Search Results (121)

Detecting a KIT D816V mutation in patients with systemic mastocytosis (SM) needs a high-sensitivity assay. Additionally, high mutant KIT transcriptional activity and multilineage involvement are strong unfavorable prognostic parameters in SM. The current frequently used quantitative PCR-based KIT D816V assays have important analytical limitations, including considerable analytical variation in the low-concentration range, a need for a calibration curve, less multiplexing capabilities, and higher susceptibility to interference by PCR inhibitors. We developed, validated, and explored the diagnostic relevance of a novel multiplex KIT D816V cDNA droplet digital PCR (ddPCR), using ABL1 and KIT as reference genes. Furthermore, we correlated the expressed allele burden (EAB) with variant allele frequency (VAF) and percentage of neoplastic mast cells (nMC%) in bone marrow samples of 79 SM patients. Our assay shows very low background signals and high assay precision, leading to a linear dynamic range spanning five orders of magnitude and very low limits of detection and quantification of 0.062% (EAB/ABL1) and 0.027% (EAB/KIT). The assay detects median 57 times more mutant cDNA copies than mutant gDNA copies in SM patients and positivity in 83% of SM-suspected patients with negative gDNA ddPCR results. Furthermore, correlations between EAB, VAF, and nMC% suggest the presence of KIT D816V in non-MCs, either with or without transcriptional activity. Using our novel high-sensitivity mRNA-based multiplex ddPCR KIT D816V assay returns high diagnostic yield in patients with suspicion for SM. We recommend incorporating our assay in routine SM diagnostics to determine low-burden KIT D816V. Full article

Background/Objectives: Beta-thalassemia (β-thal) is an inherited hemoglobin disorder caused by mutations in the HBB gene. Hb Malay (HBB:c.59A>G [NM_000518.5], p.Asn20Ser, CD19), a missense substitution (AAC→AGC; Asn→Ser) in exon 1 of the β-globin gene causing β⁺-thalassemia, has been mainly described in Southeast Asian populations but remains underreported in published Indonesian molecular epidemiology literature. This study aimed to identify the Hb Malay variant and investigate its familial segregation in a large Indonesian extended family. Methods: Cascade molecular screening was initiated following detection of the mutation in a proband referred for genetic testing. Sixteen individuals (one proband and 15 family members) consented to participate. Genomic DNA was extracted from peripheral blood and a 1.9-kb β-globin gene fragment was amplified by PCR. Mutation screening was performed using the Thalassemia GenoArray Kit HBGA-THAL-b31 (Hybribio) and confirmed by Sanger sequencing. A three-generation pedigree was constructed from molecular results and family interviews. Results: The Hb Malay mutation (HBB:c.59A>G) was identified in 13 of 16 individuals: 12 heterozygous carriers (A/G) and one homozygous individual (G/G, subject III-1, aged 15 years). Three individuals showed the normal genotype (A/A). Pedigree analysis demonstrated autosomal recessive inheritance across three generations. No α-globin deletions covered by the GenoArray panel were detected. Conclusions: To our knowledge, this is one of the few documented reports of cascade molecular screening for the Hb Malay (HBB:c.59A>G) variant in a large extended Indonesian family, confirmed by dual molecular methods. These findings illustrate the potential value of cascade molecular screening for early carrier identification in settings where hemoglobin fractionation testing is unavailable, and highlight the need for population-based epidemiological studies to guide thalassemia prevention policy in Indonesia. Full article

Head and neck mucosal melanoma (HNMM) arises in the nasal and oral cavities and has the propensity to metastasize to local and distant body sites. HNMM is also notable for its resistance to available therapeutics. The rarity of this disease makes it difficult to conduct large-scale clinical studies to develop standard treatment protocols. In contrast to cutaneous melanoma, c-Kit-dependent pathways are well studied in HNNMM and provide a potential therapeutic target. We identified and isolated genetically distinct subpopulations with stem cell characteristics in HNMM samples bearing Kit wild-type and mutations. Functional analysis of these subpopulations reveals that, in addition to expressing the stem cell marker proteins CD20, CD117, CD133, and CD166, these subpopulations are characterized by self-renewal potential, migratory capacity, and resistance to Kit inhibitors such as Imatinib. Immunofluorescence staining and inhibition experiments demonstrate that the maintenance and resistance of HHMM subpopulations to Kit inhibitors is mediated by the Kit signal to the PI3K signaling pathway. The KIT signal to the PI3K signaling pathway does not result exclusively from a KIT mutation localized to Exon 17, but can also be triggered by mutations localized to Exons 11 and 13. In the present study, we identify and characterize an HNMM subpopulation with stemness properties in patients with c-Kit wild-type and mutation, and demonstrate for the first time the mechanisms by which the CD117⁺/CD133⁺ HNMM subpopulations survive and confer resistance to the specific inhibitor of c-Kit mutation. Full article

