Targeted Next-generation Sequencing Panel Research Articles

Genetic Landscape of a Cohort of 120 Patients with Diminished Ovarian Reserve: Correlation with Infertility.

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are major causes of female infertility. We recently found a monogenic etiology in 29.3% of POI, leading to personalized medicine. The genetic landscape of DOR is unknown. A prospective study (2018-2023) of an international cohort of 120 patients with unexplained DOR was performed using a large custom targeted next-generation sequencing panel including all known POI-causing genes. The diagnostic yield, based on the American College of Medical Genetics, was 24, 2%. Genes belong to different pathways: metabolism and mitochondria (29.7%), follicular growth (24.3%), DNA repair/meiosis (18.9%), aging (16.2%), ovarian development (8.1%), and autophagy (2.7%). Five genes were recurrently found: LMNA, ERCC6, SOX8, POLG, and BMPR1B. Six genes identified in single families with POI were involved in DOR, GNAS, TGFBR3, XPNPEP2, EXO1, BNC1, ATG, highlighting their role in maintaining ovarian reserve. In our cohort, 26 pregnancies were recorded, but no pregnancy was observed when meiosis/DNA repair genes were involved, suggesting severely impaired oocyte quality. Additional studies should confirm these preliminary results. This study with a large NGS panel defines the genetic landscape of a large cohort of DOR. It supports routine genetic diagnosis. Genetics could be a biomarker predicting infertility and progression to POI.

Activation of γ-globin expression by a common variant disrupting IKAROS-binding motif in β-thalassemia

Programmed silencing of γ-globin genes in adult erythropoiesis is mediated by several chromatin remodeling complexes, which determine the stage-specific genome architecture in this region. Identification of cis- or trans-acting mutations contributing to the diverse extent of Hb F might illustrate the underlying mechanism of γ-β globin switching. Here, we recruit a cohort of 1142 β-thalassemia patients and dissect the natural variants in the whole β-globin gene cluster through a targeted next-generation sequencing panel. A previously unreported SNP rs7948668, predicted to disrupt the binding motif of IKAROS as a key component of chromatin remodeling complexes, is identified to be significantly associated with higher levels of Hb F and age at onset. Gene-editing on this SNP leads to elevation of Hb F in both HUDEP-2 and primary CD34+ cells while the extent of elevation is amplified in the context of β-thalassemia mutations, indicating epistasis effects of the SNP in the regulation of Hb F. Finally, we perform ChIP-qPCR and 4C assays to prove that this variant disrupts the binding motif of IKAROS, leading to enhanced competitiveness of HBG promoters to locus control regions. This study highlights the significance of common regulatory SNPs and provides potential targets for treating of β-hemoglobinopathy.

6492 Novel compound heterozygous mutations in the SBP2 gene

Abstract Disclosure: H. Jeong: None. Mutations in the selenocysteine insertion sequence binding protein 2 gene (SECISBP2 also known as SBP2) result in a multisystemic disorder. A 4-year-old Korean girl presented with atypical thyroid function test result, characterized by normal TSH 2.13 μIU/mL, decreased T3 0.51 ng/mL, free T3(FT3) 1.71 (pg/mL), and elevated free thyroxine (FT4) 4.28 ng/dL. Additionally, she exhibited short stature below 2 standard deviations and delayed bone age. She has not yet begun speaking, and her waking ability commenced only recently. She conceived twins through natural pregnancy, but tragically, her twin sibling passed away in utero at 8 weeks of gestation.A targeted next-generation sequencing panel revealed compound heterozygous mutation in SBP2 gene (p.Arg120Ter/p.Arg672Ter) and in TPO gene (p.Glu413Gln).Her serum selenium level was relatively low,, easuring at 6.94 ng/dL. She underwent T3 supplementation and received a daily selenium supplement of 20mcg. Subsequently, her thyroid function tests normalized. Presentation: 6/2/2024

Analytical and clinical validation of a NGS panel in detecting targetable variants from ctDNA of metastatic NSCLC patients.

Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for noninvasive cancer diagnostics, particularly in the context of metastatic non-small-cell lung cancer (NSCLC). Detecting targetable variants through ctDNA analysis offers the potential to guide treatment decisions, especially in cases where tissue samples are insufficient or unavailable. In this study, we developed and validated a next-generation sequencing panel targeting 101 cancer-related genes (101-test) to detect somatic variants in ctDNA from a large cohort of Chinese patients with metastatic NSCLC. The performance of the 101-test was assessed by evaluating its limit of detection (LOD), accuracy, and precision in identifying molecular variants. Additionally, the concordance between ctDNA and tissue samples for detecting targetable variants was analyzed in 904 patients. The 101-test demonstrated a LOD of 0.38% for single-nucleotide variants (SNVs), 0.33% for insertions and deletions (InDels), and 0.33% for fusions. Sensitivity was 98.3% for SNVs, 100% for InDels, and 100% for fusions when compared to digital droplet PCR (ddPCR)/breakpoint PCR reference methods. The by-variant sensitivity for somatic variants was 97.5%, with a specificity of 99.9% between tumor-only and tumor-normal analyses. In a real-world cohort, the concordance between ctDNA and tissue samples for identifying targetable variants was 72.2% (457/633). Notably, the EGFR S768I variant, recently recommended by clinical guidelines, achieved an 80% concordance rate. Furthermore, 4.3% of patients (27/633) with targetable variants were identified exclusively through ctDNA testing. The ctDNA-based 101-test is a highly sensitive and specific tool for detecting targetable variants in metastatic NSCLC, particularly in cases with insufficient tissue samples. The findings support the use of ctDNA testing as a reliable and complementary method to traditional tissue-based molecular analysis, enhancing the precision of treatment strategies for NSCLC patients.

Acquired gene alterations in patients treated with ribociclib plus endocrine therapy or endocrine therapy alone using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials

A prior pooled analysis of the MONALEESA-2, -3, and -7 trials identified baseline markers predictive of sensitivity or resistance to ribociclib plus endocrine therapy (ET). We report the results of an analysis of paired baseline and end-of-treatment (EOT) circulating tumor DNA (ctDNA) samples across the MONALEESA trials. Paired baseline and EOT ctDNA samples from MONALEESA-2, -3, and -7 were sequenced using a targeted next-generation sequencing panel. Genes with an EOT alteration prevalence of >5% were included. A McNemar test was carried out on paired samples and adjusted for multiple testing to control the false discovery rate. A Bayesian mixed-effects model was used to adjust for ctDNA fraction at both time points and for study differences. The analysis included 523 paired samples. At EOT, 21 genes had a >5% alteration prevalence. A trend for higher ctDNA fraction at EOT versus baseline (P= 0.08) was observed. Prevalence of alterations was higher at EOT versus baseline in RB1, SPEN, TPR, PCDH15, and FGFR2 in the ribociclib arm; PBRM1 in the placebo arm; and ESR1 in both arms. The mixed-effects model demonstrated that the same trends for increased prevalence of these alterations at EOT were observed after adjusting for ctDNA fraction and that the increased rate of RB1 and SPEN alterations at EOT were specific to ribociclib plus ET. Analysis of ESR1 indicated a similar increase at EOT in both arms. The most common acquired ESR1 mutations at EOT included Y537C/N/S/D, D538G, E380Q, and L536H/R/P/LC. The prevalence of PIK3CA hotspot mutations at baseline and EOT was similar. This analysis identified acquired gene alterations in patients with hormone receptor-positive/human epidermal growth factor receptor-2 negative advanced breast cancer treated with ribociclib plus ET or placebo plus ET. These data may support further studies on acquired resistance mechanisms and inform future systemic interventions in the post-cyclin-dependent kinase 4/6 inhibitor setting.

Molecular Profiling of KIT/PDGFRA-Mutant and Wild-Type Gastrointestinal Stromal Tumors (GISTs) with Clinicopathological Correlation: An 18-Year Experience at a Tertiary Center in Kuwait.

