Mucosal melanomas (MMs) are rare, aggressive cancers with poor outcomes and limited response to standard therapies. A significant knowledge gap exists regarding their genomic landscape and corresponding druggable targets. This study explores this issue through a multi-omic analysis, including whole exome-, RNA-, and targeted sequencing, of unique sinonasal mucosal melanoma (SN-MM) cell lines. SN-MM cell lines showed a low tumour mutational burden and low genomic instability, suggesting that their DNA repair mechanisms are largely intact. However, they exhibited a high prevalence of structural variants, likely from catastrophic genomic events. We also found novel, clinically actionable variants, including oncogenic fusion transcripts. Key findings, such as fusion transcripts and single-nucleotide variants, were validated on diagnostic biopsies. Moreover, using a break-apart FISH assay, we identified BRAF rearrangements as recurrent events in MM, supporting their biological relevance. The MAPK pathway was found to have a significant functional role, sustained by variants such as BRAF p.Asn581Ser, NRAS p.Gly12Asp, and the MKRN1::BRAF fusion. This pathway activation was effectively inhibited by the MEK inhibitor trametinib. This genomic and functional profiling unveiled novel molecular hubs in MM. The findings propose that MEK inhibitors may represent a relevant therapeutic option for BRAF fusions and non-V600 BRAF mutant tumours, providing a novel strategy for this challenging disease. Further molecular and functional validation of novel variants, and particularly of BRAF rearrangements, may reveal additional clinically relevant therapeutic targets. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BACKGROUND:Synovial sarcoma (SS) is a rare malignancy, accounting for 5%-10% of all soft tissue sarcomas, and characterized by the SS18::SSX translocation. SS predominantly affects young individuals, with a peak incidence in the fourth decade of life. Children (<15 years), and adolescents and young adults (AYAs; 15-29 years) with SS tend to have better survival rates than older adults; however, the biological basis for these age-related differences remains poorly understood. METHODS:In this study, high-throughput RNA sequencing (RNA-seq) was used to investigate biological differences in SS across age groups. Transcriptomic profiles of primary tumor tissues from 14 patients younger than 29 years were compared with those from 8 adult patients (>29 years), with the aim of identifying age-related molecular features of SS. In addition, enrichment of recently described transcriptional signatures associated with SS tumor components and infiltrating cell populations was evaluated in our dataset. RESULTS:RNA-seq analysis identified differential expression of RPL39 and GATA3 between children/AYA and adult patients. Immunohistochemical analysis confirmed increased GATA3 expression in adult tumors. Comparative analyses based on SS-related transcriptional signatures further demonstrated enrichment of actively cycling tumor cells in adult samples, as well as a trend toward increased M2 macrophage infiltration in approximately half of the children/AYA samples. CONCLUSIONS:This study reveals age-related molecular and tumor microenvironment differences in SS that may contribute to disparities in clinical outcomes. These findings underscore the need for further studies to validate these results and to support the development of age-tailored therapeutic strategies.

Purpose: Liquid biopsy has emerged as a valuable tool for detecting therapeutic targets and resistance mechanisms. This study evaluated the analytical performance and clinical utility of TruSight Oncology 500 ctDNA (TSO500) compared to the FDA-approved Guardant360 CDx (G360) in detecting actionable alterations in non-small cell lung cancer (NSCLC) patients progressing on targeted therapies. Patients and methods: We analyzed 44 plasma samples from 36 consecutive metastatic NSCLC patients with known molecular drivers ( EGFR and BRAF mutations, ALK and RET fusions) progressing on targeted therapy. The comparative analysis included 31 paired samples from 27 patients. Valid results were obtained from G360 in 42/44 cases (95.45%) and TSO500 in 32/44 samples (72.8%). Results: TSO500 demonstrated high sensitivity for G360-identified variants (81.02% overall; 85.26% for pathogenic/likely pathogenic variants). Concordance was particularly high for ESCAT I alterations (95.2% sensitivity), including 100% sensitivity for gene fusions (five detected fusions). TSO500 identified 102 additional variants not detected by G360. Among nine patients with concurrent tissue biopsy, TSO500 detected 16 potentially actionable mutations absent in tissue samples. Both assays identified resistance mutations in 12/26 patients (46.2%), with TSO500 detecting all but three G360-identified alterations. High correlation was observed between variant allele frequencies (Pearson's r = 0.89). Conclusions: TSO500 demonstrates high concordance with G360 for detecting actionable alterations and robust fusion identification. Its ability to detect additional variants and resistance mutations not found in tissue highlights its potential value in guiding personalized treatment decisions for NSCLC patients.

