Next-generation Sequencing Panel Research Articles

Molecular Review of Suspected Alport Syndrome Patients—A Single-Centre Experience

Background: Alport syndrome (AS) is a clinically and genetically heterogeneous glomerulopathy resulting from pathogenic variants in COL4A3, COL4A4, and COL4A5. Genetic diagnosis is increasingly being conducted using next-generation sequencing (NGS). Methods: Within eight years, we examined a group of 247 Polish individuals and found in total 138 unrelated probands suspected with AS based on clinical course, laboratory findings, and/or family history, as well as the total of 109 family members. We applied a targeted NGS panel to identify the genetic spectrum of AS. Known and novel variants were revealed, and detailed evaluation was performed according to ACMG/AMP guidelines to classify them as pathogenic/likely pathogenic/VUS changes. Identified genotypes were compared with clinical manifestations: hematuria, proteinuria, chronic kidney disease, sensorineural hearing impairment, ocular abnormalities, and hypertension. Results: The molecular background was established in 109/138 probands. Overall, 79 different COL4A3-COL4A5 changes (56 known and 23 novel) were revealed. About 97% were SNVs, and only two COL4A5 CNVs were identified. In total, 11 recurrent COL4A3-COL4A5 variants were observed, including the most frequent COL4A5:p.Gly624Asp, accounting for 31% of X-linked AS. Conclusions: The use of NGS panel has shown considerable promise in the field of AS, increasing diagnostic rate to 79% and reducing time to diagnosis. The phenotype-driven gene panel, specific for genetic diseases in the pediatric population, is an affordable alternative to WGS and WES, offering comparable diagnostic efficacy and supporting its implementation as a first-line genetic test in rare diseases, including AS. Based on the obtained genotype–phenotype correlation, we assessed that NGS allows us to avoid invasive renal biopsy in AS diagnosis. It provides AS confirmation/exclusion, atypical AS identification, symptomatic/asymptomatic monoallelic COL4A3-COL4A5 carrier (especially COL4A5 females) determination, and inheritance pattern establishment. AS diagnosis confirmation enables clinical course prediction and is crucial for the early introduction of renoprotective treatment with renin–angiotensin–aldosterone system blockade, aimed at slowing the disease progression and estimating the risk in family members, which is important for genetic counselling.

MSH2, MSH6, MLH1, and PMS2 immunohistochemistry as highly sensitive screening method for DNA mismatch repair deficiency syndromes in pediatric high-grade glioma

Pediatric high-grade glioma (pedHGG) can occur as first manifestation of cancer predisposition syndromes resulting from pathogenic germline variants in the DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2. The aim of this study was to establish a generalized screening for Lynch syndrome and constitutional MMR deficiency (CMMRD) in pedHGG patients, as the detection of MMR deficiencies (MMRD) may enable the upfront therapeutic use of checkpoint inhibitors and identification of variant carriers in the patients’ families. We prospectively enrolled 155 centrally reviewed primary pedHGG patients for MMR-immunohistochemistry (IHC) as part of the HIT-HGG-2013 trial protocol. MMR-IHC results were subsequently compared to independently collected germline sequencing data (whole exome sequencing or pan-cancer DNA panel next-generation sequencing) available in the HIT-HGG-2013, INFORM, and MNP2.0 trials. MMR-IHC could be successfully performed in 127/155 tumor tissues. The screening identified all present cases with Lynch syndrome or CMMRD (5.5%). In addition, MMR-IHC also detected cases with exclusive somatic MMR gene alterations (2.3%), including MSH2 hypermethylation as an alternative epigenetic silencing mechanism. Most of the identified pedHGG MMRD patients had no family history of MMRD, and thus, they represented index patients in their families. Cases with regular protein expression in MMR-IHC never showed evidence for MMRD in DNA sequencing. In conclusion, MMR-IHC presents a cost-effective, relatively widely available, and fast screening method for germline MMRD in pedHGG with high sensitivity (100%) and specificity (96%). Given the relatively high prevalence of previously undetected MMRD cases among pedHGG patients, we strongly recommend incorporating MMR-IHC into routine diagnostics.

Rate of gene fusion/rearrangement identification in GI cancers as sequencing evolved from limited panels to whole exome sequencing.

