Search Results (94)

Background: In melanoma diagnostics key molecular markers, such as BRAF, NRAS, and KIT mutations also paved the way for targeted therapies. Immunotherapies, including immune checkpoint inhibitors like anti-CTLA-4 and anti-PD-1/PD-L1, have revolutionized treatment, improving survival outcomes for advanced-stage melanoma patients. Despite these advances, challenges such as resistance to targeted therapies and variability in patient responses to immunotherapy remain critical issues. The purpose of the project is to characterize the molecular landscape of a set of 28 malignant melanomas using next-generation sequencing, identify the prevalence and nature of class 3–5 variants (e.g., NRAS, BRAF, KIT, TP53), assess the genetic complexity and molecular patterns, and use these insights to inform personalized therapies and optimize patient stratification for potential combination strategies (targeted therapy followed by immunotherapy). Methods: We analyzed a set of malignant melanoma of the skin of 17 women (61%) and 11 men (39%) at the age of 23 to 85 years (median: 63 years) by tumor-only next generation sequencing. Results: 22/28 cases (79%) present a pathogenic or likely pathogenic variant with an allelic frequency of ≥5%. In total 42 distinct somatic pathogenic or likely pathogenic variants with an allelic frequency of ≥5% could be detected. The most frequent pathogenic molecular alteration in these melanomas were found in NRAS (25%) and BRAF (25%). The most frequent molecular alteration of unknown significance was found in FANDC2 (46%), NOTCH3 (39%), ARID1A (32%), PMS2 (32%), POLE (29%), NOTCH1 (29%), TSC2 (25%), SMARCA4 (25%), ATR (25%) and TERT (21%). Conclusions: While NRAS and BRAF were the most frequent actionable alterations (each 25%), a broad spectrum of variants of unknown significance (e.g., FANDC2, NOTCH3, ARID1A, PMS2, POLE, NOTCH1, TSC2, SMARCA4, ATR and TERT) also predominates, underscoring the genetic complexity of melanoma. These variants complicate clinical decision-making because their contribution to tumorigenesis, therapeutic response, and prognosis remains uncertain. Nevertheless, these variants also offer a valuable resource for future research, as they may uncover novel pathogenic mechanisms or therapeutic targets once their significance is elucidated. Integrating comprehensive genetic profiling with immunologic markers can enhance patient stratification and support rational, potentially synergistic strategies, such as combining targeted therapies with immunotherapy, to optimize clinical outcomes. This study is limited due to a small cohort and limited available clinical data. Larger cohort studies and prospective clinical trials are necessary to validate and explore the interplay between molecular and immune biomarkers as well as general biological mechanism in paving therapeutic way in melanoma. Full article

Background/Objectives: Targeted gene sequencing (TGS) for Comprehensive Genomic Profiling (CGP) use in sarcomas has recently increased in clinical practice. We report on TGS real-world data over a period of 3 years (2022–2025) at the IRCCS Istituto Ortopedico Rizzoli, with the aim of identifying potential actionable targets and providing therapeutic indications for advanced sarcoma patients. Methods: We analyzed 22 advanced sarcoma patients by using the VariantPlex Pan Solid Tumor kit panel, including 185 genes. In nine cases, saliva samples for germinal DNA analysis were available. Sequencing was performed on the NextSeq-500 Platform and analyzed with Archer Analysis software. The Cancer Genome Interpreter and OncoKB Database tools were used to find potential actionable targets. Results: We found the most frequent genetic variants, including missense, deletion, duplication, and delins, in the NOTCH4, AR, BARD1, MUC16, and ROS1 genes. Copy Number alterations affected the CDKN2A, CDKN2B, TP53, RHOA, MYC, CCND3, and DDR2 genes mainly in osteosarcoma samples. In four patients, longitudinal analyses of subsequent lesions showed the maintenance of most genomic alterations and enrichment in missense or splice variants in PMS2, SMARCA4, ARID1A, AKT1, BMPR1A, and PTEN, indicating the occurrence of tumor evolution. Germline variants subtraction identified the specific somatic tumor mutations. Advantages and disadvantages of our approach were considered in order to refine the analysis setting and better select possible actionable targets. Conclusions: Early access to genomic analyses, routine germline assessment, and broad gene panels would help in identifying possible targeted drugs with sufficient evidence of activity beneficial to each patient. In the clinical management of advanced sarcoma patients, when analyzing cost-effectiveness and sustainability, the role of the Molecular Tumor Board in the governance of the complexity introduced by mutational oncology should be considered. Full article

