Search Results (980)

Background/Objectives: HER2 overexpression and/or amplification defines a molecularly distinct subset of endometrial carcinomas (ECs) that may benefit from HER2-targeted therapies. However, HER2 testing algorithms remain non-standardized and vary across institutions. This study is a large single-institution audit of EC HER2 testing practices, using both gynecologic (ISGyP) and gastric cancer-specific scoring algorithms at a major academic center with a reference gynecologic oncology service and biomarker laboratory. Methods: HER2 immunohistochemistry (IHC) and whole-slide fluorescence in situ hybridization (FISH) were interpreted by subspecialty breast and gynecologic pathologists, with HER2 IHC performed on 494 tumor samples (2021–2025) and reflex FISH for equivocal cases. Results: Using ISGyP criteria, 15.0% (74/494) of tumors were HER2 IHC 3+, 44.5% (220/494) equivocal (2+), and 40.5% (200/494) were negative (0/1+). Among equivocal cases, 28.2% (58/205) demonstrated ERBB2 amplification, yielding an overall HER2-positive rate of 27.5% (132/480). Re-assessment with gastric scoring criteria demonstrated variability in HER2 classification, with high concordance in cytology specimens (100%) and resections (90.8%; K = 0.842, p < 0.001) but substantially lower concordance in biopsies (60.6%; K = 0.401, p < 0.001), mainly due to reclassification of equivocal cases. Notably, 47.9% (n = 34) of ISGyP-equivocal biopsy specimens were reclassified as HER2 IHC 3+ using gastric biopsy criteria, potentially expanding eligibility for T-DXd therapy. Conclusions: These findings highlight the evolving nature of HER2 testing in EC and demonstrate the significant impact of scoring methodology on HER2 interpretation. Our results support the development of EC-specific HER2 testing guidelines and a dual-reporting approach incorporating both ISGyP and gastric scoring criteria, with selective confirmatory FISH testing, to optimize patient selection for HER2-targeted therapies. Full article

Objective: Our objectives were to describe molecular profiling in a real-life cohort of patients with radioiodine-resistant (RAI-R) differentiated or poorly differentiated thyroid cancer (DTC or PDTC) treated with lenvatinib and to focus on factors potentially influencing the quality of tissue samples for molecular analysis, including the impact of storage time, defined as the interval between tissue collection and molecular testing. Design: We retrospectively included all lenvatinib-treated RAI-R DTC or PDTC patients tested with DNA- and/or RNA-based next-generation sequencing (NGS) in our center, also analyzing the results of fluorescence in situ hybridization (FISH) for RET fusions if the sample did not satisfy quality criteria for RNA-based NGS analysis. We investigated differences in terms of histotype, biopsy site, or storage time between adequate and inadequate samples for RNA-based NGS. Results: At least one gene alteration was detected in 50% of the cohort (18 out of 36 patients); RAS and BRAF were the most frequent mutations, while gene fusions accounted for 5.6% of cases. Tissue samples were more frequently adequate for DNA-based NGS compared to RNA-NGS analysis (93.9% vs. 58.3%, p < 0.001). The median storage time was significantly longer in the case of inadequate samples for RNA-based NGS compared with adequate specimens (41.5 vs. 9.5 months, p = 0.016); samples archived for ≥3 years led more frequently to an inadequate result. Conclusions: Advanced RAI-R TC candidates for systemic therapy often harbor gene alterations. An adequate result was less frequently achieved in cases of RNA-based NGS than in DNA-based NGS, especially if the interval between tissue collection and molecular analysis was longer; nevertheless, the limited cohort size precludes definitive conclusions. Full article