Introduction: Pathogenic mutations in leucine-rich repeat protein kinase-2 (LRRK2), particularly G2019S, constitute the most common cause of autosomal dominant PD. Methods: Mouse models encoding human mutant alpha-synuclein (SNCA A53T) and LRRK2 G2019S were treated with a brain-penetrant kinase inhibitor (BK40196). Behavior, nigrostriatal and mesolimbic dopamine (DA) pathways were examined. Results: Mice harboring LRRK2 G2019S do not show age-dependent motor symptoms, but mice encoding SNCA A53T display motor deficits, while both strains exhibit anxiety-like behavior and BK40196 improves motor and behavioral defects. BK40196, a multi-kinase inhibitor of Abelson (Abl), Discoidin domain receptor (DDR)-1, c-KIT and FYN, alters microglial morphology and alpha-synuclein levels in SNCA A53T mice and improves DA neurotransmission, primarily via the nigrostriatal system. BK40196 inhibits brain LRRK2 G2019S (IC₅₀ of 89nM) and does not affect phosphorylated or total peripheral LRRK2 levels (lungs, kidneys, liver, etc.). LRRK2 G2019S mice treated with BK40196 exhibit distinct increases in DA in mesolimbic neurons such as the nucleus accumbens (NAcc), suggesting differential mechanisms of DA neurotransmission in mutant alpha-synuclein and LRRK2 models of PD. Conclusions: LRRK2 G2019S may primarily involve mesolimbic pathways leading to nonmotor symptoms independent of the motor and behavioral manifestations associated with alpha-synuclein via the nigrostriatal system. BK40196 may provide a comprehensive and synergistic therapeutic approach that addresses multiple mechanisms to reduce the pathologies related to LRRK2 G2019S and/or SNCA in PD. The multiple pathologies of PD necessitate a holistic approach that simultaneously targets inflammation and autophagy and LRRK2 inhibition. Full article

This case highlights a novel genotype–phenotype correlation in the field of endocrinology. Specific endocrine and imaging assessment, in addition to next-generation sequencing (NGS), was performed on the Illumina MiSeq platform, using a TruSight One Sequencing Panel kit for genomic analysis of coding regions of 4813 genes. A 54-year-old female was confirmed with a papillary thyroid carcinoma after total thyroidectomy and underwent radioiodine ablative therapy. Three years later, a left femoral enchondroma of almost 3 cm was identified at computed tomography (CT) scan and magnetic resonance imaging (MRI). She experienced hypertension (in addition to obesity, dyslipidaemia and impaired glucose tolerance) and was later confirmed with ACTH-independent cortisol excess [lack of cortisol suppression at 1 mg dexamethasone testing of 13.9 (normal < 1.8 µg/dL)], noting bilateral adrenal tumors, of 4.7 cm (right), respectively, and of 1.6 cm (left) at CT. Right laparoscopic adrenalectomy was performed with post-operative adrenal insufficiency, requiring glucocorticoid replacement and stopping the anti-hypertensive medication. Pathology report confirmed an adrenocortical adenoma (a Ki67 proliferation index of 2%). Noting the unusual association of the mentioned conditions, NGS was performed in the peripheral blood and identified a heterozygote missense variant of the APC gene (c.5759G>A, p.Arg1920Gln), a heterozygote missense variant of the MSH6 gene (c.2092C>G, p.Gln698Glu), and an incidental additional finding: a heterozygote stop gain pathogenic variant of the CACNA1S gene (c.2707C>T, p.Arg903*). The first two are currently classified as variants of uncertain significance. Whether the co-presence of a triple mutation may change the clinical picture and the life-long outcomes across reciprocal influence is still an open matter. Further research will point out the clinical implications of this genotype–phenotype association, which, to our best knowledge, has not been previously reported. Full article