Background: Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs) have exhibited efficacy in EGFR-mutant non-small cell lung cancer (NSCLC) patients. However, the response is modest in patients with wild-type (wt)-EGFR, and approximately 30–40% of patients develop TKI resistance. Recently, a role for BRG1 (SMARCA4) in regulating gene expression and its frequent alteration in various cancers, including NSCLC, has been reported. Yet, its specific function in response to EGFR-TKI therapy remains elusive. Herein, we investigated the role of BRG1 in EGFR-TKI response in vitro and in vivo using lung cancer models. Methods: In vitro, A549, H358, and HCC827 cell lines that varied in their EGFR and BRG1 status were assessed for response to EGFR-TKI upon overexpression or gene silencing of BRG1 through cell viability, cell migration, and Western blotting assays. In vivo, A549 and H358 tumor xenografts that overexpressed BRG1 or had BRG1 silenced were investigated for tumor growth response to EGFR-TKI. Results: EGFRwt/BRG1mt (A549) cells were resistant to TKI, and restoration of wt-BRG1 expression reverted them to TKI sensitivity both in vitro and in vivo. In contrast, silencing of BRG1wt in H358 cells showed a tendency toward TKI resistance. Additionally, wt-EGFR and pAKT^Ser473 protein complex formation in A549 cells was disrupted with an AKT inhibitor (MK2206), resulting in enhanced cytotoxicity in vitro. Conclusions: Our study demonstrates that EGFR-TKI response in wt-EGFR cells is dictated by BRG1 status. These findings propose screening of wt-EGFR NSCLC patients for BRG1 status for identifying individuals likely to benefit from EGFR-TKI therapy versus patients who will benefit from AKT inhibitor treatment. Full article

The ovary, as the primary organ responsible for reproduction and new life, plays a central role in female development, maturation, and health. Neoplasms arising from the ovary and its associated tissues exhibit substantial heterogeneity in their histopathological and molecular profiles, many of which remain poorly understood. This review aims to summarize recent advances in the understanding of genetic alterations underlying ovarian neoplasms and to explore therapeutic strategies informed by molecular biomarkers and tumor microenvironmental factors. A comprehensive literature search was performed, focusing on genomic alterations, biomarker-guided therapies, and tumor microenvironmental modulation in ovarian cancers. Emphasis was placed on studies addressing lipid mediator pathways and their roles in immune regulation and therapeutic response. Based on diagnostic classifications, recurrent alterations in TP53, MYC, PIK3CA, and KRAS are consistently observed across epithelial and germ cell ovarian tumors, whereas non-epithelial subtypes such as sex cord–stromal tumors (SCSTs) and small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), are predominantly associated with ARID1A and SMARCA4 mutations, respectively. These findings highlight distinct pathogenic mechanisms linked to specific genetic alterations and reveal potential therapeutic vulnerabilities. Moreover, lipid metabolism has been closely implicated in immune surveillance through STING signaling cascades within innate immune cells, suggesting that lipid mediators and their associated genes may represent promising therapeutic targets in ovarian cancers (OCs). Targeting lipid mediators could be particularly effective in relapsed OCs, as modulating innate immune cells within the tumor microenvironment (TME) may enhance immune surveillance and improve antitumor responses. Integrating genetic and microenvironmental insights offers a promising direction for developing more effective and personalized therapeutic strategies in OC. Full article

Background: Ovarian cancer is one of the most lethal gynecological malignancies, often diagnosed at an advanced stage. The prognosis is generally poor, with high recurrence rates and limited long-term survival. Understanding the genetic and molecular mechanisms underlying ovarian cancer is crucial for improving early diagnosis, developing targeted therapies, and enhancing patient outcomes. Methods: In this study, the clinical molecular reports of 50 ovarian cancer patients referred to the experimental cancer unit at Herlev Hospital were analyzed. The aim was to assess the number of patients being potential candidates for targeted biological therapy. Additionally, using the reports, we aimed to identify patients with potential germline mutations in cancer-predisposing genes. The possible consequences were annotated using gene lists from four hospitals in Denmark. Each hospital had its own distinct, published gene list, reflecting the genes that it considered potential carriers of germline mutations predisposing to cancer. Results: A total of twenty out of fifty patients (40%) had targetable biomarkers for biological treatment. CCNE1 amplification was identified as the most frequent variant (43%). Seven out of fifty patients (14%) had potential germline mutations in cancer-predisposing genes. Conclusions: In conclusion, the finding of a potential germline mutation in the SMARCA4 gene highlights how differences in hospital-specific gene lists may impact patient referral for genetic counseling. Full article