809 Background: Gene fusion/rearrangements occur at a frequency of 0-15% in GI system cancers. Detection and treatment of fusion genes such as NTRK, FGFR2, NRG1 and RET fusions are included in NCCN guidelines and are commonly detected using next generation sequencing (NGS). NGS panels have continued to expand over time, which should result in increases in the identification of gene fusions. The aims of this study were to describe the percent of patients with advanced GI tumors undergoing NGS with identified gene rearrangements across time as panels expanded. Methods: The study included 7237 patients in the Kaiser Permanente Northern California Integrated Health System receiving care for advanced GI cancers from 2017 to Aug 2024, whose tumors samples were evaluated using NGS (Stomach=926, Colorectal=2792, Gallbladder=569, Esophagus=761, liver=218, Pancreas=1971). Results: A total of 231 patients (3.2%) were identified with a gene rearrangement, including 4.6% in stomach(N=43), 2.8% in colorectal(N=78), 8.9% in gallbladder(N=24), 5.3% in esophagus(N=40), and 1.9% in pancreatic (N=38 ). Rearrangements included NTRK1/2/3, FGFR2/3, NRG, ALK, ROS, CLDN18-ARHGAP, RSPO2/3, DNAJB1-PRKACA, BRAF, MET and RET. The numbers per year were (see table). Conclusions: The use of more comprehensive NGS panels such as WES did not appear to increase the detection rate for fusion/rearrangements. In resource limited settings, limited panels may suffice. The number of GI cancer patients getting NGS testing has increased over time. Gene rearrangements/fusions identified per year. YEAR 2018 2019 2020 2021 2022 2023 2024 (Jan – Aug) Gene Rearrangement identified 29 37 43 55 54 62 38 TOTAL GI Cancer Samples Sequenced 610 858 900 1047 1249 1181 785 Percentage 4.75% 4.31% 4.78% 5.25% 4.32% 5.25% 4.84%

Integrating pharmacogenetic testing in the management of gastrointestinal cancers.

113 Background: Gastrointestinal cancers are among the most prevalent malignancies, often treated with chemotherapeutic agents whose efficacy and toxicity can be influenced by genetic variations. Pharmacogenetic testing for key metabolic genes, including DPYD, UGT1A1, and G6PD, can guide personalized treatment approaches. Methods: We analyzed samples from 3,519 individuals for genotyping using semiconductor-based Next-Generation Sequencing (NGS) technology. High-quality genomic DNA was extracted and subjected to target enrichment via high multiplex PCR amplification using an NGS panel targeting variants of key metabolic genes, including DPYD, G6PD, and UGT1A1. Results: For the DPYD gene, 96% of patients were normal metabolizers. However, 4% intermediate and 0.17% individuals had poor metabolizer status. Poor metabolizers possess homozygous non-functional alleles, leading to complete dihydropyrimidine dehydrogenase (DPD) deficiency and increased toxicity risk with fluoropyrimidine drugs like 5-fluorouracil (5-FU), Capecitabine, and Tegafur. Patients with certain homozygous or compound heterozygous variants in the DPYD gene are at increased risk for acute early-onset toxicity and serious, including fatal, adverse reactions due to fluorouracil. 5-FU is not recommended for use in patients known to have certain homozygous or compound heterozygous DPYD variants that result in complete absence of DPD activity. Intermediate metabolizers exhibit approximately 50% reduced DPD activity, necessitating a 50% reduction in starting doses followed by careful titration based on toxicity. UGT1A1 analysis revealed 46% normal, 42% intermediate, and 12% poor metabolizers, affecting Irinotecan metabolism and toxicity risks. Poor metabolizers show decreased UGT1A1 activity, resulting in reduced clearance of Irinotecan and increased risk of dose-limiting toxicities. These patients are also at higher risk for neutropenia, diarrhea, and asthenia, requiring starting dose adjustments. Additionally, they may experience elevated hyperbilirubinemia with treatments like Regorafenib. Recognizing G6PD deficiency in GI cancers can prevent serious complications and ensure safer treatment options. G6PD testing indicated that 98% were homozygous wildtype, 2% heterozygous, and 0.31% homozygous/hemizygous deficient. Patients with homozygous/hemizygous deficient genotype may be at increased risk for drug-induced hemolysis, particularly when treated with Dabrafenib. Conclusions: These findings highlight the therapeutic significance of incorporating genetic testing into clinical practice to tailor treatments, enhance drug efficacy, and minimize side effects in GI cancer management. By integrating pharmacogenetic insights, the safety and effectiveness of therapies can be significantly improved, ultimately leading to better patient care in GI oncology.

