Overall precision was 100% for SNVs, 96% for indels, and 100% for fusions. The average turnaround time from nucleic acid extraction to results was 2 days. The Myeloid GX v2 panel is highly accurate and reproducible for the detection of SNVs, indels, and gene fusions in myeloid neoplasms. The ability to deliver clinically relevant results in a short time is key to providing personalized treatments.

Predictive Clinical and Biological Criteria for Gene Panel Positivity in Suspected Inherited Autoinflammatory Diseases: Insights from a Case-Control Study.

In order to assess the clinical and biological criteria that predict gene panel positivity in patients with a suspected inherited genetic autoinflammatory disease, we conducted a case-control study. These new selection criteria could replace the national multidisciplinary staff approval before performing genetic testing that has been required since 2019. The study involved 119 positive gene panels matched by panel sizes to 119 randomly selected negative gene panels. The patients were referred to our laboratory for genetic testing between June 2012, and March 2023. The clinical and biological criteria were extracted from a prospectively filled database. We focused our evaluation on accuracy and the positive predictive value. Neonatal symptom onset and deafness had the highest accuracies among all criteria associated with the positivity panel, with 92.9% (88.6; 96.0) and 92.6% (88.5; 95.6), respectively. However, it is important to note that the associated Positive Predictive Values (PPVs) cannot exceed 50%. Despite finding a statistical association between clinical and biological criteria and panel positivity, the predictive values of these criteria were not sufficient to recommend Next-Generation Sequencing (NGS) gene panel testing without the national multidisciplinary staff evaluation.

A feasible molecular diagnostic strategy for rare genetic disorders within resource-constrained environments

Timely and accurate diagnosis of rare genetic disorders is critical, as it enables improved patient management and prognosis. In a resource-constrained environment such as the South African State healthcare system, the challenge is to design appropriate and cost-effective assays that will enable accurate genetic diagnostic services in patients of African ancestry across a broad disease spectrum. Next-generation sequencing (NGS) has transformed testing approaches for many Mendelian disorders, but this technology is still relatively new in our setting and requires cost-effective ways to implement. As a proof of concept, we describe a feasible diagnostic strategy for genetic disorders frequently seen in our genetics clinics (RASopathies, Cornelia de Lange syndrome, Treacher Collins syndrome, and CHARGE syndrome). The custom-designed targeted NGS gene panel enabled concurrent variant screening for these disorders. Samples were batched during sequencing and analyzed selectively based on the clinical phenotype. The strategy employed in the current study was cost-effective, with sequencing and analysis done at USD849.68 per sample and achieving an overall detection rate of 54.5%. The strategy employed is cost-effective as it allows batching of samples from patients with different diseases in a single run, an approach that can be utilized with rare and less frequently ordered molecular diagnostic tests. The subsequent selective analysis pipeline allowed for timeous reporting back of patients results. This is feasible with a reasonable yield and can be employed for the molecular diagnosis of a wide range of rare monogenic disorders in a resource-constrained environment.

Identification of monogenic diabetes in an Australian cohort using the Exeter maturity-onset diabetes of the young (MODY) probability calculator and next-generation sequencing gene panel testing.

This study aims to describe the prevalence of monogenic diabetes in an Australian referral cohort, in relation to Exeter maturity-onset diabetes of the young (MODY) probability calculator (EMPC) scores and next-generation sequencing with updated testing where relevant. State-wide 5-year retrospective cohort study of individuals referred for monogenic diabetes genetic testing. After excluding individuals who had cascade testing for a familial variant (21) or declined research involvement (1), the final cohort comprised 40 probands. Incorporating updated testing, the final genetic result was positive (likely pathogenic/pathogenic variant) in 11/40 (27.5%), uncertain (variant of uncertain significance) in 8/40 (20%) and negative in 21/40 (52.5%) participants. Causative variants were found in GCK, HNF1A, MT-TL1 and HNF4A. Variants of uncertain significance included a novel multi-exonic GCK duplication. Amongst participants with EMPC scores ≥ 25%, a causative variant was identified in 37%. Cascade testing was positive in 9/10 tested relatives with diabetes and 0/6 tested relatives with no history of diabetes. Contemporary genetic testing produces a high yield of positive results in individuals with clinically suspected monogenic diabetes and their relatives with diabetes, highlighting the value of genetic testing for this condition. An EMPC score cutoff of ≥ 25% correctly yielded a positive predictive value of ≥ 25% in this multiethnic demographic. This is the first Australian study to describe EMPC scores in the Australian clinic setting, albeit a biased referral cohort. Larger studies may help characterise EMPC performance between ethnic subsets, noting differences in the expected probability of monogenic diabetes relative to type 2 diabetes.

Multigene panel next generation sequencing in metastatic colorectal cancer in an Australian population.