Background/Objectives: High-grade B-cell lymphomas (HGBCLs) with MYC and BCL2 or BCL6 translocations, colloquially referred to as double-hit lymphomas (and abbreviated here to DHL), are aggressive malignancies. Differentiating DHL from non-DHL HGBCLs is important, as DHL patients may benefit from more intensive treatment regimes. We aimed to identify predictive clinicopathological, morphological, and immunophenotypic features that could guide selection of HGBCLs for fluorescence in situ hybridization (FISH), which is expensive and less accessible in some centers. Methods: We conducted a PRISMA systematic review and meta-analysis on 29 studies identified from four databases (PubMed (MEDLINE), Ovid (Embase), Web of Science, and Scopus). We calculated risk ratios (RRs) to compare features between DHL and non-DHL HGBCL and between MYC/BCL2 and MYC/BCL6 DHL patients. Results: DHL patients were associated with higher Ann Arbor stage (RR 1.15, p = 0.028, I² = 38.7%), International Prognostic Index (IPI) score (RR 1.27, p = 0.047, I² = 37.9%), elevated lactate dehydrogenase (RR 1.26, p = 0.012, I² = 34.0%), and germinal center B-cell-like (GCB) immunophenotype (RR 1.21, p = 0.043, I² = 35.8%) compared to non-DHL HGBCL patients. c-Myc immunopositivity, extranodal disease, and bone marrow involvement were more likely in DHL, albeit not reaching statistical significance. Extranodal disease (p = 0.015, I² = 0.0%), central nervous system involvement (p = 0.044, I² = 0.0%), and non-GCB immunophenotype (p = 0.016, I² = 71.1%) were more likely in MYC/BCL6 compared to MYC/BCL2 DHL patients. BCL2 immunopositivity, CD10 immunopositivity, and MUM1 immunonegativity were more likely in MYC/BCL2 DHL, although the differences were not statistically significant. Conclusions: Our results have associated DHL with features of aggressive disease and found GCB immunophenotype as a histopathological feature with statistically significant predictive value for MYC/BCL2 DHL. Heterogeneity within the non-DHL HGBCL group and variation in immunohistochemical cut-off values between studies limited identification of other predictive features. Larger, consistently designed, prospective cohort studies could provide further evidence for a screening strategy for DHL. Full article

Contemporary iterations of avian phylogenies based on multiple genome sequence assemblies assign three major clades: Palaeognathae (mostly ratite birds), Galloanseres (land and waterfowl) and the largest group—Neoaves. The latter two are sister clades representing subdivisions of Neognathae, while Neoaves further subdivide into Columbaves (pigeons/doves/cuckoos/bustards, etc.), Mirandornithes (flamingos/grebes), Telluraves (“higher land birds”, including finches) and the newly recognized Elementaves (e.g., penguins/pelicans/hummingbirds/swifts/cranes/shorebirds). Molecular studies provide clade information, likely divergence timings and a framework from which gross genomic (chromosomal) changes may be mapped. In this review, we consider the patterns of chromosome change that have occurred throughout all avian clades thus far examined, citing studies from standard karyotyping through molecular cytogenetics to whole genome assemblies. Standard karyotyping led to the realization that most chromosomes (particularly the microchromosomes and dot chromosomes) could not be distinguished by classical means. Indeed, cross-species comparisons were difficult, even among the macrochromosomes, because of indistinct banding patterns. Based on fluorescence (or fluorescent) in situ hybridization (FISH), comparative genomics was thence progressed considerably by cross-species chromosome painting (Zoo-FISH) for the macrochromosomes and interspecific mapping of bacterial artificial chromosome (BAC) probes for the microchromosomes. A key finding was that the most studied species, the chicken, fortuitously, has a genomic organization somewhat akin to that of the ancestral karyotype and tends to be the standard from which all others are measured. A notable exception is the fusion of basal chromosome 4 with a smaller chromosome that convergently appears in some other Galliformes, at least one goose and one dove species. While some groups such as Falconiformes (falcons, etc.) and Psittaciformes (parrots, etc.) underwent extensive interchromosomal change, most, broadly speaking, retain a basic karyotype that differs little from bird to bird. Many, e.g., Passeriformes (finches, songbirds, etc.) and Columbiformes (pigeons, doves), do this despite multiple intrachromosomal rearrangements. The complete karyotype and fully established chromosome-level genome assembly of the chicken allow full integration of DNA sequence assembly with karyotype. They further permit cytogenetic studies to be performed using genome assemblies alone alongside cutting-edge long-read sequencing and optical mapping without the need for chromosome preparation. The classic ZW sex-determination system of birds is easily visible in most Neognathae species, but intrachromosomal change in the sex chromosomes is faster than in the autosomes; indeed, there are numerous examples of autosomal fusions and new sex chromosomes formed. Sex chromosomes aside, the classic avian karyotype represents a very successful mode of genome organization established before the emergence of the dinosaurs and perpetuated to this day in their only living descendants. Full article