The Diagnostic Yield of Panel Versus Exome Sequencing to Identify Hereditary Cancer Disorders in Pediatric Cancer

This study aimed to assess whether targeted exome sequencing (TES) outperforms next- generation sequencing (NGS) panels in detecting clinically actionable cancer predisposition syndromes (CPS) in pediatric cancer patients. Patients with cancer underwent genetic counseling and NGS panel testing (27 or 64 genes). Simultaneously, a 616-gene targeted exome, including the NGS panel genes and 552 additional potential cancer-related genes, was conducted on the patients and their parents. Out of 42 patients undergoing both tests, NGS panels identified an APC risk allele (RA) in a patient with ganglioglioma and a pathogenic RB1 variant in a patient with retinoblastoma. In addition to the variants found by NGS panels, TES detected a pathogenic MUTYH variant in a patient with acute lymphoblastic leukemia (ALL) and a likely pathogenic (LP) BLM variant in another patient with ALL. TES also revealed a variant in candidate CPS genes, MC1R (RA) and EXT2 (LP), in a patient with embryonal rhabdomyosarcoma and Ewing sarcoma, respectively. Despite identifying variants in candidate CPS genes (MC1R, EXT2) not included on common NGS panels and known CPS genes (MUTYH, BLM) absent from this study’s panels, the diagnostic yield of clinically actionable CPS variants did not substantially increase with TES compared with standard NGS panels in pediatric cancer patients. In conclusion, for most cases, panel testing remains appropriate for CPS diagnosis in pediatric cancer within typical clinical settings.

Cytologic, histologic, and clinical correlation of minor mutations in pancreatic cysts.

Major mutations (e.g., KRAS, GNAS, TP53, SMAD4) in pancreatic cyst fluid (PCF) are useful for classifying and risk stratifying certain cyst types, particularly in cases with nondiagnostic cytology. However, the significance of uncommon minor mutations in PCF has yet to be reported. In total, 127 PCF specimens (2014-2021) from 121 patients that underwent molecular analysis were identified, and detailed clinicopathologic data were recorded. Molecular testing was performed using a laboratory-developed next-generation sequencing panel. Forty-five variants other than KRAS, GNAS, RNF43, TP53, CDKN2A, and SMAD4 were detected. Variants that were detected in five or more cases included ARID1A (n=28), VHL (n=17), BRAF (n=12), ATM (n=8), APC (n=8), MEN1 (n=5), serine threonine kinase 11 (STK11; n=5), PIK3CA (n=5), and CDH1 (n=5). Thirty-eight of 121 patients (31%) had histologic confirmation on follow-up resection. Twenty-seven of 28 cysts (96%) with ARID1A mutations had concurrent KRAS/GNAS mutations; 17 (61%) were diagnosed as neoplastic mucinous cysts on cytology, and 10 (36%) were diagnosed as intraductal papillary mucinous neoplasm (IPMN) on histology (80% low grade). No patients developed disease recurrence or died of disease. Cysts with STK11 mutations had RAS co-mutations (KRAS, n=5; NRAS, n=1), and four of those five cysts (80%) were mucinous neoplasms with high-grade atypia on cytology. All three resection specimens were IPMNs with high-grade dysplasia or invasive carcinoma, and two of those patients died of disease. In PCFs, ARID1A mutations were consistently associated with IPMNs (predominantly low grade) with no recurrences or deaths from disease. STK11 mutations appeared to be associated with high-risk mucinous cysts. The detection of minor variants may provide useful preoperative information and add value beyond single-gene genotyping of major mutations.

Circulating Tumor DNA and [18F]FDG-PET for Early Response Assessment in Patients with Advanced NSCLC

Background/Objectives: Identifying treatment failure at earlier time points to could spare cancer patients from ineffective treatment and side effects. In this study, circulating tumor DNA (ctDNA) and [18F]FDG-PET/CT were investigated during the first cycle of anticancer therapy in patients with advanced non-small cell lung cancer (NSCLC) to explore their potential for early response evaluation. Methods: Patients with advanced NSCLC receiving first-line therapy with immune checkpoint inhibitors and/or chemotherapy were included. CtDNA and [18F]FDG-PET/CT assessments were conducted before treatment and at weeks 1 and 3 during the first cycle of therapy. ctDNA quantification was performed using a targeted next-generation sequencing (NGS) panel, and the least favorable change in any mutated allele frequency at a given time was used for analysis. [18F]FDG-PET/CT was quantified using sumSULpeak and metabolic tumor volume (MTV4.0). Early changes in ctDNA levels and [18F]FDG-PET parameters were compared with final treatment response, measured by RECIST after 12 weeks, as well as progression-free survival and overall survival. Results: Of the sixteen included patients, eight were non-responders. ctDNA mutations were detected in baseline blood samples in eight patients. Changes in ctDNA level, MTV4.0, and sumSULpeak at week 3 indicated response in 7 out of 8 patients, 13 out of 15 patients, and 9 out of 15 patients, respectively. At week 3, no false increases were seen with ctDNA and MTV4.0. Conclusions: These results suggest that early changes in ctDNA and [18F]FDG-PET/CT at 3 weeks of treatment could be used to early assess treatment response. Increased levels of ctDNA and MTV4.0 at week 3 were only observed in patients with treatment failure.