Liquid biopsy has emerged as a valuable tool for detecting therapeutic targets and resistance mechanisms. In this study, we aimed at evaluating the analytical performance of the TruSight Oncology 500 ctDNA (TSO500) compared to the FDA-approved Guardant360 CDx (G360) in detecting actionable alterations in NSCLC patients progressing on targeted therapies. We analysed 44 plasma samples from 36 consecutive metastatic NSCLC patients with known molecular drivers. The comparative analysis included 31 paired samples from 27 patients. TSO500 demonstrated high sensitivity in detecting G360-identified variants (81.02%). Concordance was particularly high for ESCAT I alterations (sensitivity: 95.2%), including gene fusions (100% sensitivity). Both LB assays identified resistance mutations in 12/26 patients, with TSO500 detecting all but 3 G360-identified resistance alterations. Our findings support TSO500's analytical validity and clinical utility, demonstrating high concordance with G360 for actionable alterations detection, highlighting its potential value in guiding treatment decisions.

OBJECTIVE:There is limited evidence on the economic implications of assessing patients' access to personalised treatments through Comprehensive Genomic Profiling (CGP) and Molecular Tumour Board (MTB), prompting the need to analyse their impact on the cost of the cancer diagnostic journey (from hospital admission to MTB evaluation) and accessibility to personalised therapies. DESIGN:Retrospective observational cohort. SETTING:Patients discussed from April 2020 to September 2021 by the institutional MTB operating at Fondazione IRCCS Istituto Nazionale Tumori of Milan, an Italian centre of excellence in oncology pertaining to the national health system. PARTICIPANTS:676 patients focused on: non-small cell lung cancer (NSCLC), cholangiocarcinoma (CCA), pancreatic carcinoma (PC) and gastro-oesophageal carcinoma (GEC). We defined two different scenarios: (1) patients tested with small Next-Generation Sequencing (NGS) panels (≤60 biomarkers) vs (2) patients tested with comprehensive panels (>60 biomarkers). MAIN OUTCOMES AND MEASURES:We measured (1) patients' eligibility to personalised therapies based on genomic data obtained using targeted somatic NGS panels, (2) MTB cost and the overall diagnostic journey cost and (3) the cost to find a patient eligible to access personalised treatments. RESULTS:Tumour profiling with comprehensive NGS panels improved patients' eligibility to personalised therapies compared with small panels (NSCLC: 39% comprehensive panel vs 37% small panel; CCA: 43% vs 17%; PC: 35% vs 3%; GEC: 40% vs 0%). The overall diagnostic journey cost per patient was between 3.2K and 7.4K (NSCLC: 7.4K comprehensive panel vs 6.4K small panel; CCA: 4.9K vs 3.7K; PC: 5.8K vs 4.5K; GEC: 4.2K vs 3.2K). MTB discussion accounted for only 2-3% of the diagnostic journey cost per patient (around 113€/patient). The cost to find patient eligible for personalised treatments varied significantly according to panel size and tumour setting (NSCLC: 5K comprehensive panel vs 2.8K small panel; CCA: 4.4K vs 4.4K; PC: 5.5K vs 27K; GEC: 5.2K vs not measurable since none of the patients analysed with small NGS panels were eligible). CONCLUSIONS AND RELEVANCE:MTB discussion of genomic data obtained with NGS comprehensive panels significantly increases patient eligibility to targeted therapies and optimise the cost to find a patient eligible to personalised treatments, mainly for CCA, PC and GEC patients.