Next generation sequencing (NGS) is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology (ESMO) Scale for Clinical Actionability of Molecular Targets (ESCAT[E]) Tier I-V & X. Allele frequency is also increasingly recognised as an important prognostic tool in advanced cancer. The aim of this study was to determine the genomic mutations in metastatic colorectal cancer (CRC) in an Australian multicultural population and their influence on survival outcomes. Next generation sequencing with the 50-gene panel Oncomine Precision Assay™ was used on 180 CRC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. From 180 samples, 147 (82%) had at least one gene mutation identified with 68 (38%) having two or more concurrent mutations. Tier I variants included RAS wild-type [EI] in 73 (41%) and BRAF V600E [EIA] in 27 (15%). Non-tier I variants include 2 (1%) ERBB2 amplification [EIIB], 26 (15%) PIK3CA hotspot mutations [EIIIA] and 9 (5%) MET focal amplifications [EIIIA]. NGS testing revealed an additional 22% of cases with Tier II & III mutations. 43% of patients also presented with potentially actionable Tier III & IV mutations. Patients with concurrent TP53 and RAS mutations had significantly reduced overall survival (6.1 months versus 21.1 months, p <0.01). High KRAS allele frequency, as defined by those with over 20% variant allele frequency (VAF), also demonstrated reduced overall survival (12.1 months versus 42.9 months, p = 0.04). In addition to identifying patients with genomic alterations suitable for clinically proven standard of care therapeutic options, the 50 gene NGS panel has significant potential in identifying potentially actionable non-tier 1 mutations and therefore may become future standard clinical practice.

B-391 Is Comprehensive Cancer Panel By Next-generation Sequencing (NGS) More Efficient Than Cancer-specific NGS Panel in the Management of Non-small Cell Lung Cancer Patients?

Abstract Background : Genetic tests play an integral part in the diagnosis, treatment and prognosis of lung cancer patients. Comprehensive cancer panels using next-generation sequencing (NGS) are gradually becoming the standard tool and replacing the previous approach of single-gene or cancer-specific testing. In this study, we compared the performance of two commercially available NGS gene panels of different sizes concerning their clinical utility in the management of non-small cell lung cancer (NSCLC) patients. Methods A total of 65 tumor samples were sequenced with both lung cancer NGS panel (Lung-panel), 12 genes, and comprehensive cancer NGS panel (Pan-panel), 161 genes, following the manufacturer’s protocol. Results The Pan-panel detected 159 variants in 93.8% (61/65) patient samples; 37.7% (60/159) of these variants were classified as clinically relevant (tier I or II) with 68.3% (41/60) containing therapy approved and 31.7% (19/60) eligible for a clinical trial. The Lung-panel covered 51 of the 159 (32.0%) variants reported by the larger panel in 72.3% (47/65) tumor samples and all genetic alterations detected (51/51) were indicated as clinically relevant with 78.4% (40/51) described therapy-related variants and 21.6% (11/51) with a genomically matched clinical trial. While the Pan-panel detected more variants, additional variants beyond those included in the Lung-panel had no impact on NSCLC patient management. Even more remarkably, the cancer-specific panel despite covering a smaller genomic region than the comprehensive panel (28.7 kbp vs 329.4 kbp) was more informative in the context of FDA-approved therapy or biomarkers included in NCCN or ESMO guidelines that predict response or resistance to therapies in this cancer type, 78.4% and 68.3%, respectively. Conclusion Overall, this comparative analysis of the clinical utility of NGS panels of different sizes indicates that cancer-specific NGS panels containing carefully selected genes are as informative as larger panels when the primary goal is to identify clinically actionable mutations, particularly in patients with NSCLC. Furthermore, this approach presents a better balance among accurate identification of genomic variants, sensitivity, turnaround time, and cost-effectiveness for cancers with well-characterized molecular targets as shown in this study. Larger panels should be used in patients with rare and understudied forms of cancer or in academic reference centers where clinical and translational research is conducted.

Which side are they on? Diagnosing primary ciliary dyskinesias in low- or middle-income countries: A review and case series.

Primary ciliary dyskinesia (PCD) is a rare genetic condition with a variable clinical presentation, making its diagnosis a challenge. We describe two unrelated sibling pairs with PCD: adult siblings in the first and perinatal/neonatal in the second. Both families highlight the more common and rarer clinical manifestations of PCD. We use these cases to highlight: (i) current understanding of the underlying genetic and pathophysiological mechanisms of PCD; (ii) the diversity of cardiac and respiratory features of PCD across a wide age range; (iii) aspects of the history and clinical examination that should raise suspicion of PCD; and (iv) the role of next-generation sequencing gene panel testing in confirmation of the diagnosis. We note genomic evidence predicting that PCD is relatively common in black African populations. What the study adds. This review of two sibling pairs illustrates the variable histories, presentations, diagnostic processes and clinical courses of primary ciliary dyskinesia (PCD) in low- or middle-income countries (LMICs), highlighting the diagnostic challenges faced when encountering such patients in settings where there may not be access to specialised resources. Possible diagnostic tools that can be used are discussed, weighing up their pros and cons in an LMIC setting, and a potential diagnostic approach that can be adapted to the treating clinician's own context is provided.Implications of the findings. Confirmation of the diagnosis of primary ciliary dyskinesia is no longer limited to well-resourced institutions, but can be done in less specialised environments using novel, highly accurate next-generation sequencing gene panel testing, reducing the need to transport patients as well as the overall cost to the healthcare system. Well-resourced institutions that see high volumes of patients with PCD can invest in new highly sensitive diagnostic tools such as high-speed video microscopy. There is a need for research investigating the validity of tools such as ciliary immunofluorescence in the South African population.