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.

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.

INTRODUCTION:Anti-HER2 drugs are becoming an important therapeutic option for various solid tumors, increasing the need for HER2 status testing. Comprehensive genomic profiling (CGP) panels, including FoundationOne®CDx, are commonly used to assess ERBB2 (encoding for HER2) copy number alterations. We aimed to evaluate the analytical validity of FoundationOne®CDx assay, by comparing ERBB2 copy number data with traditional HER2 status by immunohistochemistry (IHC)/in situ hybridization (ISH) assays in a heterogeneous cohort of solid tumor samples. METHODS:We retrospectively reviewed the 531 cases evaluated by FoundationOne®CDx in our Institution, and HER2 status by IHC/ISH could be internally analyzed in 68 cases, including 31 (45.5%) gastroesophageal, 17 (25.0%) colorectal, four (5.8%) breast and two (2.9%) cholangiocarcinoma patients.Tumors with estimated ERBB2 copy number ⩾ 4 by FoundationOne®CDx, and tumors with strong and complete (3+) membranous staining by IHC and/or a HER2/CEP17 ratio ⩾2 by ISH were considered NGS positive and IHC/ISH positive, respectively. RESULTS:We identified 21 NGS positive cases (30.9%); IHC/ISH analysis confirming overexpression/amplification in 16 cases (sensitivity: 76.2%), while among the 47 NGS negative cases, 45 were confirmed by IHC/ISH results (specificity: 90%), with a positive predictive value of 76.2% and a negative predictive value of 95.7%. CONCLUSIONS:FoundationOne®CDx provides an accurate evaluation of ERBB2 copy number status and may represent a cost-effective option in metastatic cancer patients for whom NGS testing is recommended.

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.

Aims:Molecular analysis of FGFR2 aberrant transcripts became crucial for clinical stratification of intrahepatic cholangiocarcinoma (iCCA) patients. Several strategies, including fluorescent in situ hybridization (FISH) and next generation sequencing (NGS), are commonly used to investigate FGFR aberrations. Here, we evaluated the technical performance of clinically implemented diagnostic strategies in 8 referral Italian institutions on artificial reference formalin-fixed paraffin-embedded (FFPE) samples. Methods:Each participating institution was requested to apply its own diagnostic testing strategy on 8 sections obtained from artificial reference specimens built to harbor FGFR3(17)-TACC3(11) rearrangement and unbalanced FGFR2. A second-round slide set hosting FGFR2(17)-BICC1(3) aberrant transcript was shared to detect clinically relevant FGFR2 fusion. Artificial reference sample was previously validated by the University of Naples Federico II before arranging the shipment. Technical procedures (e.g. extraction methods, testing platforms and assays) were recorded. Results:Overall, cell resuspension yielded higher amounts of DNA and RNA (SNU16 61.5 ng/µl, 38100.0 pg/µl; RT112 118.0/µl, 2140.0 pg/µl, respectively) in comparison with SNU16+ RT112 mixing cell block (0.7 ng/µl DNA and 412.0 pg/µl RNA). Moreover, FFPE samples showed a higher fragmentation index (DIN 1.2 and RIN not calculated) compared with cell line resuspension (DIN 2.2 and 9.5 for SNU16 and RT112; RIN 3.9 and 6.8 for SNU16 and RT112). All participating institutions identified FGFR2(17)-BICC1(3) and FGFR3(17)-TACC3(11) aberrant transcripts. Moreover, ID#2, ID#4, ID#7 institutions also detected FGFR2(3)-CD44(1) rearrangement on RNA, whereas institutions ID#1, ID#2, ID#3, ID#5, ID#6, ID#8 identified FGFR2 CNVs on DNA. Conclusions:NGS represents the most suitable approach in molecular profiling of FGFR aberrant transcripts. Rings trial based on artificial reference samples play a pivotal role in optimizing routine diagnostic procedures filling the gap in clinical stratification of iCCA patients.

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.