IntroductionOvarian cancer (OC) is one of the leading causes of cancer-related death in women worldwide. Treatment with PARP-inhibitors has significantly improved survival in patients with high-grade serous cancer (HGSC) bearing BRCA1/2 mutations (~22% of the cases), and/or homologous recombination deficiency (HRD, ~50%). Unfortunately, limited therapeutic alternatives are available for BRCA1/2 wild type/HR proficient HGSC patients, who usually exhibit resistance to standard treatments and poor prognosis.MethodsHerein, we present the results of a comprehensive genomic profiling (CGP) analysis using the Oncomine Comprehensive Assay® (OCA) Plus in a consecutive retrospective cohort of 102 HGSC patients characterized in our institution.ResultsGenomic instability, measured by Genomic Instability Metric (GIM) >16, was found in 40% of the cases and was significantly associated with BRCA1/2 mutations (p=0.009), with a better prognosis in terms of recurrence-free survival (p=0.01). CCNE1 amplification was observed in 29% of cases and was negatively correlated with BRCA1/2 mutations (p=0.001), without any association with GIM, supporting CCNE1 as a strong and independent driver of tumorigenesis. Additionally, CCNE1 amplification was validated with fluorescent in situ hybridization (FISH), supporting the analytical robustness of NGS data (rho=0.93), and investigated by immunohistochemistry (IHC), revealing that CCNE1 protein overexpression was observed in the absence of gene amplification in 45% of cases.DiscussionOur real-world study supports the clinical utility of the GIM metric and the analytical validity of CCNE1 amplification, a new promising biomarker for personalizing treatment in HR proficient HGSC patients. The discordance between CCNE1 amplification and protein expression raises intriguing questions about the mechanisms of CCNE1-driven tumorigenesis and warrants further investigation.

Copy number alterations (CNAs) play a fundamental role in cancer development and constitute a potential tool for tailored treatments. The CNAs recognition in formalin fixed paraffin embedded (FFPE) material for diagnostic purposes has relied for years mainly on fluorescence in situ hybridization. The introduction of other procedures, such as Next-Generation Sequencing has dramatically improved CNAs discovery at genome-wide level. The detection of CNAs by NGS in FFPE material is, nonetheless, a complex issue, which still requires validation studies. Herein, the CNAs detection by a widely used NGS assay (Oncomine Comprehensive Assay plus®, OCA+) were evaluated in 14 FFPE samples mirroring diagnostic daily practice and compared to a whole-genome assay. OCA+, a targeted DNA panel, showed lower CNAs detection sensitivity and equal specificity for gains and losses. According to proprietary software pipeline, OCA+ accurately identified gains characterized by CN ≥ 5,2. No significant threshold maximizing the difference between true and false positive losses was found. Orthogonal FISH tests validated seven CNAs characterized by CN gain ≥ 6 or complete loss. Considering the CNAs growing significance in precision medicine, our findings further prompt towards a robust validation of NGS detection in FFPE materials.

PURPOSE The Molecular Tumor Board (MTB) is an important tool for the selection of molecularly targeted agents (MTAs), but its clinical utility is unconfirmed in real-world practice. MATERIALS AND METHODS We explored the clinical relevance of MTAs in patients discussed by the MTB of the National Cancer Institute of Milan. Patients were stratified into eligible (ie, at least one actionable molecular alteration identified) and treated with MTA (group A), eligible but not treated (group B), or not eligible (group C). Cases for whom a European Society for Medical Oncology Scale for Clinical Actionability of Molecular Targets IA tier target was identified were excluded from this analysis and managed as per clinical practice. The study end points were overall survival (OS), progression-free survival (PFS), and PFSratio, defined as PFS on MTA/PFS on the last previous treatment in group A. RESULTS As of November 2022, 1,813 cases were discussed, of which 458 (25.2%) were potentially eligible to MTA and 117 (6.4%) started treatment. The median PFS on MTA was 12.0 months and the median PFSratio in patients receiving MTA as >1 line of treatment was 2.71, with a benefit rate of 64%. OS was significantly longer in group A than in B and C metastatic patients (adjusted hazard ratio, 2.21 and 1.71 in group B and C, respectively; P < .001). CONCLUSION Efforts should be pursued to offer MTB-driven therapies, as eligible patients who did not receive MTA showed a significantly poorer prognosis.

