Mismatch repair deficiency (dMMR)/Microsatellite instability (MSI) plays a central role in colorectal cancer (CRC) as a predictive and prognostic biomarker and as a “red flag” for diagnosis of Lynch Syndrome (LS). HER2 is emerging as a therapeutic target and clinical trials are ongoing. Several reports suggested that HER2 amplification could be mutually exclusive with dMMR/MSI, which is mostly associated with HER2 mutations or other alterations. Clinical and molecular aspects in this field mostly derive from subgroup analyses of trials and real-life data are lacking. We retrospectively collected data on 350 dMMR/MSI CRC patients who underwent HER2 testing in two Italian referral centers from 2016 to 2024. HER2 amplification was evaluated with immunohistochemistry (IHC) and FISH for IHC 2 + patients. Median age was 75 years, 84% were stage II and stage III. BRAF and RAS mutation occurred in 60% and 14% of the patients with available molecular data, respectively. No patients harbored HER2 amplification, suggesting that the co-occurrence of HER2 amplification and dMMR/MSI is essentially anecdotal, making therefore HER2 testing in this subgroup less compelling.

Mismatch repair deficiency (dMMR)/Microsatellite instability (MSI) plays a central role in colorectal cancer (CRC) as a predictive and prognostic biomarker and as a "red flag" for diagnosis of Lynch Syndrome (LS). HER2 is emerging as a therapeutic target and clinical trials are ongoing. Several reports suggested that HER2 amplification could be mutually exclusive with dMMR/MSI, which is mostly associated with HER2 mutations or other alterations. Clinical and molecular aspects in this field mostly derive from subgroup analyses of trials and real-life data are lacking. We retrospectively collected data on 350 dMMR/MSI CRC patients who underwent HER2 testing in two Italian referral centers from 2016 to 2024. HER2 amplification was evaluated with immunohistochemistry (IHC) and FISH for IHC 2+ patients. Median age was 75 years, 84% were stage II and stage III. BRAF and RAS mutation occurred in 60% and 14% of the patients with available molecular data, respectively. No patients harbored HER2 amplification, suggesting that the co-occurrence of HER2 amplification and dMMR/MSI is essentially anecdotal, making therefore HER2 testing in this subgroup less compelling.

Patients with colorectal cancer (CRC) and Lynch syndrome (LS) have elevated cancer risks and require personalized treatment and targeted surveillance. At-risk relatives can also benefit from preventive measures. However, LS remains largely undiagnosed. Integration of universal tumor screening (UTS) with mainstream genetic testing in patients with CRC has been recommended, but implementation of this approach remains inconsistent. This scoping review mapped evidence on barriers and facilitators in diagnostic pathways that integrate UTS and mainstream genetic testing for LS in CRC. A search of MEDLINE, Scopus, CINAHL and PsycINFO was conducted, and five articles were selected. Barriers and facilitators were mapped to the Theoretical Domains Framework (TDF). The most frequently mapped domains were Environmental Context and Resources (in 5/5 articles) Social/Professional Role and Identity, Beliefs about Consequences, Social Influences, and Behavioral Regulation (all in 4/5 articles). Barriers included insufficient clinician education, difficulties in navigating consent processes and interpreting testing results, and perceived lack of evidence demonstrating the benefit of genetic testing. Unclear and conflicting perceptions among different professional groups about who should be responsible for essential components of genetic testing and follow-up also emerged. Social Influences such as strong leadership, collaboration, and networked support from “champions” acted as key facilitators. Mechanisms of Behavioral Regulation, including audit, feedback, and ongoing education, as well as appropriate resourcing, were identified as essential for sustaining practice change. Future studies could usefully explore patients’ views and preferences regarding novel diagnostic pathways for LS in CRC that align UTS and mainstream genetic testing, as well as strategies developed and implemented in low-resource settings.