LBA3515 Background: Retreatment (re-tx) with anti-EGFR monoclonal antibodies offers a promising approach to extend the continuum of care of patients (pts) with RAS and BRAF wild-type (wt) metastatic colorectal cancer (mCRC) with no mutations of resistance in their ctDNA at the time of treatment re-exposure. Methods: PARERE (NCT04787341) is an open-label, multicenter, randomized phase II trial investigating the optimal sequencing of panitumumab (pani) and regorafenib (rego) in the chemorefractory setting of RAS and BRAF wt mCRC pts, who previously derived benefit from first-line anti-EGFR-containing regimens, then received at least one intervening anti-EGFR-free line of treatment, and were prospectively selected for the absence of RAS and BRAF mutations in their ctDNA. Eligible pts were randomized 1:1 to receive pani followed by rego after progression (arm A) versus the reverse sequence (arm B). Primary endpoint was overall survival (OS). 155 events were required to detect a hazard ratio (HR) of 0.69 in favor of arm B, using a two-sided unstratified log-rank test, with type I error of 0.15 and 80% power. Secondary endpoints included 1 st and 2 nd objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and safety. Results: From December 2020 to December 2024, 428 pts from 37 Italian centers underwent molecular screening, and 213 with RAS and BRAF wt ctDNA were randomized (arm A/B = 106/107). Median age was 61 and 64 years (A/B), most pts had left-sided primary tumors (92/88%, A/B), and had received a median of 2 prior lines of therapy in both tx arms. After a median follow-up of 23.5 months (mos), 194 and 135 1 st and 2 nd disease progression events were recorded, while OS data were not mature yet. Key efficacy outcomes are summarized in the table. Adverse events occurred with the expected frequency and grade in both treatment arms. Conclusions: PARERE is the largest randomized trial demonstrating an ORR and PFS advantage in favor of liquid biopsy-guided anti-EGFR re-tx, compared to regorafenib, in the late-line setting of RAS / BRAF wt mCRC. Clinical trial information: NCT04787341 . ARM A a pani, N = 106 ARM Brego, N = 107 p* p** 1 st PFS, median mos 4.1 2.4 HR: 1.23 (95% CI: 0.93 – 1.64) 0.15 0.12 1 st ORR, % 16.0 1.9 OR: 0.1 (95% CI: 0.01 – 0.44); p < 0.01 1 st DCR, % 59.4 31.8 OR: 0.32 (95% CI: 0.18 – 0.56); p < 0.01 ARM A a rego, N = 75 ARM B pani, N = 70 2 nd PFS, median mos 2.7 3.7 HR: 0.76 (95%CI: 0.54 – 1.07) 0.12 0.07 2 nd ORR, % 0 17.4 OR: NA (95% CI: 3.44 - NA); p < 0.01 2 nd DCR, % 37.3 55.7 OR: 2.17 (95% CI: 1.11 – 4.27); p = 0.02 Per protocol population b 2 nd PFS, median mos 2.7 3.9 HR: 0.71 (95%CI: 0.5 – 1.01) 0.06 0.03 OR, Odds Ratio; CI, confidence interval; NA, not assessable; *Cox proportional hazards model; **stratified log-rank test according to ECOG PS; a reference; b pts actually treated with rego and pani (A/B, N = 68/69).

AIM OF THE STUDY:Incidental small testicular masses (STMs) pose the dilemma of invasive Vs surveillance. To test miRNA 371 kit (M371, CE approved) in predicting germ cells tumor (GCT) in patients with STM. METHODS:Thirty-two consecutive men with a STM between 5 and 20 mm have been prospectively accrued. All patients had miRNA 371 blood testing before surgery. Serum miRNA processation was standardized and certified and following transcription into cDNA with an endogeneous control. The Relative Quantity (RQ) was determined by quantitative polymerase chain reaction (qPCR) following preamplification. Different RQ cut-offs were tested. The results of pre-surgery 371 miRNA were compared to definitive pathology. RESULTS:Median lesion size was 14 mm (IQR 8-18 mm). 21 of 32 patients (65.6 %) had GCTs: 14 seminomatous (S)GCTs, 6 non-seminomatous (NS)GCTs, 1 in-situ germ cell neoplasia (GCNIS). Median RQ was 57.50 (IQR 18.50, 223.75) among GCT patients and 5.00 (IQR 1.00, 13.50) in patients without GCTs. We tested for sensitivity (SE), specificity (SP), positive (PPV) and negative predictive value (NPV) at different RQ cut-offs of 5 and 10. SE, SP, PPV and NPV were 100 %, 45 %, 78.1 %, 100 % 95.2 %, 63.7 %, 83.6 %, 87.3 %, respectively. CONCLUSIONS:Serum miRNA 371 can predict GCTs in selected patients with 5-20 mm STM. These results need to be refined by standardizing the analytical process.