PURPOSE:This study used a deidentified nationwide (US-based) high-grade serous ovarian cancer (HGSOC) clinicogenomic database (CGDB) to enrich our understanding of HGSOC's genomic heterogeneity and assess the utility of comprehensive genomic profiling (CGP) in clinical settings. PATIENTS AND METHODS:We conducted a retrospective observational analysis on 856 patients with HGSOC profiled with CGP genomic tests, retrieved from CGDB from January 2011 to September 2023. RESULTS:In addition to BRCA1 (11.7%) and BRCA2 (6.5%) variants, CGP revealed further potentially actionable alterations (amplifications and/or mutations) in CCNE1 (16%), FGFR1/2/3/4 (6.5%), PIK3CA (3.9%), TP53Y220C (3.7%), ERBB2 (3.5%), CDK12 (2.3%), ARID1A (2.2%), KRAS (2.1%), and BRAF (1%) genes. Then, 439 patients were selected, presenting both CGP test performed on specimen collected at the time of surgery and initiation of first-line therapy within ±8 months from surgery, and categorized into no (NS, n = 74), interval (IS, n = 157), and upfront surgery (n = 208) groups, each comparable by clinical features. The CGP revealed BRCA mutations, at similar frequency in the three groups, in 54/439 patients (12.3%). Patients with pathogenic BRCA mutations had better event-free survival (EFS) compared with those with BRCAwt. Loss-of-heterozygosity (LOH) ≥16 (LOH-positive patients) was found in 142/433 (32.8%) patients, with different prevalence across treatment groups (12.8% NS; 9% IS; 8.8% upfront surgery). Patients treated with poly (ADP-ribose) polymerase inhibitors (PARPi) had improved EFS (hazard ratio for other drugs v PARPi 1.77 [95% CI, 1.21 to 2.58]). Interestingly, in 206 BRCAwt and LOH-negative patients, not eligible for PARPi, CGP detected potentially targetable alterations in 99 of them (48%). CONCLUSION:Overall, our study provides evidence that CGP significantly improves the identification of molecular targets in HGSOC, supporting its importance in the clinical practice to provide patients with more therapeutic options.

BackgroundRetreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening.MethodsPatients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing.ResultsA total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients.ConclusionsThis is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.

Background: Metastatic Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors characterized by high morbidity and limited systemic treatment options, mainly based on radiometabolic treatments or chemotherapy. Based on the preclinical rationale that PGGLs carcinogenesis relies on angiogenesis, treatment with tyrosine kinase inhibitors (TKI) may represent another viable therapeutic option. Methods: We conducted a prospective phase II study in patients with metastatic or unresectable PGGLs. Patients received sunitinib (50 mg daily for 4 weeks, followed by a 2-week rest period) until progressive disease (PD), unacceptable toxicity or consent withdrawal. The primary endpoint was 12-month progression-free survival (PFS) rate; secondary endpoints were safety overall response rate (ORR) according to RECIST 1.1 criteria and overall survival (OS). EudraCT Number: 2011-002632-99. Results: Fifty patients were included. At a median follow-up of 71.7 months (IQR 35.4-100.1), the 1 year-PFS rate was 53.4 % (95 %CI 41.1-69.3) and median PFS was 14.1 months (95 % CI 8.9-25.7). ORR was 15.6 %, the median OS was 49.4 months (95 %CI 21.2-NA), and grade 3 or higher treatment-related adverse events were reported in 34 % patients. No significant correlation was found between specific genetic alterations or genomic clusters and sunitinib efficacy. Conclusion: Sunitinib is an active drug in patients with advanced PGGLs, capable of inducing prolonged disease control with a manageable toxicity profile.

Introduction: Tumour BRCA1/2 sequencing has progressively increased along with the expanding indications for poly(ADP-ribose) polymerase inhibitors. In our study, we investigated the feasibility and outcomes of a workflow for the identification of germline carriers based on tumour sequencing results. Methods: Between April 2020 and December 2022, BRCA1/2 tumour testing results from 2020 patients were reviewed. Analysed tumours included: 323 ovarian, 104 breast, 314 pancreas-biliary, 87 prostate, 374 gastrointestinal, 309 lung, and 509 less common histologies. Testing was performed through small (only BRCA1/2, 16%) or comprehensive (>50 genes) next-generation sequencing panels (84%). Patients with pathogenic/likely pathogenic variants were referred for genetic counselling and germline testing. Results: Tumour BRCA1/2 pathogenic variants were identified in 145 patients (7%). The pathogenic variant frequency ranged between 23% (75/323 ovarian) and 3.5% (11/314 pancreas-biliary). The highest frequency was observed in high-grade ovarian carcinomas (27%, 64/235). By 30 June 2023, 79 out of 145 patients (54%) underwent subsequent genetic counselling and germline testing. In these patients, mostly affected with ovarian carcinoma (67%, 53/79), 48 were confirmed germline pathogenic variants (61%). Conclusions: In our tumour-to-germline testing approach, we observed the BRCA1/2 pathogenic variant frequency reported in other large unselected ovarian cancer cohorts, thus confirming its effectiveness in identifying putative germline carriers irrespective of eligibility for germline testing. As the range of tumours subjected to genetic testing broadens, this approach is expected to also be effective in other tumour settings for enhancing the identification of carriers, reducing the burden on genetic services, and avoiding unnecessary concerns related to germline testing.