Characteristics of genomic alterations and heavy metals in hypertensive patients with non‑small cell lung cancer.

Both lung cancer and cardiovascular disease (CVD) are prevalent diseases that contribute to global mortality rates. Although individuals with CVD may face an elevated risk of cancer based on the presence of shared risk factors (such as tobacco smoking and excessive body weight), the roles of somatic mutations and heavy metal distributions remain unknown. The present study aimed to explore the differences in somatic mutations and heavy metal distributions between hypertensive patients and non-hypertensive patients in a cohort of patients with non-small cell lung cancer (NSCLC). Tumor tissue samples from 64 patients were analyzed using a next-generation sequencing panel consisting of 82 tumor-related genes through hybrid capture. Serum samples were also analyzed to determine the levels of 18 heavy metals using inductively-coupled plasma mass spectrometry. Among the 16 hypertensive patients, all patients (16/16; 100.00%) harbored 47 somatic mutations in 14 mutant genes, whereas 45 patients without hypertension (45/48; 93.75%) harbored 113 somatic mutations across 26 mutant genes (no mutations were detected in the remaining 3 patients). Among the 32 identified mutant genes in these two groups, FBXW7, CBR3, CDKN2A, HRAS, SMO and UGT1A1 were exclusively observed in patients with hypertension, while 18 mutant genes were only observed in patients without hypertension. No significant mutually exclusive interactions were found in hypertensive patients, but mutually exclusive interactions were observed between EGFR and STK11 (P=0.0240) and between STK11 and KRAS (P=0.0169) in non-hypertensive patients. 'Non-small cell lung cancer' was the top Kyoto Encyclopedia of Genes and Genomes pathway in hypertensive patients, whereas 'central carbon metabolism in cancer' was the top pathway in patients without hypertension. Moreover, the proportions of altered key signaling pathways and biological function categories shared between these two groups were 54.37% (56/103) and 21.62% (8/37), respectively. Furthermore, the levels of chromium (Cr) in the serum of hypertensive patients were notably elevated compared with those in patients without hypertension. In addition, significant negative correlations were observed between Cr and CEA, between CYFRA21-1 and Zn, and between NSE and As in hypertensive patients but not in non-hypertensive patients, indicating differing interactive profiles among the traditional serum biomarkers and heavy metals between these two patient groups. In summary, there were differences in genomic alterations, somatic interactions and the serum levels of Cr between patients with NSCLC with hypertension and patients with NSCLC without hypertension. Furthermore, patients with hypertension exhibited significant negative correlations between Cr and CEA, between CYFRA21-1 and Zn, and between NSE and As, suggesting that heavy metals may contribute to the occurrence of NSCLC with different hypertensive status.

A next-generation sequencing-based universal target panel and algorithm for one-stop detection of copy number alterations and single-nucleotide variations in the HBB gene cluster for rapid diagnosis of β-thalassemia.

This study aimed to develop and validate a targeted next-generation sequencing (NGS) panel along with a data analysis algorithm capable of detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) within the beta-globin gene cluster. The aim was to reduce the turnaround time in conventional genotyping methods and provide a rapid and comprehensive solution for prenatal diagnosis, carrier screening, and genotyping of β-thalassemia patients. We devised a targeted NGS panel spanning an 80.4 kb region on chromosome 11, encompassing the beta-globin gene cluster and 5' locus control region. We also developed an advanced data analysis algorithm consisting of variant calling and depth plot analysis that facilitates simultaneous detection of SNVs and CNVs in a single run. The test panel and algorithm were validated with 14 in-house β-thalassemia carrier/patient samples and cross-checked against the HbVar database. We identified seven pathogenic SNVs and five CNVs within the beta-globin gene cluster in various combinations, such as heterozygous, homozygous, and compound heterozygous conditions. Additionally, the coordinates of 169 rare deletions and 11 fusion mutations reported in the HbVar database were checked to verify the theoretical ability of our developed gene panel to detect all the CNVs within the target region. The developed panel and NGS technology can detect both SNVs and CNVs in a single run and can also be utilized for prenatal diagnosis and carrier screening for hemoglobinopathies, underscoring its versatility and clinical utility.

Diagnosis of hereditary ataxias: a real-world single center experience.