Telomerase reverse transcriptase (TERT) plays a key role in tumorigenesis by maintaining telomere length, promoting chromosomal stability, and enabling cells to evade replicative senescence. TERT promoter mutations have been detected in various types of tumor; however, their prevalence in ameloblastoma has not been verified. This study aimed to determine the frequency of TERT promoter mutations in ameloblastoma. This retrospective study included formalin-fixed, paraffin-embedded (FFPE) tissue specimens and corresponding medical records from patients who underwent surgical treatment for jaw ameloblastoma at the Department of Oral and Maxillofacial Surgery, Kyungpook National University (Daegu, Republic of Korea) between January 2011 and December 2024. Clinical data were reviewed through January 2026. Of the 49 patients included, genomic DNA was extracted from two 5 μm thick FFPE tissue sections using the PANAMAX™ FFPE Plus DNA Extraction Kit (HLB PANAGENE, Daejeon, Republic of Korea), according to the manufacturer’s instructions. Hotspot TERT promoter mutations (C228T and C250T) were analyzed using the PNAClamp™ TERT Mutation Detection Kit (HLB PANAGENE, Daejeon, Republic of Korea). From a total of 73 TERT promoter mutation analyses performed in 49 patients, one of the recurrent cases harbored both C228T and C250T hotspot mutations. In the non-recurrent group, one case exhibited a C250T mutation. These findings indicate that TERT promoter mutations are rare in ameloblastoma. Full article

Despite being a commensal bacterium, Cutibacterium acnes has been widely considered a major opportunistic pathogen due to its capacity for biofilm production and inflammatory induction, causing device-related, post-implant infections, and skin inflammatory diseases. In this study, we isolated and characterised the novel bacteriophage Cutibacterium acnes phage KIT09 as a potential antimicrobial candidate for the treatment of Cutibacterium acnes-related infections such as acne vulgaris and postsurgical infections. Subsequently, phage-resistant bacterial mutants were generated through phage KIT09 exposure and characterised. Wastewater samples were collected for the isolation of C. acnes phages, followed by their characterisation using C. acnes National Institute of Technology and Evaluation (NITE) Biological Resources Center (NBRC) 107605 (phylotype IA1). Resistant mutants were isolated after prolonged exposure of the newly isolated phage to host bacteria and then characterised. A novel C. acnes phage, designated KIT09, was isolated, demonstrating prolonged bacteriolysis lasting up to 96 h at a multiplicity of infection of 10, and exhibiting high thermal and pH stability. Following sustained selective pressure by phage KIT09, three phage-resistant bacterial isolates were obtained, forming smaller colonies than the wild-type strain, but maintaining a high phage adsorption capacity (>90% after 20 min). Whole-genome sequencing revealed 12 nucleotide mutations across five genes, including six non-synonymous substitutions. Three genes encoding a two-component histidine kinase, DNA processing protein A (DprA), and a ThuA-containing domain protein were mutated in all resistant isolates. Characterisation of the novel phage KIT09 demonstrated its robust lytic activity and environmental stability against C. acnes phylotype IA1. Isolated resistant mutants retained high phage adsorption, accompanied by recurrent mutations in genes encoding a two-component histidine kinase, DprA, and a ThuA-domain protein, suggesting the presence of alternative, CRISPR-Cas–independent resistance mechanisms in C. acnes. Full article