In gastrointestinal stromal tumors (GISTs), identifying prototypical mutations in the KIT/PDGFRA oncogenes, or in rare alternate genes, is essential for prognostication and predicting response to tyrosine kinase inhibitors. Conversely, wild-type GISTs (WT-GIST), which lack known mutations, have limited treatment options. Data on the mutational landscape of GISTs and their impact on disease progression are very limited in Kuwait. Using a targeted next-generation sequencing panel, we investigated the spectrum and frequency of KIT, PDGFRA, and RAS-pathway-related mutations in 95 out of 200 GISTs diagnosed at Kuwait Cancer Center from 2005 to 2023 and assessed their correlation with clinicopathological parameters. Among the 200 tumors (median age 55 years; 15-91), 54% originated in the stomach, 33% in the small bowel, 7% in the colorectum, 1.5% in the peritoneum, and 4.5% had an unknown primary site. Of the 95 molecularly profiled cases, 88% had a mutation: KIT (61%), PDGFRA (25%), NF1 (2%), and one NTRK1 rearrangement. Ten WT-GISTs were identified (stomach = 6, small bowel = 2, and colorectum = 2). WT-GISTs tended to be smaller (median 4.0 cm; 0.5-8.0) (p = 0.018), with mitosis ≤5/5 mm2, and were of lower risk (p = 0.019). KIT mutations were an adverse indicator of disease progression (p = 0.049), while wild-type status did not significantly impact progression (p = 0.934). The genetic landscape in this cohort mirrors that of global studies, but regional collaborations are needed to correlate outcomes with genetic variants.

The K9 lymphoma assay allows a genetic subgrouping of canine lymphomas with improved risk classification

We present here the K9 lymphoma assay, a novel 31-gene targeted next-generation sequencing panel designed for genomic profiling of canine lymphoid neoplasms. Addressing the growing demand for advanced diagnostics in veterinary oncology, this assay enables sensitive identification of known and actionable mutations specific to canine lymphomas, while evaluating its prognostic potential to facilitate diagnosis and prognosis. Our analysis, spanning several B- and T-cell lymphoma histotypes, unveiled distinct mutational landscapes distinguishing tumors derived from immature versus mature lymphocytes. Clustering analysis revealed a shared genetic origin between diffuse large B-cell lymphoma and marginal zone lymphoma, aligning with findings in human lymphomas, with TRAF3 emerging as the most frequently mutated gene across B-cell lymphoma subtypes. Significantly, TP53 mutations demonstrated universal adverse prognostic implications across B-cell lymphomas. Additionally, SETD2 mutations contributed to shorter time-to-progression, underscoring the role of epigenetic dysregulation in B-cell tumors. In T-cell lymphomas, SATB1 and FBXW7 were frequently mutated, warranting further investigation in larger cohorts. Our findings advocate for tailored therapeutic approaches based on the genetic profile, impacting treatment decisions and outcomes in canine lymphoma management. This study provides pivotal insights bridging veterinary and human oncology, paving the way for comprehensive genomic diagnostics and therapeutic strategies in comparative oncology.

Isolated intraductal carcinoma of the prostate: a clinicopathological and molecular analysis

Objective: To study the clinicopathological features, immunohistochemical phenotypes, molecular changes, differential diagnosis and prognosis of isolated intraductal carcinoma of the prostate (iIDC-P). Methods: Three iIDC-P cases were collected retrospectively from 2016 to 2022 at Ningbo Clinical Pathology Diagnosis Center, Ningbo, China. The clinicopathologic features and immunophenotypic profiles were studied using light microscopy and immunohistochemistry. A targeted next-generation sequencing panel was used to analyze cancer-associated mutations. Follow-up and literature review were also performed. Results: The patients' ages were 61, 67 and 77 years, and their preoperative prostate specific antigen (PSA) levels were 7.99, 7.99 and 4.86 μg/L, respectively. Case 1 and 2 were diagnosed on needle biopsy and radical prostatectomy (RP) specimens, and case 3 was diagnosed on a specimen of transurethral resection of the prostate (TURP). The RP specimen was entirely submitted for histologic examination. In the case 1, iIDC-P was found in one tissue core (involving two ducts) in the biopsy specimen, and in 6 sections (diameter, 0.3-1.1 cm) from the radical prostatectomy specimen, and one section had separate foci of low-grade acinar adenocarcinoma (diameter, 0.05 cm). In the case 2, 6 tissue sections from the biopsy specimens showed iIDC-P, and 13 sections from RP specimen showed iIDC-P (diameter, 0.5-1.6 cm), and the other 3 sections had separate low grade acinar adenocarcinoma (diameter, 0.6 cm). In the case 3, 5 tissue blocks from the TURP specimen showed iIDC-P. The case 1 and 2 showed solid architecture with expansile proliferation of neoplastic cells in native ducts and acini. The case 3 showed dense or loose cribriform pattern, with marked cytological atypia, and frequent mitotic figures. Comedonecrosis was found in solid or dense cribriform glands in the case 2. Immunohistochemically, surrounding basal cells were highlighted using high-molecular-weight cytokeratin (34βE12 and CK5/6) and p63, while P504s was positive in the tumor cells. The tumor cells were also positive for AR and prostate markers (NKX3.1, PSA and PSAP), and negative for GATA3. The iIDC-P and acinar adenocarcinoma both showed weak PTEN expression and no ERG (nuclear) expression. In case 2 and 3, targeted sequencing revealed activated oncogenic driver mutations in MAPK and PI3K pathway genes (KRAS, MTOR and PTEN). In addition, pathogenic mutation in TP53 and FOXA1 mutation were found in the case 2 and 3, respectively. No case demonstrated TMPRSS2::ERG translocation. All cases were microsatellite stable and had lower tumor mutation burdens (range, 2.1-3.1 muts/Mb). The patients showed no biochemical recurrence or metastasis after follow-up of 16-91 months. Conclusions: iIDC-P is a special type of intraductal carcinoma of the prostate and differs from intraductal carcinoma within high-grade prostate cancer. iIDC-P has unique molecular characteristics and may represent as a molecularly unique in situ tumor of prostate cancer.