This study aims to evaluate our experience in the diagnosis of hereditary ataxias (HAs), to analyze data from a real-world scenario. This is a retrospective, cross-sectional, descriptive study conducted at a single Italian adult neurogenetic outpatient clinic, in 147 patients affected by ataxia with a suspicion of hereditary forms, recruited from November 1999 to February 2024. A stepwise approach for molecular diagnostics was applied: targeted gene panel (TP) next-generation sequencing (NGS) and/or clinical exome sequencing (CES) were performed in the case of inconclusive first-line genetic testing, such as short tandem repeat expansions (TREs) testing for most common spinocerebellar ataxias (SCA1-3, 6-8,12,17, DRPLA), other forms [Fragile X-associated tremor/ataxia syndrome (FXTAS), Friedreich ataxia (FRDA) and mitochondrial DNA-related ataxia, RFC1-related ataxia/CANVAS] or inconclusive phenotype-guided specific single gene sequencing. A definitive diagnosis was reached in 36.7% of the cases. TREs testing was diagnostic in 30.4% of patients. The three most common TREs ataxias were FRDA (36.1%), SCA2 (27.8%), and RFC1-related ataxia/CANVAS (11.1%). In five patients, the molecular diagnosis was achieved by single gene sequencing and causative mutations were identified in POLG (2), SACS (1), DARS2 (1), MT-ATP6 (1). Of 94 patients with a suspicion of HAs of indeterminate genetic origin, 68 underwent new molecular evaluation using the NGS approach. In 28 of these cases, CES was performed after the TP sequencing resulted negative. In 13 patients, the diagnosis was achieved by NGS approach. In 7 of these 13 patients, the diagnosis was made by CES. Genes mutations identified as causative of HAs were found in SPG7 (4), SACS (1), CACNA1A (1), CACNA1G (1), EEF2 (1), PRKCG (1), KCNC3 (1), ADCK3 (1), SYNE1 (1), ITPR1 (1). A positive family history of ataxia and early onset of symptoms were associated with a higher likelihood of obtaining a definite diagnosis. The molecular diagnosis of HAs remains a significant challenge for neurologists. Our data indicate that, in most cases, a diagnosis of HA can be established through first line genetic testing, particularly TREs testing. However, for patients with a clinical diagnosis of HA who do not achieve a molecular diagnosis through initial genetic tests, the use of NGS proves to be a valuable tool, providing a definitive diagnosis in approximately 20% of cases. Therefore, when feasible in clinical practice, integrating NGS testing, especially exome sequencing, into the diagnostic decision-making process for unsolved cases is crucial.

Novel and recurrent genetic variants associated with male and female infertility.

Recently, the knowledge of the genetic basis of fertility disorders has expanded enormously, mainly thanks to the use of next-generation sequencing (NGS). However, the genetic cause of infertility, in the majority of patients, is still undefined. The aim was to identify novel and recurrent pathogenic/likely pathogenic variants in patients with isolated infertility or puberty delay using a targeted NGS technique. We have enrolled 41 patients (36 males and 5 females) with infertility problems or delayed puberty. We included the patients with hypogonadotropic hypogonadism (n = 12), hypergonadotropic hypogonadism (n = 15), abnormal sperm parameters (n = 10), androgen insensitivity syndrome (n = 3) and 46,XY gonadal dysgenesis (n = 1). Genetic tests were performed using targeted NGS panel of 35 genes implicated in fertility. Pathogenic or likely pathogenic variants potentially explaining the clinical phenotype were identified in 12 of 41 patients (29%). These included 9 of 12 patients (75%) with hypogonadotropic hypogonadism, 2 of 3 patients (66%) with androgen insensitivity syndrome, and the single patient with 46,XY gonadal dysgenesis. Among the 18 identified variants, 4 were novel (FGF8:p.Ala147Thr; SEMA3A:p.Arg544Cys; FGFR1:p.Thr141IlefsTer10; NSMF: p.Tyr242Cys), while 14 were recurrent. Our study expands the knowledge of the genetic basis of the infertility disorders and highlights the importance of genetic testing for proper diagnosis making and genetic counselling.

Prognostic Value of Residual Circulating Tumor DNA in Metastatic Pancreatic Ductal Adenocarcinoma.