Background and Clinical Significance: Tyrosine kinase inhibitors (TKIs) have transformed Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) into a largely manageable chronic disease. However, off-target toxicities are increasingly recognized; rarer complications such as bone marrow edema (BME) remain underreported. BME is a radiological syndrome characterized by excess intramedullary fluid on fat-suppressed T2/STIR magnetic resonance imaging sequences and may progress to irreversible osteochondral damage if unrecognized. We report a case series of TKI-associated BME and propose a practical diagnostic-therapeutic framework. Case Presentation: We describe three patients with Ph+ CML who developed acute, MRI-confirmed BME of the lower limb during TKI therapy. Case 1 developed unilateral then bilateral knee BME, temporally associated first with dasatinib and subsequently with imatinib; symptoms improved after TKI interruption, bisphosphonate therapy, and supportive measures, and did not recur after switching to bosutinib. Case 2 presented with proximal femoral BME during long-term imatinib; imatinib was stopped, intravenous neridronate administered, and bosutinib initiated with clinical recovery and later near-complete radiological resolution. Case 3 experienced multifocal foot and ankle BME during imatinib; symptoms resolved after drug discontinuation and bisphosphonate therapy, and disease control was re-established with bosutinib without recurrence of BME. All patients underwent molecular monitoring and mutational analysis to guide safe therapeutic switching. Discussion: Temporal association across cases and the differential kinase profiles of implicated drugs suggest PDGFR (and to a lesser extent, c-KIT) inhibition as a plausible mechanistic driver of TKI-associated BME. PDGFR-β blockade may impair pericyte-mediated microvascular integrity, increase interstitial fluid extravasation, and alter osteoblast/osteoclast coupling, promoting intramedullary edema. Management combining MRI confirmation, temporary TKI suspension, bone-directed therapy (bisphosphonates, vitamin D/calcium), symptomatic care, and, when required, therapeutic switching to a PDGFR-sparing agent (bosutinib) led to clinical recovery and preservation of leukemia control in our series. Conclusions: BME is an underrecognized, potentially disabling, TKI-related adverse event in CML. Prompt recognition with targeted MRI and a multidisciplinary, stepwise approach that includes temporary TKI adjustment, bone-directed therapy, and consideration of PDGFR-sparing alternatives can mitigate morbidity while maintaining disease control. Prospective studies are needed to define incidence, risk factors, optimal prevention, and management strategies. Full article

Melanoma is a highly heterogeneous disease, with unique genetic subtypes that influence clinical behavior and treatment response. While targeted therapies and immunotherapy have transformed care for more common types of melanoma, optimal strategies for KIT-mutant melanoma are less well-defined. In this review, we summarize the associations between KIT mutations and specific clinico-pathologic patterns, including their enrichment in acral, mucosal, and chronically sun-damaged melanomas. We detail the spectrum of KIT mutations across relevant exons, noting how mutation subtype influences sensitivity to targeted therapies. We will then analyze several therapeutic trials and case reports that describe the use of c-KIT inhibitors as well as immune checkpoint inhibitors in melanoma treatment. We also discuss early findings supporting combination strategies that may enhance therapeutic outcomes. Finally, we identify critical gaps in understanding the mechanisms of resistance, CNS progression, and the immunogenic landscape of KIT-driven melanoma. Deeper insight into the function of KIT and its interaction with therapeutic pathways is essential to optimizing treatment sequencing and tailoring personalized strategies that improve outcomes in this patient population. Full article

Melanoma is an aggressive form of skin cancer marked by unique genetic alterations that promote tumor growth and resistance to therapy. Advances in targeted therapy have markedly improved clinical outcomes by selectively inhibiting key oncogenic pathways. This review focuses on three clinically relevant agents—vemurafenib, trametinib, and imatinib—analyzing their mechanisms of action, clinical applications, efficacy, and limitations. Vemurafenib, a selective BRAFV600E inhibitor, significantly extends progression-free and overall survival in BRAF-mutant melanoma but is limited by acquired resistance and frequent cutaneous toxicities. Trametinib, a MEK1/2 inhibitor, acts downstream in the MAPK pathway and is typically combined with BRAF inhibitors to enhance efficacy and delay resistance. Imatinib, targeting c-KIT and PDGFR mutations, demonstrates therapeutic benefit primarily in acral and mucosal melanoma subtypes, though with lower response rates than BRAF-directed therapies. Adverse events associated with these drugs are generally manageable with appropriate monitoring. Despite substantial advances, secondary mutations and reactivation of oncogenic signaling remain major challenges. This narrative review integrates data from clinical, preclinical, and real-world studies to update the current understanding of targeted therapies in cutaneous melanoma and highlight ongoing research aimed at overcoming resistance and optimizing personalized treatment strategies. Full article