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.

Targeted next-generation sequencing panel to investigate antiplatelet adverse reactions in acute coronary syndrome patients undergoing percutaneous coronary intervention with stenting

Antiplatelet therapy, the gold standard of care for patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI), is one of the therapeutic approaches most associated with the development of adverse drug reactions (ADRs). Although numerous studies have shown that pharmacological intervention based on a limited number of high-evidence variants (primarily CYP2C19*2 and *3) can reduce the incidence of major adverse cardiovascular events (MACEs), ADRs still occur at variable rates (10.1 % in our case) despite personalized therapy.This study aimed to identify novel genetic variants associated with the endpoint of MACEs 12 months after PCI by designing and analyzing a targeted gene panel. We sequenced 244 ACS-PCI-stent patients (109 with event and 135 without event) and 99 controls without structural cardiovascular disease and performed an association analysis to search for unexpected genetic variants.No single nucleotide polymorphisms reached genomic significance after correction, but three novel variants, including ABCA1 (rs2472434), KLB (rs17618244), and ZNF335 (rs3827066), may play a role in MACEs in ACS patients. These genetic variants are involved in regulating high-density lipoprotein levels and cholesterol deposition, and as they are regulatory variants, they may affect the expression of nearby lipid metabolism-related genes. Our findings suggest new targets (both at the gene and pathway levels) that may increase susceptibility to MACEs, but further research is needed to clarify the role and impact of the identified variants before these findings can be incorporated into the therapeutic decision-making process.

Racial Disparities in Glioblastoma Genomic Alterations: A Comprehensive Analysis of a Multi-Institution Cohort of 2390 Patients

Although molecular biomarkers have significantly advanced precision oncology in glioblastoma, the prevalence of these biomarkers by race remains underexplored. This study aims to characterize the genomic alterations in glioblastoma across Asian, Black, and White patients, offering insights into racial disparities that may influence treatment outcomes and disease progression. Analyzing data from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange database V13.0, this study examined 2390 primary glioblastoma samples from unique patients. Genomic alterations in 566 cancer-related genes were assessed using targeted next-generation sequencing panels from 3 large cancer institutes. The patient cohort included 112 Asians, 67 Blacks, and 2211 Whites. Statistical significance of associations between genomic alterations and race was evaluated using the chi-squared test, with the Benjamini-Hochberg method applied to control for multiple testing adjustments. Significant racial differences were observed in the frequency of genomic alterations. Asians exhibited a higher frequency of TP53 alterations (52.68%, P < 0.001), Blacks showed more frequent alterations in NRAS (7.46%, P < 0.001), MTOR (10.45%, P= 0.039), and TET2 (8.96%, P=0.039), and Whites had a higher occurrence of PTEN alterations (48.67%, P= 0.045). Additionally, Black patientshad an elevated rate of RET deletions (14.29%, P<0.001). This study identifies significant racial disparities in the alteration frequencies of 6 key glioblastoma genes: NRAS, TP53, MTOR, TET2, PTEN, and RET. These findings underscore the need for racial considerations in glioblastoma treatment strategies and highlight potential avenues for targeted therapeutic interventions. Further research is needed to explore the clinical implications of these genomic disparities.