10618 Background: International guidelines recommend universal Lynch syndrome (LS) screening in colorectal cancer (CRC) using tumor mismatch repair deficiency (dMMR) testing. However, LS remains underdiagnosed worldwide, largely due to inconsistent referral for genetic counseling and germline testing. We report the final feasibility results of a novel, mainstreamed, oncologist-led diagnostic algorithm for LS. Methods: ItaLynch is a prospective, observational, multicenter Italian study on patients with dMMR CRC (May 2021–Dec 2025; 23 high-volume centers). The pathway includes: (1) universal dMMR screening by immunohistochemistry (IHC) in all CRC; (2) reflex testing and “Lynch alert”: MLH1-deficient (dMLH1) tumors undergo BRAFV600E testing and, if wild-type, MLH1 promoter methylation; a Lynch alert is added to all dMMR pathology reports (positive for high LS risk based on reflex testing or non-MLH1 loss; negative for BRAFV600E-mutated or MLH1-hypermethylated cases); (3) oncologist-led mainstream germline testing for patients with positive alert, followed by post-test genetic counseling for carriers of pathogenic variants (PV) or clinically suspicious cases. Results: Up to Dec. 31 2025, we enrolled 1,482 pts with dMMR CRC. Overall, 1,316 were eligible for analysis (14 not eligible; 152 not yet filled out). Excluding 104 cases with incomplete data, 1,212 patients were evaluable from somatic to germline testing. Overall, 1033 (85%) completed the ItaLynch diagnostic pathway, demonstrating the strong feasibility of this this algorithm. Of patients with IHC data, 975 (78%) were dMLH1 and 281 (22%) had non-MLH1 loss. BRAF testing was performed in 904/975 (93%) dMLH1 cases; 386/904 (43%) were BRAFwt. MLH1 methylation testing was done in 329/386 (86%); 146/329 (44%) were not hypermethylated. Germline testing was performed in 108/146 (74%), identifying 21 LS-associated PV (19%). Among non-MLH1 loss, 232/281 (83%) underwent germline testing and 115 (50%) were diagnosed with LS. Overall, germline testing was performed in 361/1,033 adherent patients (35%), identifying 143 PV (40% of tests) and 24 VUS; PV prevalence was 14% (143/1,033). Mainstreaming reduced genetic pre-test counseling visits by 54% (196/361 avoided) and, compared with universal germline testing, avoided 672/1,033 tests (65%). Post-test counseling uptake was 83% among PV carriers. Median turnaround time from somatic to germline diagnosis was 217 days for dMLH1 and 73 days for non-MLH1. Among non-adherent patients, 67 underwent germline testing (14 PV, 3 VUS). In the full cohort, 157 PV were identified (MLH1 37, MSH2 57, MSH6 43, PMS2 20). Conclusions: This oncologist-led, mainstreamed diagnostic pathway for LS is feasible and optimized use of healthcare resources. Analyses of pathway deviations (15%), cost-effectiveness, and clinical/translational outcomes are ongoing.