Circulating tumor DNA (ctDNA) is a potential biomarker in pancreatic ductal adenocarcinoma (PDAC). However, studies on residual ctDNA in patients post-chemotherapy are limited. We assessed the prognostic value of residual ctDNA in metastatic PDAC relative to that of carbohydrate antigen 19-9 (CA19-9). ctDNA analysis using a targeted next-generation sequencing panel was performed at baseline and during chemotherapy response evaluation in 53 patients. Progression-free survival (PFS) and overall survival (OS) were first evaluated based on ctDNA positivity at baseline. For further comparison, patients testing ctDNA-positive at baseline were subdivided based on residual ctDNA into ctDNA responders (no residual ctDNA post-chemotherapy) and ctDNA non-responders (residual ctDNA post-chemotherapy). Additional survival analysis was performed based on CA19-9 levels. The baseline ctDNA detection rate was 56.6%. Although clinical outcomes tended to be poorer in patients with baseline ctDNA positivity than in those without, the differences were not significant. Residual ctDNA post-chemotherapy was associated with reduced PFS and OS. The prognosis of ctDNA responders was better than that of non-responders but did not significantly differ from that of ctDNA-negative individuals (no ctDNA both at baseline and during post-chemotherapy). Compared with ctDNA responses to chemotherapy, a ≥ 50% decrease in the CA19-9 level had less effect on both PFS and OS based on hazard ratios and significance levels. ctDNA could be monitored in half of the patients whose baseline CA19-9 levels were within the reference range. Residual ctDNA analysis post-chemotherapy is a promising approach for predicting the clinical outcomes of patients with metastatic PDAC.

Multicenter In-House Evaluation of an Amplicon-Based Next-Generation Sequencing Panel for Comprehensive Molecular Profiling.

Predicting response to targeted cancer therapies increasingly relies on both simple and complex genetic biomarkers. Comprehensive genomic profiling using high-throughput assays must be evaluated for reproducibility and accuracy compared with existing methods. This study is a multicenter evaluation of the Oncomine™ Comprehensive Assay Plus (OCA Plus) Pan-Cancer Research Panel for comprehensive genomic profiling of solid tumors. A series of 193 research samples (125 DNA and 68 RNA samples) was analyzed to evaluate the correlation and concordance of the OCA Plus panel with orthogonal methods, as well as its reproducibility (n = 5 DNA samples) across laboratories. The success rate for DNA and RNA sequencing was 96.6% and 89.7%, respectively. In a single workflow, the OCA Plus panel provided a detailed genomic profile with a high success rate for all biomarkers tested: single nucleotide variants/indels, copy number variants, and fusions, as well as complex biomarkers such as microsatellite instability, tumor mutational burden, and homologous recombination deficiency. The concordance for single nucleotide variants/indels was 94.8%, for copy number variants 96.5%, for fusions 94.2%, for microsatellite instability 80.8%, for tumor mutational burden 81.3%, and for homologous recombination deficiency 100%. The results showed high reproducibility across the five European research centers, each analyzing shared pre-characterized tissue biopsies (average of 1890 single nucleotide variants/indels per sample). This multicenter evaluation of the OCA Plus panel confirms the results of previous single-center studies and demonstrates the high reproducibility and accuracy of this assay.

Hereditary Pancreatic Cancer: Advances in Genetic Testing, Early Detection Strategies, and Personalized Management.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a five-year survival rate of approximately 13% for advanced stages. While the majority of PDAC cases are sporadic, a significant subset is attributable to hereditary and familial predispositions, accounting for approximately 25% of cases. This article synthesizes recent advancements in the understanding, detection, and management of hereditary pancreatic cancer (PC). Results: Our review highlights the critical role of genetic testing (GT) in identifying high-risk individuals (HRIs), with germline pathogenic variants (PVs) found in up to 20% of hereditary PDAC cases. Since the implementation of next-generation sequencing (NGS) panels in 2014, detection capabilities have been significantly enhanced. HRIs can be included in screening programs that facilitate the early detection of PDAC. Early detection strategies, including the use of microribonucleic acid (miRNAs) signatures and novel imaging techniques like hyperpolarized 13C-magnetic resonance spectroscopy (MRS) have shown promising results. The identification of germline pathogenic variants (PVs) or mutations in homologous recombination (HR) genes plays a predictive role in the response to various treatments, prolonging patient survival. Discussion: Universal germline testing for PDAC, as recommended by the National Comprehensive Cancer Network (NCCN), is now a standard practice, facilitating the identification of at-risk individuals and enabling targeted surveillance and intervention. Multidisciplinary management, integrating genetic counseling, imaging, and gastrointestinal services, is essential for optimizing outcomes. Conclusions: Advances in genetic testing and biomarker research are transforming the landscape of hereditary PC management. Early detection and personalized treatment strategies are pivotal in improving survival rates. Ongoing multi-institutional research efforts are crucial for validating biomarkers and developing preventive measures, ultimately aiming to reduce the burden of this aggressive cancer.