Imaging-based spatial transcriptomics has advanced from low-plex single-molecule fluorescence in situ hybridization to a diverse set of highly multiplexed platforms, with recent multimodal and pathology-compatible capabilities. Despite major differences in chemistry, coding, and imaging strategies across different platforms, their biological interpretation often converges on a few notable computational biology problems. This review examines imaging-based spatial transcriptomics through the lens of data interpretation and applications, focusing on the analytical framework that converts raw fluorescence signals or accompanying in situ sequencing data into molecule-, cell-, and tissue-level representations. We discuss the key challenges in preprocessing, registration, restoration, feature detection, barcode decoding, molecule calling, cell segmentation, transcript assignment, probabilistic cell typing, spatial-domain inference, and atlas integration. We also highlight how optical crowding, tissue thickness, panel bias, and multimodal complexity increase computational difficulty. Finally, we summarize applications of imaging-based spatial transcriptomics techniques, ranging from subcellular RNA localization to atlas-scale and pathology-aware spatial analysis. Full article

Background and Objectives: Comprehensive real-world data on dose-adjusted EPOCH-R (DA-EPOCH-R) incorporating molecular prognostic stratification remain limited. We evaluated the long-term efficacy, safety, and prognostic determinants of DA-EPOCH-R in a multicenter Turkish cohort. Materials and Methods: This retrospective study included 140 patients with aggressive B-cell lymphoma (diffuse large B-cell lymphoma [DLBCL], n = 81; primary mediastinal B-cell lymphoma [PMBL], n = 39; other, n = 20) treated with DA-EPOCH-R at five academic centers (2015–2020). Molecular profiling included immunohistochemistry (MYC, BCL-2, BCL-6) and fluorescence in situ hybridization (FISH). Survival was estimated by Kaplan–Meier analysis with Cox regression for prognostic factors. Results: At a median follow-up of 50.1 months, 5-year overall survival (OS) and event-free survival (EFS) rates were 71.3% and 66.3%, respectively (complete response rate: 68.6%). Molecular subtypes included double-expressor (DEL; n = 39), triple-expressor (TEL; n = 21), double-hit (DHL; n = 17), and triple-hit lymphoma (THL; n = 11). Five-year OS by IPI risk group ranged from 88.6% (low) to 49.4% (high) (p = 0.005). DEL status did not confer inferior OS (p = 0.738), whereas DHL and THL had markedly poor outcomes (p < 0.001). In multivariate analysis, IPI ≥ 3 (HR 2.54; p = 0.007) and MYC FISH rearrangement (HR 3.62; p < 0.001) independently predicted inferior OS. Grade 3–4 neutropenia occurred in 57.1%, with no grade 3–4 cardiotoxicity. Conclusions: DA-EPOCH-R provides favorable long-term outcomes in aggressive B-cell lymphomas. DEL status did not confer a survival disadvantage, an association that is hypothesis-generating and requires confirmation, as the present design cannot establish a causal mechanism. FISH-defined DHL/THL remain associated with dismal outcomes, warranting novel therapeutic strategies. Full article