10580 Background: International guidelines recommend universal screening for LS through somatic DNA mismatch repair deficiency (dMMR) testing in CRC. However, LS remains largely underdiagnosed, often due to inconsistent referrals from oncologists to genetic counseling and germline testing. We report preliminary results of the ItaLynch study, proposing a mainstream, oncologist-led diagnostic pathway for LS. Methods: ItaLynch is a prospective, observational, multicenter, multidisciplinary Italian study on pts with dMMR CRC. It started in May 2021, and is ongoing in 23 high-volume centers. The ItaLynch diagnostic pathway is based on three key steps. The first step is universal screening through dMMR testing by immunohistochemistry (IHC) in all CRC pts. The second step consists of reflex testing and a Lynch alert: MLH1-deficient (dMLH1) pts undergo reflex testing for BRAF V600E and, if wild-type, for MLH1 promoter methylation. A Lynch alert is added to the pathology report of all dMMR CRC pts. The alert is positive for pts with a high risk of LS as per reflex testing results or loss of non-MLH1 proteins; it is negative for pts not likely to have a hereditary predisposition (i.e. BRAF V600E mut or MLH1 promoter hypermethylated dMLH1). The third and most innovative step is the oncologist-led mainstreaming germline testing for pts with a positive Lynch alert. Carriers of a germline pathogenic variant (PV) as well as those who have non-informative test results but are clinically suspicious are then referred to post-test genetic counselling. Results: Up to Dec. 19 2024, we enrolled 1,146 pts with dMMR CRC. Among the 937 pts eligible for the current analysis, 714 (76%) were dMLH1, and 223 (24%) displayed loss of non-MLH1 proteins. Reflex testing was carried out in 653 (91%) of the dMLH1 pts and 271 (41%) were BRAF wt. Of these, 219 (80%) subsequently underwent MLH1 promoter methylation testing, and 98 (45%) were not hypermethylated. Of these, 60 (61%) underwent oncologist-led germline genetic testing, and 11 (18%) were carriers of an LS-associated PV. Among the 223 cases with loss of non-MLH1 proteins, 157 (70%) underwent genetic testing, and 86 (55%) were diagnosed with LS. At the time of writing, the overall proportion of LS cases is 10% (97/937 pts with data available for the current analysis). These 97 LS cases represent 45% of the 217 pts who were flagged with a positive Lynch alert and underwent oncologist-led genetic testing (60 of which with dMLH1 and 157 with non-MLH1 loss). Conclusions: Overall, our large cohort is representative of the population of pts with dMMR CRC. Our novel diagnostic algorithm, through the implementation of the Lynch Alert flagging system that identifies dMMR CRC pts with a high likelihood of LS and of an oncologist-led germline genetic testing, obtained a high diagnostic yield. Further analyses are ongoing on the entire cohort to evaluate the feasibility of the proposed diagnostic pathway.

At the end of the past century, the introduction of Total Mesorectal Excision (TME), preceded by either short-course radiotherapy (SCRT) or chemoradiation (CRT), established the new standard of care for locally advanced rectal cancer (LARC). Recently, significant advancements were achieved for both dMMR/MSI and pMMR/MSS LARC patients. For the 2–3% of dMMR/MSI LARCs, ablative immunotherapy emerged as a curative approach, offering the possibility of avoiding chemotherapy (CT), radiotherapy, and surgery altogether. In pMMR/MSS LARCs, the intensification of preoperative treatments with Total Neoadjuvant Treatment (TNT) afforded three outcomes: (a) a reduction of distant metastases, positively impacting on survival endpoints, (b) a significant increase of complete clinical response (cCR) rate, paving the way for non-operative management (NOM), and (c) the selective omission of radiotherapy following induction CT. The choice of the most appropriate therapeutic strategy can only be made through the shared decision-making process between physician and patient based on risk stratification and patient preferences.

BACKGROUND:Management of gastrointestinal (GI) cancers has shifted from conventional chemotherapy to biomarker-based precision oncology. Biomarker assessment requires adequate endoscopic biopsy tissue both in gastro-esophageal/gastric and colorectal carcinomas. AIMS:This study evaluated real-world endoscopic biopsy adequacy, focusing on tissue quality and suitability for biomarker analysis. METHODS:We retrospectively reviewed 819 endoscopic procedures (274 upper-GI and 545 lower-GI; time-window: January 2021-2024). Gastrointestinal pathologists reviewed 4,908 biopsies to assess diagnostic yield, number of invasive carcinoma-containing biopsies, and tumor cellularity. Biopsy adequacy was evaluated against European Society of Gastrointestinal Endoscopy (ESGE) recommendations and biomarker-specific cellularity thresholds. RESULTS:A histologic diagnosis of invasive carcinoma was established in 96 % of upper-GI and 84 % of lower-GI procedures (p<0.001). However, 41-43 % of procedures yielded fewer than six biopsies, which is below ESGE guidance. Importantly, only 66.7 % of upper-GI and 49.7 % of lower-GI biopsies contained invasive carcinoma, while the rest were composed of samples inadequate for biomarker testing (such as non-invasive lesions, mucin, necrosis, granulation tissue, and normal mucosa). Low neoplastic cellularity (<1000 tumor cells) was observed in 27 % of upper-GI and 5 % of lower-GI cases, while <20 % tumor cellularity was present in 41.7 % of colorectal biopsies. CONCLUSION:Optimizing sampling strategies and ensuring representative, high-cellularity specimens are essential to support precision oncology in GI cancers.