Gene Panel-Based Genotyping of 279 Turkish Maturity-Onset Diabetes of the Young Patients from Eastern Anatolia

Introduction: The aim of this study was to analyze the genotypic and clinical characteristics of Turkish patients with maturity-onset diabetes of the young (MODY) in Eastern Anatolia in order to improve the understanding of the genetic diversity and clinical manifestations of MODY. The findings should help to improve the diagnosis and management of monogenic diabetes. Methods: This retrospective study included 279 patients with suspected MODY, selected for age at presentation, family history, no pancreas autoantibodies, and minimal/no need for insulin. A next-generation sequencing panel was used for genetic analysis. The panel included 15 different genes. Results: Pathogenic variants were identified in 94% of the patients, with the majority being in GCK, HNF1A, and HNF4A. Variants in GCK were the most common (65%), followed by HNF1A (20%) and HNF4A (9%). Patients with GCK mutations exhibited mild fasting hyperglycemia, whereas HNF1A and HNF4A mutations were associated with progressive hyperglycemia. Conclusion: This study provides significant insights into the genotypic and clinical spectrum of MODY within a Turkish population. The detection of pathogenic variants in genes such as GCK, HNF1A, and HNF4A underscores the importance of comprehensive genetic testing in patients suspected of MODY. The results emphasize the need for personalized approaches to optimize the diagnosis and treatment of MODY. Detailed genetic and clinical assessments play a pivotal role in identifying at-risk individuals and tailoring management strategies. These findings could guide the development of targeted therapies and enhance the clinical outcomes of patients with monogenic diabetes.

Prognosis of TP53 and Its Concomitant EGFR Mutation in Lung Cancer Especially Non-Small Cell Lung Cancer.

Human Lung Carcinoma (LC) is among the most diagnosed cancers across the world among those non-small cell lung cancer (NSCLC) comprises about 85%. Next Generation Sequencing based detection of mutations are now well established in molecular oncology. With the advent of modern diagnostic methods, it is now well known that there are several mutations and gene rearrangements which are associated with the development of LC. Among those mutations, TP53 is the most prevalent with the concomitant EGFR mutation. In this retrospective study, a total number of 414 patients have been incorporated who have attended RGCIRC in the period between November 2015 to March 2024. Clinical stage has been determined as per NCCN Guideline version 2.2024. Nucleic Acid (DNA and RNA) from FFPE samples were extracted and detection of mutation was performed by Next generation sequencing (NGS) method. All 414 patients opted for the customised NGS panel for lung cancer among those 203 patients were TP53 mutation and 87 patients were EGFR mutation positive. 62 patients were TP53-EGFR double mutation positive. The results of this study have shown that TP53 mutated patients show poor prognosis with conventional therapy. However, TP53-EGFR co-mutated patient's recovery rates are comparatively promising due to the availability of the targeted therapy of EGFR. Studies have shown that TP53 mutation is unlikely to derive clinical benefit in LC patients and shows poorer prognosis when compared to TP53 wild type and EGFR mutated patients show improved recovery due to availability of the Kinase Inhibitor (KI) treatment. In this study we have observed and concluded that TP53-EGFR co-mutated group also shows promising prognosis for the application of KI treatment. A further large cohort study will establish this clinical observation and enlighten more therapeutically relevant information.

Targeted Next-Generation Sequencing of Cell-Free DNA to Detect MYC-Immunoglobulin Translocation and Epstein-Barr Virus DNA in Plasma of Burkitt Lymphoma Patients in East Africa.

Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) affects children in sub-Saharan Africa, but diagnosis via tissue biopsy is challenging. We explored a liquid biopsy approach using targeted next-generation sequencing to detect the MYC-immunoglobulin (MYC-Ig) translocation and EBV DNA, assessing its potential for minimally invasive BL diagnosis. The panel included targets for the characteristic MYC-Ig translocation, mutations in intron 1 of MYC, mutations in exon 2 of MYC, and three EBV genes: EBV-encoded RNA (EBER)1, EBER2, and EBV nuclear antigen 2. It was first tested in a small derivation cohort of four precharacterized BL-derived cell lines with known translocation status and eight precharacterized plasma samples with known EBV DNA status by quantitative polymerase chain reaction (qPCR). These different data modalities were combined to assess the accuracy of this approach in the diagnosis of BL in 20 patient plasma samples in Tanzania and Uganda. The next-generation sequencing panel detected three of four MYC-Ig translocations in the BL-derived cell lines. EBV viral load by targeted sequencing correlated strongly with qPCR results (Spearman's rho = 0.94) in precharacterized plasma samples. Using the patient plasma samples, mutations in MYC intron 1 were associated with the presence of a MYC translocation with 25 or more mutations being predictive of a translocation with AUC, sensitivity, and specificity of 1. Overall, liquid biopsy parameters associated with a diagnosis of BL (P < .05) included cell-free DNA concentration, circulating tumor DNA concentration, MYC intron 1 mutations, MYC-Ig translocation, and autosome entropy. Integrating these parameters into a diagnostic model demonstrated excellent performance with an AUC of 0.95, sensitivity of 0.9, and specificity of 1. This analysis demonstrates the potential of liquid biopsy to improve BL diagnosis in settings with limited pathology resources. Validation of our approach in a larger data set is needed.