Human epidermal growth factor receptor 2 (HER2) remains the most recognized and clinically established molecular biomarker in gastric cancer; however, the regulatory mechanisms underlying its dysregulation are not fully understood. This study aimed to identify microRNAs associated with HER2 gene amplification, chromosome 17 centromere copy number increase (CNI), or alternative mechanisms driving HER2 protein overexpression. We analyzed 115 gastric cancer patients treated surgically at a single institution, with available material for immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and microRNA profiling. Among 11 candidate microRNAs, four demonstrated significant associations with HER2-related alterations. hsa-miR-128-3p expression was positively associated with HER2 gene amplification, while hsa-miR-145-5p expression showed an inverse relationship with centromere enumeration probe 17 (CEP17) signal count and correlated with membranous HER2 protein expression. hsa-miR-27b-5p expression was linked to CEP17 CNI, whereas hsa-miR-552-3p expression was associated with both increased HER2 amplification and CEP17 signal count. Importantly, hsa-miR-27b-5p upregulation independently predicted worse overall survival, whereas hsa-miR-128-3p upregulation independently predicted improved survival outcomes. These findings identify distinct microRNA signatures associated with HER2 pathway alterations and prognosis in gastric cancer, highlighting their potential as biomarkers and contributors to HER2-driven tumor biology. Full article

Plant genetic resources are vital for crop improvement, ecological resilience, and agrobiodiversity conservation, making their characterization through morphological and cytogenetic methods essential for breeding and germplasm management. This study comparatively analyzed two herbaceous cultivars Hibiscus moscheutos cv. ‘Carousel Jolly Heart’ and cv. ‘Carousel Pink Passion’ and two woody cultivars, Hibiscus syriacus cv. ‘Sukim’ and cv. ‘Freedom’, to assess interspecific diversity and hybridization potential. Morphological assessments revealed notable differences in flower size and leaf shape between species, with ‘Carousel Pink Passion’ exhibiting the largest flower diameter (16.70 cm) and ‘Freedom’ exhibiting the smallest (10.20 cm). Chromosome analysis confirmed diploidy (2n = 38) in H. moscheutos and polyploidy (2n = 84) in H. syriacus, highlighting a fundamental genomic distinction between the two species. Fluorescence in situ hybridization (FISH) consistently identified two 5S rDNA loci across all cultivars; however, species-specific variation in 18S rDNA loci was detected with four loci in H. syriacus and six in H. moscheutos, suggesting divergent rDNA evolution and distinct genomic organization in the two species. Flow cytometry confirmed significant differences in nuclear DNA content corresponding to ploidy levels: ‘Carousel Jolly Heart’ and ‘Carousel Pink Passion’ measured 2.06 pg and 2.05 pg, respectively, while ‘Sukim’ and ‘Freedom’ measured 4.18 pg and 4.27 pg, respectively. Full article

IDH-wildtype glioblastoma remains the most aggressive primary brain tumor, with a median overall survival (OS) of 14–16 months despite maximal treatment. A small subset of patients, however, survive beyond 30 months, suggesting distinct underlying biological features. The aim of this pilot study was to explore whether selected molecular alterations detectable by FISH show differing distribution patterns between patients with prolonged and poor survival in IDH-wildtype glioblastoma. We retrospectively analyzed 20 patients with newly diagnosed primary IDH-wildtype glioblastoma who underwent gross-total resection followed by standard radiotherapy and temozolomide treatment between 2016 and 2022. Patients were categorized into two predefined groups according to survival outcomes: long-term survivors (OS > 30 months) and short-term survivors (OS < 10 months). Fluorescence in situ hybridization (FISH) was used to evaluate alterations in ATRX, BRAF, and PIK3CA. MGMT promoter methylation, EGFR amplification, and TERT promoter mutation status were obtained from routine diagnostic reports. Because survival groups were intentionally pre-selected as extreme phenotypes, time-to-event analysis was not appropriate. Therefore, statistical comparisons were performed using Fisher’s exact test and multivariable logistic regression with long-term versus short-term survival as a binary outcome. Short-term survivors had a significantly higher median age (57.5 vs. 46.5 years, p = 0.043) and a higher rate of EGFR amplification (100% vs. 50%, p = 0.033). Strikingly, combined BRAF and PIK3CA alterations (predominantly polysomy) were detected in 8 out of 10 (80%) long-term survivors, compared to 0 out of 10 (0%) short-term survivors (p = 0.0007). In multivariable logistic regression adjusted for age and MGMT promoter methylation, BRAF/PIK3CA alteration remained strongly associated with long-term survival, though the effect size was mathematically inflated due to perfect separation (0 events in Group B). BRAF and PIK3CA copy number alterations were observed exclusively in long-term survivors in this small exploratory cohort, suggesting a possible association with prolonged survival. However, given the limited sample size, the selection of extreme survival groups, and the predominance of chromosomal polysomy detected by FISH, these findings should be interpreted as hypothesis-generating only. Further validation in larger cohorts using high-resolution genomic methods is warranted. Full article