Background Universal screening of colorectal cancer (CRC) patients for Lynch syndrome (LS) through MisMatch Repair (MMR) testing is recommended. BRAFV600E mutation and/or MLH1 promoter methylation (Reflex Testing, RefT)generally rule out LS in MLH1-deficient (dMLH1) patients. We estimated the impact of RefTon genetic counseling (GC) and on the diagnostic yield of genetic testing (GT). Methods Overall, 3199 CRC patients were referred to our center between 2011 and 2021. Patients referred until January 2019 (n=2536) underwent universal MMR testing and were termed ‘Cohort A’; among patients after February 2019 (n=663), ‘Cohort B’, RefT was also performed in dMLH1 patients. Results Overall, 401/3199 patients (12.5%) were MMR-deficient (dMMR); 312 (77.8%) in cohort A and 89 (22.2%) inB; 346/401 were dMLH1 (86.3%), 262/312 (83.9%) in cohort A and 84/89 (94.3%) in B. In Cohort A, 91/312 (29.1%) dMMR patients were referred to GC, 69/91 (75.8%) were in the dMLH1 group; 57/69 (82.6%) dMLH1 patients underwent GT and 1/57 (1.7%) had LS. In Cohort B, 3/84 dMLH1 patients did not undergo BRAF testing. Three BRAF wt and not hypermethylated of the remaining 81 dMLH1 patients were referred to GC and GT, and one had LS. This diagnostic pathway reduced GC referrals by 96% (78/81) in Cohort B and increased the diagnostic yield of GT by about 20 times. Conclusion Our findings support RefT in dMLH1 CRC patients within the LS diagnostic pathway, as it reduces the number of GC sessions needed and increases the diagnostic yield of GT.

BackgroundPatients with metastatic colorectal cancer (mCRC) carrying a deficit in the mismatch repair system/microsatellite instability (dMMR/MSI) show great responses to immune checkpoint inhibitors. However, 30% of patients with dMMR/MSI are primarily immunoresistant, and another 30% develop secondary resistance. Thus several combinations such as anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) are being pursued. The combination of radiotherapy and immunotherapy is another avenue of research that can increase the release of neoantigens resulting in the abscopal effect. This phenomenon has demonstrated promising potential activity in colon cancer preclinical studies; nevertheless, clinical results are limited to just a few case series.Patients and methodsWe conducted a prospective interventional single-institution study to assess the feasibility, safety, and disease control rate of the combination of pembrolizumab and stereotactic ablative radiotherapy (SABR) in a cohort of 14 consecutive patients with dMMR/MSI mCRC.ResultsAmong the 14 patients enrolled, 11 received SABR in combination with pembrolizumab as the first to the fourth line. The disease control rate was 50% in the intention-to-treat population, with six patients still maintaining the response after >15 months. Any-grade treatment-related adverse events occurred in 50% of patients, with grade 3 (G3) events in three patients; no treatment-related death occurred.ConclusionsOur findings convey no signal of enhanced systemic efficacy compared with historical data on pembrolizumab alone even if the local control rate is high. To our knowledge, this represents the largest study conducted in this population; further studies could extend the knowledge on the toxicity profile of this combination.