Background: Seminoma is a malignant germ cell tumor that most commonly involves the testicles but may involve the mediastinum, the retroperitoneum, and other extra-gonadal sites as well. This study aims to investigate the somatic genomic landscape of seminoma. Methods: Data for a retrospective observational analysis of seminoma was acquired from the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) with clinical and genomic data from 2017 and beyond. Using the R and R Studio software (R 4.5.0), analyses for common somatic mutations and copy number alterations were run with a statistical significance of p < 0.05. Results: The most mutated genes included KIT (22.6%), KRAS (17.1%), and MTOR (5.1%), with significant copy number alterations in CDKN1B (17.2%), KRAS (14.7%), CCND2 (10.3%), and H3F3C (9.8%). These suggest involvement within the KIT/RAS/MAPK and PI3K/AKT/mTOR (PAM) pathways for seminoma development. A novel finding within comparative evaluation of PMS1 and AMER1 mutations were found in Black individuals. Additionally, our findings were consistent with a lower testicular cancer rate among individuals with African ancestry than European ancestry. BRD4 mutations were found only in metastatic samples while KMT2C, STAG2, ALK, AXL, and EGFR were only found in primary samples, suggesting a possible association. Conclusions: This study provided a comprehensive molecular and genetic profiling of seminoma including key genetic alterations, affected pathways, and potential therapeutic strategies. Moreover, overlap between pathways and gene mutations provides the potential for alternative treatment options for seminoma via multiple pathways. Full article

Background: Medullary thyroid cancer (MTC), a neuroendocrine tumor originating from thyroid parafollicular C-cells, presents therapeutic challenges, particularly in advanced stages. While RET proto-oncogene mutations are known drivers, a comprehensive understanding of the broader somatic mutation landscape is needed to identify novel therapeutic targets and improve prognostication. This study leveraged the extensive AACR Project GENIE dataset to characterize MTC genomics. Methods: A retrospective analysis of MTC samples from GENIE examined recurrent somatic mutations, demographic/survival correlations, and copy number variations using targeted sequencing data (significance: p < 0.05). Results: Among 341 samples, RET mutations predominated (75.7%, mostly M918T), followed by HRAS (10.0%) and KRAS (5.6%), with mutual exclusivity between RET and RAS alterations. Recurrent mutations included KMT2D (5.3%), CDH11 (5.3%), ATM (5.0%), and TP53 (4.1%). NOTCH1 mutations were enriched in metastatic cases (p = 0.023). Preliminary associations included sex-linked mutations (BRAF/BRCA1/KIT in females, p = 0.028), and survival (ATM associated with longer survival, p = 0.016; BARD1/BLM/UBR5/MYH11 with shorter survival, p < 0.05), though limited subgroup sizes warrant caution. Conclusions: This large-scale genomic analysis confirms the centrality of RET and RAS pathway alterations in MTC and their mutual exclusivity. The association of NOTCH1 mutations with metastasis suggests a potential role in disease progression. While findings regarding demographic and survival correlations are preliminary, they generate hypotheses for future validation. This study enhances the genomic foundation for understanding MTC and underscores the need for integrated clinico-genomic datasets to refine therapeutic approaches. Full article

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that develop in the gastrointestinal tract; KIT mutations are present in both canine and human GISTs. In this study, genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) sections of 55 canine GIST cases, and mutation searches were performed for exons 8, 9, and 11. The results revealed novel mutations, A434T and F436S, in exon 8. In contrast to the A434T mutation without functional changes, the F436S mutant retained its dimerization ability, but lost its phosphorylation function and attenuated downstream Akt signaling, which is reflected in wound healing and migration activities. A comparison of the subcellular localization of WT KIT and the F436S mutant revealed no differences. In silico simulations indicated that the F436S mutation alters the structure of the near-membrane region and that its effects may extend to the transmembrane and intracellular domains compared to the WT. F436S is a point mutation that affects the entire molecule because co-mutation with the F436S mutation and the known autophosphorylation mutation reduces the autophosphorylation abilities. Full article