Colorectal cancer (CRC) is a global health concern, and the incidence of early onset (EO) CRC, has an upward trend. This study delves into the genomic landscape of EO-CRC, specifically focusing on pediatric (PED) and young adult (YA) patients, comparing them with adult (AD) CRC. In this retrospective monocentric investigation, we performed targeted next-generation sequencing to compare the mutational profile of 38 EO-CRCs patients (eight PED and 30 YA) to those of a 'control group' consisting of 56 AD-CRCs. Our findings reveal distinct molecular profiles in EO-CRC, notably in the WNT and PI3K-AKT pathways. In pediatrics, we observed a significantly higher frequency of RNF43 mutations, whereas APC mutations were more prevalent in adult cases. These observations suggest age-related differences in the activation of the WNT pathway. Pathway and copy number variation analysis reveal that AD-CRC and YA-CRC have more similarities than the pediatric patients. PED shows a peculiar profile with CDK6 amplification and the enrichment of lysine degradation pathway. These findings may open doors for personalized therapies, such as PI3K-AKT pathway inhibitors or CDK6 inhibitors for pediatric patients. Additionally, the distinct molecular signatures of EO-CRC underscore the need for age-specific treatment strategies and precision medicine. This study emphasizes the importance of comprehensive molecular investigations in EO-CRCs, which can potentially improve diagnostic accuracy, prognosis, and therapeutic decisions for these patients. Collaboration between the pediatric and adult oncology community is fundamental to improve oncological outcomes for this rare and challenging pediatric tumor.

PDF file - 76K, Swiss GIST case material.

PDF file - 69K, Italian GIST case material.

Supplementary Figure 1. Evidence of tumor regression and inflammatory infiltration in IM-treated metastatic FS-DFSP. IHC staining of a metastatic FS-DFSP lesions derived from patient #4 in Table 1 and 2. Panel A and B show haematoxylin and eosin stains of (a, b) pre-IM and (c, d, e, f) post-IM lesions. Micrograph picture c shows the presence of a cellular depleted area replaced by hyalinized stroma. Panel B displays the intra-tumor presence of inflammatory cells (indicated by the arrows). Original magnification: a and c 25x; d and e 50x; b and f 100x.

Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended.

Supplementary Table 1. Supervised analysis highlighted the differential expression of 250 genes, with a p value <0.01 and two-fold difference

Introduction: During the last few years it has been shown that an anaplastic T cell lymphoma can develop as a rare and late sequelae of implant-based breast reconstruction. This malignancy was recognized in the 2017 by WHO and named breast implant associated anaplastic large T cell lymphoma (BIA-ALCL). BIA-ALCL usually presents as abundant effusion around the implant, thus, in addition to cytology smears, its diagnosis also requires immunohistochemistry, T cells clonality and cytometry. Due to the increasing attention of clinicians, it is likely that the number of the BIA-ALCL suspected cases will grow in the future, implying the necessity of a reliable and cost-effective diagnostic procedure. Methods: To achieve this goal, we retrospectively analyzed the results of laboratory investigations performed at our Institute (Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Italy) on 44 effusions obtained from 31 women suspected for BIA-ALCL. Results: Through cytology, eight out of 44 effusions showed the presence of BIA-ALCL cells. Lymphoma cells were than confirmed in seven samples by immunohistochemistry and/or T cell clonality and/or cytometry. Overall, cytology showed 100% sensitivity, 97% specificity and positive and negative predictive values of 87.5% and 100% respectively. Further analyses were particularly useful in effusions showing small percentages of BIA-ALCL cells. Moreover, an extended cytometric profile that can be applied when fast confirmation of the cytologic result is required was also identified. Conclusions: Our results evidenced a central role of cytopathology in the management of BIA-ALCL suspected effusions and suggested that further laboratory investigations might be applied only in cases showing atypical/activated lymphoid cells through cytology.