Feline herpesvirus-1 (FHV-1) is taxonomically classified within the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus, and species Felid alphaherpesvirus 1. The genome of FHV-1 is 135,797 bp in length and encodes 74 proteins. Among these proteins, serine/threonine protein kinase (pK) and thymidine kinase (TK) have been identified as potential virulence factors in alphaherpesviruses, although these kinases are dispensable for viral replication. As kinases, regulating phosphorylation modification is one of their functions, while the mechanism by which phosphorylation modification affects cell physiological functions and thereby influences viral replication remains unclear. In this study, we generated pK- and TK-deficient FHV-1 mutants by CRISPR/Cas9-mediated homologous recombination. The pK-deficient virus produced significantly smaller plaques than the TK-deficient virus. The replication kinetics of the pK-deficient virus were attenuated in multistep growth compared to the TK-deficient virus. These results indicate that deletion of the pK gene markedly reduces the replicative capacity of FHV-1. We applied data-independent acquisition (DIA) quantitative proteomics to profile changes in global protein expression and phosphorylation in F81 cells upon infection with TK⁻, pK^−, and wild-type FHV-1 strain. The pK-deficient virus exhibited 3632 differentially phosphorylated proteins containing 11,936 modification sites; the TK-deficient virus showed 4529 differentially phosphorylated proteins with 19,225 phosphorylation sites. Functional characterization through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified significant involvement of phosphoproteins in spliceosome pathways in pK-deficient virus and ATP-dependent chromatin remodeling pathway in TK-deficient virus. Notably, several splicing regulators—including Ess-2 and CDK13, which modulate host spliceosomal function—displayed significantly reduced phosphorylation levels in pK-deficient viruses. A significant enrichment of ATP-dependent factors, such as SMARCA5 and RSF1, was observed in the TK-deficient virus. To our knowledge, this is the first investigation into the effects of FHV-1 infection on the host cell phosphoproteome. These data offer new insights into the phosphoregulatory circuits and signaling networks triggered by FHV-1 and may enhance our understanding of the FHV-1 replication mechanism. Full article

Atypical Teratoid/Rhabdoid Tumor (AT/RT) is a highly aggressive neoplasm of the central nervous system (CNS), most commonly affecting infants and young children. Originally recognized as a distinct entity following cytogenetic identification of monosomy 22 in renal Rhabdoid Tumors, AT/RT now encompasses CNS tumors characterized by SMARCB1 (INI-1) or SMARCA4 (BRG-1) alterations within the SWI/SNF chromatin-remodeling complex. The integration of immunohistochemical markers with advanced molecular diagnostics—including next-generation sequencing, DNA methylation profiling, and gene enrichment analyses—has facilitated robust tumor classification and the identification of three molecular subgroups: TYR, SHH, and MYC. Despite its distinctive histopathologic features, AT/RT remains diagnostically challenging in adolescent and adult populations due to age-related bias and potential morphologic heterogeneity. Differential considerations, including epithelioid sarcoma, poorly differentiated chordoma, CRINET, choroid plexus carcinoma, and rare composite tumors, further complicate the diagnostic landscape. A comprehensive, multimodal diagnostic approach combining histologic, immunophenotypic, and molecular data is essential to accurately identify AT/RT and guide clinical management, particularly in diagnostically ambiguous or atypical cases. Full article