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy in which prognosis is strongly influenced by cytogenetic abnormalities. Recent updates from the International Myeloma Working Group (IMWG), along with the European Hematology Association (EHA) and European Myeloma Network (EMN), have refined the definition of high-risk (HR) disease by integrating TP53 alterations, chromosome 1 abnormalities, and specific combinations of cytogenetic lesions. However, validation of these criteria in real-world patient populations remains limited. We conducted a retrospective, single-center study including 738 patients diagnosed with MM between 2017 and 2025, of whom 408 had available fluorescence in situ hybridization (FISH) data at diagnosis. Patients were reclassified according to the latest IMWG/IMS high-risk criteria proposed in international literature. Cytogenetic abnormalities, treatment patterns, and clinical outcomes, including overall survival (OS), progression-free survival (PFS), response rates, and relapse, were analyzed. Survival was estimated using the Kaplan–Meier method. A total of 103 patients (25%) were reclassified as high-risk according to IMWG/IMS high-risk criteria. Cytogenetic HR abnormalities were identified in 17.2% of cases, with del(17p) being the most frequent (14.7%). Median OS and PFS in HR patients were 52.4 months and 16 months, respectively, compared with 68.4 months and 28 months in standard-risk patients (log-rank test p values of 0.0197 and 0.0004, respectively). Although overall response rates were high (83% in HR vs. 91% in standard-risk), relapse remained frequent in HR patients. Outcomes varied significantly according to cytogenetic complexity. Isolated del(17p) was associated with improved survival compared with cases harboring additional abnormalities, while double-hit and triple-hit profiles demonstrated inferior outcomes. The presence of chromosome 1 abnormalities, particularly in combination with IGH translocations, further worsened prognosis. Among HR patients, 44% underwent autologous stem cell transplantation (ASCT), including 10 cases of TANDEM ASCT. No survival benefit was observed for TANDEM compared with single ASCT, with median OS of 52.9 vs. 78.3 months, respectively (log-rank test p values of 0.2516). Our real-world analysis supports the prognostic relevance of the updated IMS/IMWG high-risk criteria in MM. Cytogenetic complexity, rather than individual abnormalities alone, is a key determinant of outcome. Despite high response rates achieved with modern therapies, survival remains inferior in HR patients. TANDEM ASCT did not confer additional benefit in this cohort, supporting a more individualized approach to treatment intensification. Full article

Self-grooming is a fundamental and evolutionarily conserved behavior and is essential for removing Varroa destructor in honeybees (Apis mellifera). However, its molecular and neural regulation remains poorly understood. This study identifies a microRNA-mediated neuromodulatory pathway that governs the intensity of self-grooming behavior in A. mellifera. Comparative behavioral analyses revealed pronounced differences in grooming intensity between bees exhibiting strong (MS) and weak (MW) grooming phenotypes. Liquid chromatography detection and pharmacological experiments demonstrated that octopamine (OA) levels are significantly different between MS and MW bees, and OA enhances grooming behavior. Small RNA sequencing revealed differentially expressed miRNAs in the brains of MS versus MW bees, leading to the identification of miR-281-x as a candidate associated with OA regulation. Functional validation showed that overexpression of miR-281-x significantly reduces grooming behavior, whereas inhibition of miR-281-x enhances grooming. Mechanistically, bioinformatics prediction and experimental validation confirmed that miR-281-x directly targets the tdc2, which encodes tyrosine decarboxylase 2 in OA biosynthesis. Fluorescence in situ hybridization (FISH) revealed co-localization of miR-281-x and tdc2 in Kenyon cells of the mushroom bodies. RNAi-mediated knockdown of tdc2 significantly reduced grooming intensity and rescue experiments confirmed the miR-281-x–tdc2–OA regulatory axis. Together, our findings uncover a post-transcriptional modulatory mechanism linking a microRNA to neuromodulator-dependent behavioral plasticity, providing insight into the control of grooming intensity in honeybees. Full article