Tissue Prior to the Initial Hematoxylin-Eosin Section Demonstrates Value as an Alternative Source of DNA for Molecular Testing.

Small biopsies are used for histologic, immunophenotypic, cytogenetic, molecular genetic, and other ancillary studies. Occasionally, this diagnostic tissue is exhausted before molecular testing can be performed. To investigate a simple banking protocol for currently discarded tissues trimmed off prior to the initial hematoxylin-eosin section, as an alternative source of DNA for molecular studies. Mock biopsies of lung adenocarcinomas, benign testes, and B-cell lymphomas were constructed from biobank blocks; these simulated biopsies were assessed via epidermal growth factor receptor (EGFR) p.L858R droplet digital polymerase chain reaction (PCR), Biomed B-cell clonality testing by PCR, or a custom next-generation sequencing panel for lymphomas. For each cancer mock biopsy, DNA amounts and molecular test results from the "trimmings" samples were compared to data from corresponding molecular samples acquired via a "standard" clinical protocol. The data show that although trimmings samples usually contained less DNA than standard samples, both sample classes generally had sufficient DNA for testing and produced essentially identical molecular results. A single sample showed low-level carryover contamination on droplet digital PCR testing. Tissue trimmings banked by using the studied protocol demonstrated value as a potential alternative sample for molecular testing.

Mutational disparities in colorectal cancers of White Americans, Alabama African Americans, And Oklahoma American Indians

AbstractThe high incidence and mortality rates of colorectal cancer (CRC) in Alabama African Americans (AAs) and Oklahoma American Indians (AIs) are recognized as cancer disparities, yet the underlying causes have been poorly demonstrated. By evaluating CRC whole-exome sequencing and mutational profiles, here we report sets of mutated genes whose frequencies differed significantly (p &lt; 0.05) in a race-specific manner. Secondary screening with cancer database identified “survival-critical genes (SCGs)” (i.e., genes whose mutations/alterations are associated with significant differences in the patients’ survival rates) among the differentially mutated genes. Notable SCGs with race-pronounced variants were different from DEGs and their involved pathways included nucleotide catabolism and cell cycle checkpoints for AAs, and extracellular matrix organization for AIs. The inclusion of these SCGs with race-pronounced variants in the clinical CRC next-generation sequencing panels and the development of targeting drugs will serve as refinements for precision medicine to overcome racial disparities in health outcomes of CRC.

MET Exon 14 Skipping and Novel Actionable Variants: Diagnostic and Therapeutic Implications in Latin American Non-Small-Cell Lung Cancer Patients.

Targeted therapy indications for actionable variants in non-small-cell lung cancer (NSCLC) have primarily been studied in Caucasian populations, with limited data on Latin American patients. This study utilized a 52-genes next-generation sequencing (NGS) panel to analyze 1560 tumor biopsies from NSCLC patients in Chile, Brazil, and Peru. The RNA sequencing reads and DNA coverage were correlated to improve the detection of the actionable MET exon 14 skipping variant (METex14). The pathogenicity of MET variants of uncertain significance (VUSs) was assessed using bioinformatic methods, based on their predicted driver potential. The effects of the predicted drivers VUS T992I and H1094Y on c-MET signaling activation, proliferation, and migration were evaluated in HEK293T, BEAS-2B, and H1993 cell lines. Subsequently, c-Met inhibitors were tested in 2D and 3D cell cultures, and drug affinity was determined using 3D structure simulations. The prevalence of MET variants in the South American cohort was 8%, and RNA-based diagnosis detected 27% more cases of METex14 than DNA-based methods. Notably, 20% of METex14 cases with RNA reads below the detection threshold were confirmed using DNA analysis. The novel actionable T992I and H1094Y variants induced proliferation and migration through c-Met/Akt signaling. Both variants showed sensitivity to crizotinib and savolitinib, but the H1094Y variant exhibited reduced sensitivity to capmatinib. These findings highlight the importance of RNA-based METex14 diagnosis and reveal the drug sensitivity profiles of novel actionable MET variants from an understudied patient population.