Targetable gene alterations have become increasingly important in the treatment of cancers. Thirty STK11-mutated lung cancers from 199 cases with molecular profiling performed during 2016–2024 were studied for clinical, morphologic, immunohistochemical (IHC) and molecular features. Of the 30 STK11-mutated lung cancers, 29 were lung adenocarcinomas (LADCs) and 1 was large cell neuroendocrine carcinoma (LCNEC). STK11 mutation was not found in other subtypes of lung cancers. Of the 29 STK11-mutated LADCs, 6 (21%) were mucinous and 23 (79%) were non-mucinous. Of the 19 non-mucinous LADCs with sufficient material for IHC, 9 (47%) displayed acinar/papillary/lepidic patterns, 8 (42%) were poorly differentiated (solid/trabecular/basaloid/complex glandular), and 2 (11%) had mixed solid and acinar patterns. The most common concurrent altered genes were KRAS (52%), followed by TP53 (38%), KEAP1 (34%), and DNA repair genes (BRCA2/ATM) (21%). A total of 6/15 (40%) LADCs with a KRAS mutation presented with mucinous morphology. Concurrent EGFR, ROS, or ALK alterations with STK11 mutation were rare or non-existent. Of the 3 LADCs with SMARCA4 deficiency, 2 were mucinous and 1 had basaloid/adenoid cystic-like features. All the cases were microsatellite stable (MSS). The majority (55%) had low TMB (<10). Most (86%) had PD-L1 TPS 0 or <5%. Among the 14 non-mucinous LADCs with IHC performed, 5 (36%) were TTF-1-negative and all displayed poorly differentiated morphology. Overall, 8/10 (80%) of poorly differentiated components in non-mucinous LADCs were negative for TTF-1. In contrast, all LADCs with better differentiated patterns (acini/papillary/lepidic) were positive for TTF-1. The majority (14/21, 67%) of patients with available follow-up presented with metastasis. Full article

Multiple Endocrine Neoplasia Type 1 (MEN1) is an autosomal dominant disorder commonly associated with tumor development in the parathyroid glands, pancreas, and pituitary gland. While pituitary adenomas are frequently observed in MEN1 patients, the presence of additional tumors within the pituitary gland is unusual. Moreover, the co-occurrence of a pituitary adenoma with an atypical teratoid/rhabdoid tumor (ATRT) has not been previously documented. ATRT is a rare, aggressive neoplasm predominantly affecting young children and is typically associated with inactivating mutations in the SMARCB1 or SMARCA4 tumor suppressor genes. These mutations result in uncontrolled cellular proliferation, which underlies the malignancy’s rapid progression. In adults, ATRT is exceedingly rare, making this case particularly noteworthy for its uniqueness in both tumor type and patient demographics. ATRTs are now classified into three molecular subgroups—MYC, SHH, and TYR—each with distinct epigenetic and clinical features, further refining diagnostic and prognostic assessments. In this case report, we describe a case of a female patient with MEN1 who experienced several recurrences of pituitary adenoma, ultimately necessitating surgical resection. Detailed pathological evaluation of the resected tissue revealed two distinct neoplasms within the pituitary gland: one typical of a pituitary adenoma, and the other confirmed as ATRT. The diagnosis of ATRT was established through extensive workup including immunohistochemical analysis, next-generation sequencing and methylation profiling, which served as essential tools in distinguishing ATRT from other potential differential diagnoses. This case illustrates the complex diagnostic journey and challenges encountered in identifying ATRT in the context of MEN1, underscoring the importance of using advanced molecular and immunohistochemical techniques in atypical presentations. Furthermore, it expands the understanding of potential tumor associations within MEN1, providing insight for pathologists and clinicians into the rare possibility of concurrent tumors in addition to pituitary adenoma in MEN1 patients. Raising awareness of such co-occurrences could prompt earlier diagnostic considerations by refining the differential diagnosis in patients with MEN1 presenting with unusual tumor types. Full article

PARP inhibitors exploit synthetic lethality in BRCA1/2-mutated ovarian cancers but are limited by emerging therapeutic resistance. Therefore, novel biomarkers predicting PARP inhibitor response are urgently needed. In this study, we performed integrative analysis using drug sensitivity, patient survival, gene dependency, and expression data to identify biomarkers associated with PARP inhibitor response in ovarian cancer. Mutations in BRCA1, MLL2, NF1, and SMARCA4 were associated with increased sensitivity to PARP inhibitors, suggesting potential synthetic lethality with PARP1. In contrast, SMAD4 mutations were linked to PARP inhibitor resistance, and low SMAD4 expression was associated with poor overall survival in patients with ovarian cancer. Further gene dependency score (GDS)-based screening revealed 51 candidate genes potentially involved in SMAD4-mediated resistance. Functional enrichment revealed associations with stress response, tumor-associated signaling pathways, and additional processes. Subsequent correlation and survival analyses nominated ACACA, PRPF4B, and TUBD1 as potential therapeutic targets. Notably, low ACACA expression in patients with low SMAD4 expression was associated with improved survival, indicating its relevance in overcoming PARP inhibitor resistance. This study contributes to predicting clinical outcomes in ovarian cancer and developing personalized treatment strategies. Full article