3552 Background: The amount of ctDNA is a proxy for metastatic colorectal cancer (mCRC) disease burden, with potentials for prognostic stratification and treatment monitoring. We investigated two methods based on low-pass whole genome and methylome sequencing (WGS and WMS) for ctDNA detection and quantification in the VALENTINO trial. Methods: All patients from the VALENTINO trial - a phase II trial comparing the addition of 5FU to Panitumamb-based maintenance after first line FOLFOX+Panitumamb induction in RAS wild-type mCRC - were eligible. Baseline (BL) and 8-week (8w) plasma samples were collected for low-pass WGS and WMS analysis. Two methods for ctDNA quantification based on DNA methylation (METER) and copy number alterations (ichorCNA) were assayed. Chi-squared, Wilcoxon and Cox regression tests were used. Performances of WMS and variant allele fraction (VAF) of a 14-gene panel were compared. Results: A BL liquid biopsy was available for 154 patients, with 142 also having an 8w assessment. METER and ichorCNA detected ctDNA in 112 (72.7%) and 94 (59.7%) BL samples, respectively; all discordant cases were METER+ but ichorCNA-. Detection rate increased in the presence of liver metastases (86.0% vs 42.6% for METER, 75.7% vs 23.4% for ichorCNA; both p < 0.001) and decreased with peritoneal metastases (55.6% vs 78.0% for METER, 38.9% vs 66.1% for ichorCNA; p = 0.011, p = 0.006). Tumor fraction (TF) of both BL METER and ichorCNA correlated with the diameter of measurable lesions (both p < 0.001) and CEA (p < 0.001 and p = 0.010). Both PFS and OS were shorter after baseline ctDNA detection with METER (mPFS: 10.6 vs 18.6 months, HR: 1.65, p = 0.010; mOS: 28.7 vs 62.2 months; HR: 2.24, 95%CI: 1.37-3.66; p = 0.001) or ichorCNA (mPFS: 10.6 vs 15.0 months, HR: 1.42, 95%CI: 1.00-2.00, p = 0.047; mOS: 27.8 vs 48.4 months, HR: 1.35, 95%CI: 1.29-2.95; p = 0.002). In the multivariate analysis, METER ctDNA detection was the strongest predictor of both PFS and OS (p = 0.005 and p = 0.001) while ichorCNA ctDNA detection was significantly associated with OS but not with PFS (p = 0.002 and p = 0.093). METER ctDNA TF decreased significantly at 8w in patients with CR, PR, or SD (paired Wilcoxon p = 0.015, p < 0.001, p < 0.001) but not PD (p = 0.560) as the best radiological response. Patients without METER ctDNA clearance at 8w had a higher risks of progression (HR: 2.70, 95%CI: 1.63-4.49; p < 0.001) and death (HR: 3.37, 95%CI: 2.00-5.69; p < 0.001). Among 123 patients with both METER and VAF available, concordance was 78.0% and in 10 and 17 patients, respectively, ctDNA was detected only with METER or only with VAF. The mPFS and mOS of discordant cases were longer than METER+ / VAF+ cases and shorter than METER− / VAF− cases. Conclusions: CtDNA quantification with low-pass WMS by METER retains a prognostic significance, can be used for disease monitoring during treatment and refines ctDNA detection based on a restricted gene panel assay. Clinical trial information: NCT02476045 .

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.

