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Purpose:BRAF mutations are grouped in activating RAS-independent signaling as monomers (class 1–V600E) or as dimers (class 2–codons 597/601), and RAS-dependent with impaired kinase activity (class 3–codons 594/596). Although clinical, pathologic, and molecular features of V600EBRAF-mutated metastatic colorectal cancer (mCRC) are well known, limited data are available from the two other classes.Experimental Design:Data from 117 patients with BRAF (92 class 1, 12 class 2, and 13 class 3)-mutated mCRC were collected. A total of 540 BRAF wt mCRCs were included as control. IHC profiling was performed to determine the consensus molecular subtypes (CMS), cytokeratin 7/20 profiles, tumor-infiltrating lymphocyte infiltration, and BM1/BM2 categorization. Overall survival (OS) and progression-free survival were evaluated by Kaplan–Meier and log-rank test.Results:Class 3 BRAF-mutated mCRC was more frequently left sided (P = 0.0028), pN0 (P = 0.0159), and without peritoneal metastases (P = 0.0176) compared with class 1, whereas class 2 cases were similar to class 1. Hazard ratio for OS, as compared with BRAF wt, was 2.38 [95% confidence interval (CI), 1.61–3.54] for class 1, 1.90 (95% CI, 0.85–4.26) for class 2, and 0.93 (95% CI, 0.51–1.69) for class 3 (P < 0.0001). Class 2 and 3 tumors were all assigned to CMS2-3. A higher median CD3/CD8-positive lymphocyte infiltration was observed in BRAF-mutated class 2 (P = 0.033) compared with class 3 cases.Conclusions:For the first time, different clinical and pathologic features and outcome data were reported according to the three BRAF mutation classes in mCRC. Specific targeted treatment strategies should be identified in the near future for such patients.

Colorectal adenosquamous carcinoma (ASC) is exceedingly rare, comprising less than 0.1% of all colorectal malignancies, and is characterized by an aggressive disease course, with a higher metastatic rate and worse outcome than conventional colorectal adenocarcinoma. A comprehensive molecular profile of this group of neoplasms is still lacking. A total of 22 cases of colorectal ASCs (with 22 primary lesions and 7 metastases matched with 4 primaries) were subject to NGS targeting 67 cancer-related genes (VariantPlex solid tumor; Archer). Mismatch repair (MMR), p53, and V600EBRAF status were also investigated by immunohistochemistry. In 28 of 29 (96.6%) ASC samples, at least one single-nucleotide variant (SNV) or copy number variation (CNV) was detected. Among the 22 primary tumors, the most frequently mutated genes were TP53 (59.1%), APC (40.9%), KRAS (27.3%), BRAF (13.6%), and GNAS (9.1%). Only 1/22 (4.5%) primary ASC was MMR-deficient (MMRd) and harbored a BRAF mutation. Limited differences in SNVs were observed between primary and metastatic diseases. This study sheds light on the molecular landscape of colorectal ASCs. According to our data, the genomic profile of colorectal ASC is similar to that of conventional colorectal carcinoma, with significant druggable genetic alterations. Further studies are required to understand the more aggressive clinical behavior of this neoplasm.

The inability to efficiently repair DNA double-strand breaks using the homologous recombination repair pathway is defined as homologous recombination deficiency (HRD). This molecular phenotype represents a positive predictive biomarker for the clinical use of poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors and platinum-based chemotherapy in ovarian cancers. However, HRD is a complex genomic signature, and different methods of analysis have been developed to introduce HRD testing in the clinical setting. This review describes the technical aspects and challenges related to HRD testing in ovarian cancer and outlines the potential pitfalls and challenges that can be encountered in HRD diagnostics.

AimsPoly (ADP-ribose) polymerase (PARP) inhibitors (PARPIs) represent a standard of care for the clinical management of high-grade serous ovarian cancer (HGSOC). The recognition of homologous recombination deficiency (HRD) has emerged as a predictive biomarker of response for first-line PARPIs treatment in patients with HGOSC. On the other hand, this test is extremely complex and therefore it is often externalised. Regrettably, the reliability of outsourced HRD testing can be troubled by inconclusive results and high rejection rates. In this methodological study, we assessed the technical feasibility, interassay and interlaboratory reproducibility of in-house HRD testing using three different commercially available next-generation sequencing assays.MethodsA total of n=20 epithelial ovarian cancer samples previously analysed with MyChoice CDx were subjected to HRD retesting using three different platforms in three different major pathology laboratories, that is, SOPHiA DDM HRD Solution, HRD focus and Oncomine homologous recombination repair pathway predesigned panel. Concordance was calculated by Cohen’s (dual) and Fleiss (triple) κ coefficients.ResultsIn-houseBRCA1/2molecular testing yielded a concordance rate >90.0% among all participating centres. HRD scores were successfully calculated by each institution with a concordance rate of 76.5%. Concerning the external gold standard test, the overall percentage of agreement ranged from 80.0% to 90.0% with a positive percentage agreement ranging from 75.0% to 80.0% and a negative percentage agreement ranging from 80.0% to 100%.ConclusionsIn-house testing for HRD can be reliably performed with commercially available next-generation sequencing assays.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

Background: Peritoneal dissemination represents a poor prognostic indicator in gastric cancer. Despite a comprehensive molecular characterization of this disease, no peritoneal dissemination-specific signature has been identified, limiting the tailoring of the surgical and oncological treatments. In this review, we outline the available literature focusing on the role of the different molecular pathways involved in the acquisition of peritoneal metastatic dissemination. Summary: According to our results, several molecular determinants are associated with peritoneal carcinomatosis and are involved in several cellular and molecular carcinogenetic processes. However, a comprehensive understanding of the complex molecular landscape of gastric carcinosis is still lacking. Key Messages: More efforts should be made toward the integration of molecular and histologic data to perform a risk prediction assessment of peritoneal dissemination based on molecular profiling and histological evaluation.