Background/Objectives: The BRG1 loss-of-function (LOF) mutation is found in ~10% of non-small cell lung cancer (NSCLC) cases, but its role in lung tumorigenesis is unclear and so it is investigated in this study. Methods: BRG1-knockout (KO) lines were generated from various non-malignant, pre-malignant, and malignant human lung epithelium-derived cell lines using CRISPR. The effects of BRG1-KO on cell growth, the transcriptome, the methylome, and epigenetic therapy were compared with those of wild-type (BRG1-WT) isogenic controls using standard in vitro and in vivo assays. Results: The BRG1 protein was expressed in all non-/pre-malignant lung epithelial cells but lost in 47% (14/30) of NSCLC cell lines. BRG1-KO and cigarette smoke (CS) exposure individually transformed human bronchial epithelial cell lines (HBECs), as evidenced by anchorage-independent growth. BRG1-KO and CS produced additive to synergistic effects on sensitivity to transformation that differed across HBECs. RNA-seq analysis revealed that BRG1-KO significantly changed the expression of over 8500 genes on average, impacting lung development, function, damage repair, and cancer pathways, including axonal guidance, pulmonary wound healing, and epithelial-to-mesenchymal transition (EMT). BRG1-KO also led to the hypermethylation of >47,000 promoter CpGs within ~9500 genes on average in different HBECs, including silencing of epithelial genes involved in EMT and tumor suppressor genes. BRG1-KO also moderately increased the in vitro and in vivo sensitivity of NSCLC cells to some epigenetic drugs. Conclusions: BRG1-LOF is frequent in NSCLC; can drive the transformation of lung epithelial cells such that they acquire properties of pre-malignant cells, indicating a potential role in lung cancer initiation; and sensitizes lung tumors to epigenetic therapy. Full article

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. Also, no unified computational framework exists to specifically link miRNA expression with functional gene targets during RV infection. This study aimed to first analyse the impact of RV16 on miRNA expression across the viral life cycle to identify a small panel with altered expression. We then developed a novel bioinformatics pipeline that integrated time-resolved miRNA profiling with multi-database gene-phenotype mapping to identify diagnostic biomarkers and their regulatory networks. Our in-house Python-based tool, combining mirDIP, miRDB and VarElect APIs, predicted seven genes (EZH2, RARG, PTPN13, OLFML3, STAG2, SMARCA2 and CD40LG) implicated in antiviral responses and specifically targeted by RV16 and regulated by our miRNAs. This method therefore offers a scalable approach to interrogate miRNA-gene interactions for viral infections, with potential applications in rapid diagnostics and therapeutic target discovery. Full article

Background/Objectives: KRAS mutations are among the most prevalent oncogenic drivers in non-small cell lung cancer (NSCLC), with their impact on survival influenced by co-mutations. SMARCA4 mutations are increasingly associated with poor prognosis and can be classified as class 1 or class 2 mutations. This study evaluates the prognostic implications of KRAS and SMARCA4 mutations, including their co-mutations and their impact on NSCLC patients by utilizing real-world evidence. Methods: A retrospective analysis was conducted using the AACR GENIE Biopharma Collaborative (BPC) NSCLC 2.0 dataset. NSCLC patients with KRAS mutations, SMARCA4 mutations, or KRAS/SMARCA4 co-mutations were identified. Survival outcomes were assessed using univariate and multivariate Cox proportional hazards models, incorporating key clinical variables such as sex, race, smoking history, and stage. Results: Among 659 NSCLC patients with KRAS or SMARCA4 mutations analyzed, KRAS mutations were the most prevalent (79%, n = 518). SMARCA4 mutations were identified in 14% of cases (n = 95) across two classes. Six percent (n = 41) with class 1 mutations and 8% (n = 54) with class 2. Neither SMARCA4 class was associated with worse survival outcomes compared to KRAS-mutated patients (p = 0.438 & 0.720). Patients harboring KRAS/SMARCA4 class 1 co-mutations (3%, n = 18) had significantly worse overall survival compared to those with KRAS mutations alone (hazard ratio [HR] = 3.23, p < 0.001). In contrast, KRAS/SMARCA4 class 2 co-mutations (4%, n = 28) did not significantly impact survival compared to KRAS-mutated patients (HR = 1.34, p = 0.205). Conclusions: KRAS/SMARCA4 class 1 co-mutations are associated with significantly worse overall survival compared to KRAS-mutated NSCLC patients. Our multivariate analysis demonstrates the critical need to incorporate routine next-generation sequencing (NGS) testing in managing NSCLC patients at the time of metastatic diagnosis, with particular emphasis on identifying SMARCA4 mutation class as a potential prognostic biomarker in those with KRAS co-mutations. Full article