Background: Helicobacter pylori is a Gram-negative bacterium responsible for various gastrointestinal diseases, including peptic ulcers and gastric cancer. Despite available antibiotic therapies, increasing resistance to clarithromycin—a key antibiotic in eradication regimens—poses a significant challenge. This resistance is primarily linked to point mutations in the 23S rRNA gene, particularly A2143G, A2142G, and A2142C, which hinder clarithromycin binding, reducing its bacteriostatic efficacy. This study aimed to assess the prevalence and variability of clarithromycin resistance mutations in pediatric patients from Bydgoszcz, Poland. Methods: A total of 45 gastric biopsy samples from pediatric patients were analyzed using the Bosphore^® Helicobacter pylori Genotyping Kit v1 to detect clarithromycin resistance-associated mutations. Results: Among the 45 tested samples, 30 were classified as wild-type, while 12 contained resistance-associated mutations. The most frequently detected mutation was A2143G (58.3%), followed by A2142G (33.3%). One sample exhibited both A2142G and A2143G mutations, and another contained a mixture of wild-type and mutant strains. The A2142C mutation was not detected in any sample. Conclusions: Our findings confirm the predominance of A2143G among clarithromycin-resistant H. pylori strains, consistent with global trends. The detection of both mutant and wild-type strains in a single patient highlights potential co-infections or subpopulations with varying resistance profiles. Continuous surveillance and improved diagnostic tools are crucial for optimizing treatment strategies. Tailored eradication protocols based on resistance profiling are necessary to enhance treatment efficacy and mitigate the spread of resistant strains. Further research is needed to understand the clinical implications of mixed infections and double mutations in H. pylori resistance development. Full article

Background/Objectives: Massively parallel sequencing technologies have advanced chronic lymphocytic leukemia (CLL) diagnostics and precision oncology. Illumina platforms, while offering robust performance, require substantial infrastructure investment and a large number of samples for cost-efficiency. Conversely, third-generation long-read nanopore sequencing from Oxford Nanopore Technologies (ONT) can significantly reduce sequencing costs, making it a valuable tool in resource-limited settings. However, nanopore sequencing faces challenges with lower accuracy and throughput than Illumina platforms, necessitating additional computational strategies. In this paper, we demonstrate that integrating publicly available short-read data with in-house generated ONT data, along with the application of machine learning approaches, enables the characterization of the CLL transcriptome landscape, the identification of clinically relevant molecular subtypes, and the assignment of these subtypes to nanopore-sequenced samples. Methods: Public Illumina RNA sequencing data for 608 CLL samples were obtained from the CLL-Map Portal. CLL transcriptome analysis, gene module identification, and transcriptomic subtype classification were performed using the oposSOM R package for high-dimensional data visualization with self-organizing maps. Eight CLL patients were recruited from the Hematology Center After Prof. R. Yeolyan (Yerevan, Armenia). Sequencing libraries were prepared from blood total RNA using the PCR-cDNA sequencing-barcoding kit (SQK-PCB109) following the manufacturer’s protocol and sequenced on an R9.4.1 flow cell for 24–48 h. Raw reads were converted to TPM values. These data were projected into the SOMs space using the supervised SOMs portrayal (supSOM) approach to predict the SOMs portrait of new samples using support vector machine regression. Results: The CLL transcriptomic landscape reveals disruptions in gene modules (spots) associated with T cell cytotoxicity, B and T cell activation, inflammation, cell cycle, DNA repair, proliferation, and splicing. A specific gene module contained genes associated with poor prognosis in CLL. Accordingly, CLL samples were classified into T-cell cytotoxic, immune, proliferative, splicing, and three mixed types: proliferative–immune, proliferative–splicing, and proliferative–immune–splicing. These transcriptomic subtypes were associated with survival orthogonal to gender and mutation status. Using supervised machine learning approaches, transcriptomic subtypes were assigned to patient samples sequenced with nanopore sequencing. Conclusions: This study demonstrates that the CLL transcriptome landscape can be parsed into functional modules, revealing distinct molecular subtypes based on proliferative and immune activity, with important implications for prognosis and treatment that are orthogonal to other molecular classifications. Additionally, the integration of nanopore sequencing with public datasets and machine learning offers a cost-effective approach to molecular subtyping and prognostic prediction, facilitating more accessible and personalized CLL care. Full article