218 Background: Immune checkpoint inhibitors (ICI) showed high efficacy in both first and subsequent lines in metastatic colorectal cancer with mismatch repair deficiency (dMMR-mCRC); however, they still fail in a minority of patients (pts). In non-preplanned analyses from previous studies, RAS mutations ( RASm) have been related to limited activity of ICI monotherapy (ICIm) as compared to ICI doublets (ICId) in dMMR-mCRC. Emerging data suggest different immunological features in presence of RASm, resulting in lower immunogenicity. Methods: NERDY is a retrospective, monocentric study designed to investigate the effect of ICIm and ICId on dMMR-mCRC basing on RASm. Pts with dMMR-mCRC treated with ICIm/ICId at our Institute were included. Clinical-pathological features for each patient were collected. On primary tumor specimens, proinflammatory pathways and CMS subgroup will be assessed by Nanostring and RNA-Seq respectively. The study is exploratory and no formal hypothesis has been postulated. Both PFS and OS were calculated with Kaplan-Meier. Cox proportional hazard model was adopted in the interaction tests. Primary objective was to assess OS and PFS according to RAS in dMMR-mCRC pts treated with ICIm or ICId. Secondary objectives were to describe the inflammatory infiltrate and TMB in dMMR-mCRC according to RAS status. Results: From June 2015 to January 2022, a total of 126 consecutive dMMR-mCRC pts treated with ICI were included, 33 RASm/93 RASwt. RASm pts were more frequently males (p=0.015) and younger (median age 47 vs 65). An imbalance was observed in sidedness (more right-CRC in RASwt than in RASm as BRAF effect, p=0.001) and timing of ICI (administered in later lines in RASm, p=0.013). No differences in ECOG-PS, histotype, disease burden, stage at diagnosis, treatment with ICIm vs ICId and best response. At a median follow up of 53.5 months (95%CI 34.7-56.9), a trend toward longer PFS in pts treated with ICId over ICIm was found in the overall population (HR 0.62; 95%CI 0.36-1.05; p=0.055), being significantly longer in RASm-only pts (HR 0.41; 95%CI 0.13-1.28; p=0.047) but not in RASwt-only pts (HR 0.64; 95%CI 0.34-1.20; p=0.139). No difference was observed in OS between ICId and ICIm in the overall population (HR 0.64; 95%CI 0.36-1.16; p=0.121), in RASwt (HR 0.59; 95%CI 0.29-1.20; p=0.132) nor in RASm pts (HR 0.59; 95%CI 0.19-1.78; p=0.275). Interaction test for RAS and ICI treatment type was not significant for PFS (HR 0.63; 95%CI 0.21-1.94; p=0.423) nor for OS (HR=1.00; 95%CI 0.29-3.41; p=0.999). Conclusions: Preliminary clinical results of the NERDY study suggest enhanced activity of ICId compared to ICIm in pts with RASm dMMR-mCRC. Further data are expected from pending translational analyses and a pre-planned adjunctive cohort from two other Italian centers will be used for external validation.

The introduction of next-generation sequencing (NGS) in the molecular diagnostic armamentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

In sessile serrated lesions (SSLs) with adenomatous dysplasia, the dysplastic component and the serrated component without dysplasia should be considered as part of the same lesion, classified as SSL with dysplasia. However, some of these lesions may actually represent collisions between a serrated polyp and a conventional adenoma. Further supporting the "collision theory", conventional adenomatous dysplasia may be found in association with hyperplastic polyps (HPs). In order to determine the molecular and biological landscape of conventional type dysplasia in serrated lesions, we collected 17 cases of colorectal serrated lesions with adenomatous dysplasia, classifying them as SSL with dysplasia (n = 10) or as mixed lesions comprising a HP component and a conventional adenomatous component (n = 7). We characterized the dysplastic and the non-dysplastic component of each lesion, after microdissection, through the targeted mutational analysis of 11 commonly altered genes in colorectal cancer (AKT1, APC, BRAF, CTNNB1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, PTEN and TP53). We also characterized MMR and p53 status by immunohistochemistry. Overall, 14/17 (82.4 %) cases harbored a mutation in at least one of the two components. The most altered genes were BRAF in 10/17 (58.8 %) cases, APC in 2/17 (11.8 %) and TP53 in 4/17 (23.5 %). Among the SSL with dysplasia, the mutational profile was concordant between the two components in 7/10 (70 %) cases, while among the mixed lesions, the mutational profile was concordant in 1/7 (14.3 %). In all but two cases of SSL with dysplasia, MMR status was concordant between the two components of the serrated lesions. Our findings suggest that adenomatous dysplasia may develop in SSL as part of the serrated lesion, even if some SSL with dysplasia may actually be collision lesions. On the other hand, the polyps that are morphologically classifiable as mixed lesions composed of a HP and a conventional adenomatous component are more likely to be collision lesions.

2025 Background: Brain metastases (BM) from primary colorectal cancer (prCRC) are rare (1-3%); few is known about CRC BM predictive factors, prognosticators and molecular pathways. High rate of HER2 amplification ( HER2+) in CRC with BM was previously described, being HER2+ overall rare in CRC (