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 &lt;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 &gt;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.

Genomic profiling of non-small cell lung cancer with rare aberrations in EGFR codon L858 and the survival outcome under real-world first-line EGFR tyrosine kinase inhibitor treatment compared to classic EGFRL858R.

8596 Background: Rare genomic aberrations in EGFR codon L858 causing L858R (rare EGFRL858R) or non-L858R mutations have been reported, besides the predominant c.2573T&gt;G genomic mutation leading to classic EGFRL858R. However, molecular features, survival benefit under EGFR tyrosine kinase inhibitors (TKIs), and resistance mechanisms of these rare mutations lack comprehensive investigation in non-small cell lung cancer (NSCLC). Methods: We retrospectively included 489 NSCLCs with treatment-naive tumor tissues/plasma biopsies harboring rare EGFRL858R (N=124), EGFRL858Q/M (N=17), or classic EGFRL858R mutations (N=348). Mutational landscapes, tumor mutation burden (TMB), and chromosome instability (CIN) scores, were investigated using tissue samples subject to a targeted next-generation sequencing panel covering 437 cancer-related genes. Advanced NSCLCs receiving EGFR TKIs as first-line therapy were sub-grouped to study survival benefit and resistance mechanisms. Results: NSCLCs with rare EGFRL858R had less-frequently mutated TP53 (59% vs. 72%, P=0.04) and lower CIN scores (0.29 vs. 0.23, P=0.02) than those with classic EGFRL858R, while age, sex, stage at diagnosis, histology subtype, and TMB were comparable. We also detected the cooccurrence of EGFRL858Q/M with EGFRL861Q ( P&lt;0.01), particularly in patients carrying EGFRL858M (100%). Compared to 233 advanced NSCLCs harboring classic EGFRL858R, 58 patients with rare EGFRL858R exhibited superior first-line progression-free survival (PFS) [median: 13.0 vs. 10.0 months, hazard ratio (HR): 0. 57, 95% confidence interval (CI): 0.41–0.80], with a similar association observed in approximately 80% of included patients who received first-generation EGFR TKIs (median: 12.0 vs. 10.0 months, HR: 0.64, 95% CI: 0.45–0.92). After controlling for age, sex, histological subtype, EGFR TKI category, and anti-vascular therapy, rare EGFRL858R remained associated with improved PFS (adjusted HR: 0.55, 95% CI: 0.39–0.77). Patients with EGFRL858Q/M also had better PFS than those with classic EGFRL858R (adjusted HR: 0.26, 95% CI: 0.10–0.67). Of note, NSCLCs with combined EGFRL858M and EGFRL861Q appeared to have lower progression risks when receiving afatinib than using gefitinib/icotinib as first-line therapy. Furthermore, matched samples at baseline and progression revealed similar resistance mechanisms between NSCLCs with rare and classic EGFRL858R. Conclusions: NSCLCs carrying rare genomic aberrations in EGFR codon L858 were likely to get more survival benefits from first-line EGFR TKI treatment than those with classic EGFRL858R, giving scientific evidence supporting the clinical application of EGFR TKIs in these patients.

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. To assess the rate and pathogenicity of CHEK2 variants amongst individuals with pituitary adenomas (PAs). We assessed 165 individuals with PAs for CHEK2 variants. The study population comprised a primary cohort of 29 individuals who underwent germline and tumor whole-exome sequencing, and a second, independent cohort of 136 individuals who had a targeted next-generation sequencing panel performed on both germline and tumor DNA (n = 52) or germline DNA alone (n = 84). We identified rare, coding, nonsynonymous germline CHEK2 variants amongst 3 of 29 (10.3%) patients in our primary cohort, and in 5 of 165 (3.0%) patients overall, with affected patients having a range of PA types (prolactinoma, thyrotropinoma, somatotropinoma, and nonfunctioning PA). 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 = .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 PAs. As PAs are relatively common and typically lack classic autosomal dominant family histories, risk alleles-such as these variants found in CHEK2-might be a significant contributor to PA 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.