BackgroundLoco-regional recurrence (LRR) poses a clinical challenge for the follow-up of patients treated with curative intent for early-stage breast cancer (EBC). While circulating tumor DNA (ctDNA) has been shown to predict distant metastases, its value for LRR is less characterized.MethodsStarting from an index case with documented LRR and available tumor and plasma samples, we report the analysis of the prospective phase III fenretinide prevention trial, which primarily aimed to assess the incidence of second malignancy in women with T1-T2 N0 EBC. Patients were eligible if they had FFPE and/or frozen tissue from primary or recurrent invasive tumor for next generation sequencing, and at least three serial plasma samples for ctDNA analysis by digital PCR.ResultsThe TP53 R196* mutation was identified in the primary tumor of the index case with a variant allele frequency (VAF) of 29%, and in the LRR with a VAF of 58%. The same mutation was also detected in plasma prior to both the primary and LRR surgeries with VAFs of 0.19% and 0.12%, respectively. Following treatment, the mutation became undetectable in plasma samples during follow-up, consistent with the absence of recurrence. Among 40 eligible patients from the fenretinide prevention trial, 27 (67.5%) had primary tumor somatic variants trackable in plasma. Median age was 55 years (range, 35-78); stage I (16, 59%) and stage II (11, 41%); mostly luminal-like (19, 70%); median follow-up 173 months (range, 98-193); common mutations included PIK3CA (50%), TP53 (30.7%), and PTEN (5.9%). Six patients developed LRR as first event; 4 distant metastases. In all LRR cases, except one, ctDNA was detected prior to surgery and anticipated the clinical diagnosis up to 28 months. Three patients with LRR developed distant metastases 1 to 2 years later.ConclusionThese findings show the potential of ctDNA for the early detection of LRR in EBC, and its promise as a tool for timely interventions and personalized surveillance strategies.

Next-Generation Sequencing (NGS) has an increasing role in patients with advanced non-small-cell lung cancer (aNSCLC) and, in parallel, the use of targeted therapy dramatically improves the outcome of those with actionable alterations. In this scenario, the possible prognostic significance of some features provided by NGS testing and, among these, of Variant Allele Frequency (VAF), is still unclear. Herein, we report a real-world single-center prospective cohort of 88 consecutive patients with aNSCLC profiled by NGS and harboring a classic EGFR mutation, who were treated with the standard first-line tyrosine kinase inhibitor osimertinib. A subset of patients with shorter progression-free survival (PFS) was characterized by extremely high VAF/tumor cellularity, >1.7 adjusted VAF (aVAF). Median PFS was 5.3 months shorter in the high aVAF cohort; a similar trend was observed in overall survival too. No significant association between aVAF and TP53 mutations, Tumor Mutational Burden or other clinical, pathological or molecular features was found. Our findings suggest that NGS could improve the traditional binary classification of EGFR mutations in aNSCLC, highlighting a correlation between high EGFR mutations aVAF and worse outcome.

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.

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.

Durable remissions are observed in 10%-20% of treated patients with advanced metastatic melanoma but the factors associated with long-term complete clinical responses are largely unknown. Here, we report the molecular characteristics of tumor evolution during disease progression along a 9-year clinical course in a patient with advanced disseminated melanoma who received different treatments, including trametinib, ipilimumab, radiation, vemurafenib, surgical tumor debulking and a second ipilimumab course, ultimately achieving complete long-term disease remission.Longitudinal analyses of therapies-resistant metastatic tumors revealed the effects of different treatments on tumor's microenvironment and immunogenicity, ultimately creating a milieu favorable to immunotherapy response. Monitoring of the temporal dynamics of T cells by analysis of the T cell receptor (TCR) repertoire in the tumor and peripheral blood during disease evolution indicated that T-cell clones with common TCR rearrangements, present at low levels at baseline, were maintained and expanded after immunotherapy, and that TCR diversity increased. Analysis of genetic, molecular, and cellular components of the tumor depicted a multistep process in which treatment with kinase inhibitors strongly conditioned the immune microenvironment creating an inflamed milieu converting cold into hot tumors, while ipilimumab impacted and increased the TCR repertoire, a requirement for tumor rejection.Since the optimal sequencing of treatment with antibodies targeting immune checkpoints and kinase inhibitors for advanced melanoma is still clinically debated, this case indicates that immunotherapy success is possible even after progression on targeted therapy.

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.