Background/Objectives: Atypical teratoid/rhabdoid tumor (AT/RT) is a rare and aggressive pediatric embryonal tumor of the central nervous system with marked histological and immunophenotypic heterogeneity, which can make diagnosis difficult in some cases. This study aimed to summarize the clinicopathological and molecular features of pediatric AT/RT and to evaluate an HE–IHC dual-path deep learning model as an auxiliary diagnostic approach. Methods: Clinical, histopathological, immunophenotypic, ultrastructural, and fluorescence in situ hybridization (FISH) data were retrospectively collected from 18 children with AT/RT treated at Beijing Children’s Hospital between February 2010 and April 2021. A total of 361 pathological images were used to train and test a ResNet50-based dual-path classification model with transfer learning and feature fusion. An additional independent test set of 175 histological and immunohistochemical images from six newly collected patients was used for supplementary validation. Results: The mean age at diagnosis was 2 years and 3 months. All cases showed loss of INI1 expression, positivity for CK and EMA, and a high Ki-67 index. FISH analysis identified SMARCB1 deletion in 7 of 15 tested cases. In the original image-based test set, the dual-path model achieved an accuracy of 90.91%, compared with 81.82% for the model without transfer learning, 86.36% for the single-path immunohistochemistry model, and 50.00% for the single-path histological model. In the additional independent test set, the trained model correctly classified all 175 images. Conclusions: Pediatric AT/RT shows diverse clinicopathological features and complex SMARCB1 alteration patterns. The HE–IHC dual-path model showed encouraging preliminary performance for auxiliary pathological assessment; however, larger multicenter cohorts with molecular subgroup annotation are needed for further validation before routine clinical application. Full article

Background: Leukemic non-nodal variant mantle cell lymphoma (nnMCL) is an uncommon subtype of mantle cell lymphoma that lacks lymphadenopathy and generally follows an indolent clinical course. Adverse genetic alterations such as TP53 inactivation and del(13q) may have prognostic significance. Clinical findings such as splenomegaly may serve as a clue to the diagnosis and should prompt further evaluation. Case Presentation: We describe a 91-year-old woman who presented with a one-month history of anemia (hemoglobin 12.3 g/dL), mild thrombocytopenia (platelets 136 × 10⁹/L), isolated splenomegaly and no palpable lymphadenopathy. Despite a normal total white blood cell count, intermittent relative lymphocytosis with atypical lymphocytes (4%) and smudge cells was detected on the complete blood count. Peripheral blood flow cytometry demonstrated a monoclonal kappa-restricted B-cell population negative for CD5 and CD10, comprising approximately 20% of lymphocytes. Conventional karyotyping and fluorescent in situ hybridization (FISH) analysis identified del(13q), del(17p)/TP53, and IGH-CCND1 rearrangement in 8–19.5% of peripheral blood leukocytes. A month after the initial assessment, the patient presented following a fall. CT imaging of the abdomen revealed marked splenomegaly, a large subcapsular/perisplenic hematoma, and moderate-to-large hemoperitoneum. Emergent laparotomy showed an enlarged spleen (1490 g, 23 × 16 × 7.5 cm) with laceration. Histologic evaluation showed atypical lymphoid cells positive for CD20 and cyclin D1, with strong p53 expression, negative for CD5 and SOX11, and a low Ki-67 index. Similar involvement was identified in the small bowel and appendix. Targeted sequencing of splenic tissue, performed as part of a retrospective molecular characterization, identified a pathogenic TP53 variant (p.His179Gln). Conclusions: This case provides a rare opportunity to evaluate splenic and small intestinal involvement by nnMCL at both the gross and histologic levels. It highlights the importance of integrating clinical findings with flow cytometry, imaging, cytogenetic, and molecular data in establishing the diagnosis. Even when peripheral blood findings suggest a low disease burden, imaging may better define the extent of disease and support appropriate clinical assessment, particularly in elderly patients at risk for complications related to splenomegaly. Full article