Human Papillomavirus (HPV), particularly high-risk strains such as HPV16 and HPV18, is a leading cause of cervical cancer and a significant risk factor for several other epithelial malignancies. While the oncogenic mechanisms of viral proteins E6 and E7 are well characterized, the broader effects of HPV infection on host transcriptional regulation remain less clearly defined. This study explores the hypothesis that conserved genomic motifs within the HPV genome may act as molecular decoys, sequestering human transcription factors (TFs) and thereby disrupting normal gene regulation in host cells. Such interactions could contribute to oncogenesis by altering the transcriptional landscape and promoting malignant transformation.We conducted a computational analysis of the genomes of high-risk HPV types using MEME-ChIP for de novo motif discovery, followed by Tomtom for identifying matching human TFs. Protein–protein interactions among the predicted TFs were examined using STRING, and biological pathway enrichment was performed with Enrichr. The analysis identified conserved viral motifs with the potential to interact with host transcription factors (TFs), notably those from the FOX, HOX, and NFAT families, as well as various zinc finger proteins. Among these, SMARCA1, DUX4, and CDX1 were not previously associated with HPV-driven cell transformation. Pathway enrichment analysis revealed involvement in several key biological processes, including modulation of Wnt signaling pathways, transcriptional misregulation associated with cancer, and chromatin remodeling. These findings highlight the multifaceted strategies by which HPV may influence host cellular functions and contribute to pathogenesis. In this context, the study underscores the power of in silico approaches for elucidating viral–host interactions and reveals promising therapeutic targets in computationally predicted regulatory network changes. Full article

Background: Clear cell renal cell carcinoma with rhabdoid features (ccRCC-R) is a highly aggressive variant of renal cell carcinoma that carries a poor prognosis and limited treatment options. Methods: To better define the clinicopathologic and molecular landscape of ccRCC-R, we conducted an integrated clinicopathologic and molecular study of 17 tumors of ccRCC-R, utilizing comprehensive histomorphologic evaluation, immunohistochemistry, and targeted next-generation sequencing (NGS). Results: Histologically, all tumors demonstrated classic clear cell renal cell carcinoma morphology with focal to extensive rhabdoid differentiation, characterized by eccentrically located nuclei, prominent nucleoli, abundant eosinophilic cytoplasm, and paranuclear intracytoplasmic inclusion. Architectural alterations, including solid/sheet-like, alveolar/trabecular, and pseudopapillary growth patterns, were frequently observed. Immunohistochemically, tumors commonly exhibited loss of PAX8 and Claudin4 expression, preserved cytokeratin AE1/AE3 staining, and diffuse membranous CAIX expression. Frequent loss of SMARCA2 with retained SMARCA4 supported aberrations in chromatin remodeling. Unsupervised hierarchical clustering based on pathway-specific somatic mutations identified four distinct molecular subgroups defined by recurrent alterations in (1) DNA damage repair (DDR) genes, (2) chromatin remodeling genes, (3) PI3K/AKT/mTOR signaling components, and (4) MAPK pathway genes. Clinicopathologic correlation revealed that each subgroup was associated with unique biological characteristics and suggested distinct therapeutic vulnerabilities. Conclusions: Our findings underscore the molecular heterogeneity of ccRCC-R and support the utility of pathway-based stratification for guiding precision oncology approaches and biomarker-informed clinical trial design. Full article