Background: Evaluation of gene-level copy number variants (CNVs) for diagnosis and therapeutic decision making has become standard of care with next-generation sequencing (NGS), immunohistochemistry (IHC), and/or fluorescence in situ hybridization (FISH) being used to detect gene amplifications/deletions in tumor tissue. In contrast to most solid tumors, CNS cancers are challenging to evaluate by resection and/or biopsy due to the associated risks with invasive brain surgery that can also result in death or associated morbidity and therefore alternate methods are required.Methods: This study presents the analytical validation of using quantitative PCR (qPCR) to detect gene CNVs directly from cerebrospinal fluid (CSF)-derived DNA and from the Ascent^TM low-pass whole genome sequencing (LP-WGS) libraries, demonstrating concordance with the gold standard of NGS/IHC/FISH used in tumor tissue. Results: The analytical sensitivity of qPCR to detect gene amplification calls for ERBB2 (erb-b2 receptor tyrosine kinase 2) was demonstrated to be 100% and that of EGFR (epidermal growth factor receptor) was 83%, with specificities of 96% and 100%, respectively. The analytical sensitivity of qPCR to detect gene deletions for CDKN2A/2B (cyclin-dependent kinase inhibitor 2A/2B) was 60% and that for MTAP (methylthioadenosine phosphorylase) was 100% with a specificity of 100% for all three genes. Ascent^TM was demonstrated to have a higher sensitivity (100%) when compared to qPCR for the same genes evaluated and demonstrated 100% positive agreement and 100% negative agreement with known CNV status. Conclusions: The results demonstrate that given the paucity of cells in CSF limiting the use of IHC and FISH, qPCR and Ascent^TM provide highly sensitive, novel, minimally invasive methods for the evaluation of gene copy number (CN) status to inform the diagnosis and management of CNS cancers. Full article

Background/Objectives: Genomic alterations play a central role in diagnosis, prognostication, and therapeutic planning for hematolymphoid malignancies. At our tertiary care center, frontline genomic testing relies on optical genome mapping, karyotyping, and fluorescence in situ hybridization, with targeted next-generation sequencing (NGS) performed externally. To provide more comprehensive genomic profiling, we evaluated two large-panel NGS platforms and subsequently performed a clinical validation of the selected assay. Methods: The Illumina PanHeme DNA panel and the SOPHiA Genetics Community Myeloid Solution were compared using 24 bone marrow aspirate specimens with previously characterized alterations, including single nucleotide variants (SNVs), insertions/deletions (indels), and copy number variants (CNVs). The selected panel underwent full analytical validation using 60 specimens. Results: Both panels demonstrated excellent concordance for SNVs and indels, with comparable analytical performance and workflow. CNV calling with SOPHiA was notably strong. Platform selection was influenced by practical considerations, including panel content and cost, leading to a preference for further evaluation of the Illumina assay. Clinical validation of the Illumina PanHeme DNA panel, along with a complementary RNA Exome panel, was subsequently performed. Sequence variant detection showed 100% concordance with orthogonal testing, while CNV detection was variable, reflecting known limitations of targeted NGS. The RNA panel detected all expected fusion transcripts. Conclusions: These findings demonstrate robust analytical performance of both evaluated DNA panels. Clinical validation of the Illumina PanHeme DNA and RNA Exome assays supports their use for comprehensive molecular profiling of hematologic malignancies. Full article