Abstract Background: The backbone chemotherapy of first-line standard of care (SOC) for microsatellite stable (MSS) metastatic colorectal cancer (mCRC) combines 5-Fluorouracil to oxaliplatin and/or irinotecan. There are no biomarkers to predict response, which is complete or long-lasting (CR/LLR) in 20-25% of patients, while 10-15% are primary refractory. The aim of this work is to develop a predictive biomarker, based on digital pathology images, that can help stratify patients according to their risk of resistance. Methods: We trained a supervised bag-of-words artificial intelligence (AI)-based model on a cohort of response outliers mCRC patients classified as “really sensitive” (RS) if they achieved CR or LLR >10 months to any SOC, or “really resistant” (RR) if progression occurred at first disease reassessment. Whole-slide Imaging (WSI) of the resected primary tumors were tiled into patches of 224 × 224 pixel (0.5μm/pixel). First-order and texture features were subsequently extracted from all tumoral tiles and grouped into homogenous clusters through a k-means algorithm (k=6). For each patient, the percentage of tiles belonging to each tiles’ cluster was computed to represent new features (called bag of words) with whom different machine learning classifiers were trained. Main clinicopathological features were matched to treatment response by Fisher’s exact test. Results: To date, we analyzed 82 response outlier patients, of whom 35 were classified as RS and 46 RR. Of them, patients identified at Italian centers were used as construction cohort (N=47; 27 RR and 20 RS) and those identified at Spanish centers were used a validation cohort (N=35; 19 RR and 16 RS). The best result was obtained using a stepwise logistic regression, reaching a negative predictive value (NPV) of 90% (18/20; 95% CI=70-97%) and 71% (10/14; 95% CI=49-87%), in the construction and validation sets. No standard clinicopathological features (including stage, RAS/BRAF status, histology and sidedness) was associated with the chance of being RR. Conclusions: We demonstrated that a pathomics signature has the potential to predict resistance to SOC in MSS mCRC. Further validation of these preliminary findings on a larger cohort of response outlier patients is ongoing. Citation Format: Luca Lazzari, Gianluca Mauri, Valentina Giannini, Debora Cafaro, Giulia Nicoletti, Caterina Marchiò, Andrea Sartore-Bianchi, Federica Marmorino, Maria Nieva Munoz, Nadia Saoudi Gonzalez, Alberto Puccini, Chiara Cremolini, Clara Montagut, Elena Elez, Stefania Sciallero, Enrico Berrino, Martina Carullo, Pietro Paolo Vitiello, Maria Costanza Aquilano, Martina Di Como, Emanuela Bonoldi, Salvatore Siena, Alberto Bardelli, Daniele Regge, Silvia Marsoni. Development and validation of an artificial-intelligence-based pathomics biomarker to predict resistance to first-line treatment in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 900.

BACKGROUND AND AIM:Germline BRCA1-2 test is routinely recommended in Pancreatic Cancer (PC) patients, due to its clinical-epidemiological relevance. Data on the prevalence of germline pathogenic variants (gPV) in other cancer predisposition and DNA Damage Repair (DDR) system-related genes in unselected PC cases are sparce in Italy. We assessed this prevalence in a multicentre cohort, to derive recommendations for PC patients. METHODS:Clinical data of 1200 consecutive PC patients, of any age and stage, tested with a multigene germline panel were collected. A descriptive analysis of gPV frequency and clinical variables was performed both in 1092 patients tested for an 18 genes core-panel (CP-18 cohort) and in 869 patients screened only for CDKN2A. RESULTS:11.5 % (126/1092) of CP-18 cohort patients harbored a gPV in ≥ 1 gene. Highest gPV frequencies were detected in ATM (3.1 %), BRCA2 (2.9 %), BRCA1 (1.6 %), CHEK2 (1.1 %). Patients harboring any CP-18 gene and BRCA1-2 gPV were younger and with a higher rate of personal (PH) or family history (FH) of cancer when compared to no gPV patients. The risk of having a gPV was ≥ 7 % in all subgroups of patients, including those aged > 73, with tumor stage I-III and negative FH/PH. CDKN2A gPV were detected in 2.6 % (23/869) of patients. CONCLUSIONS:A remarkable prevalence of gPV in cancer predisposition and DDR genes is reported in this large multicentre cohort of consecutive and unselected PC patients. Therefore, we recommend multigene germline testing (at least including BRCA1-2, ATM, CDKN2A, PALB2) for all PC patients, irrespective of age, stage, PH/FH.

Aims: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS-associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAF(V600E)IHC and BRAF molecular analysis in a large, mono-institutional CRC whole-slide, case series with laboratory validation.Methods and results: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAF(V600E)IHC were performed on all unselected cases of surgically resected CRCs (2018-2023). An in-house validation study for BRAF(V600E)IHC was performed in order to obtain optimal IHC stains. BRAFV(V600E)IHC was considered negative (score 0), positive (scores 2-3), and equivocal (score 1). Interobserver differences in BRAF(V600E)IHC scoring were noted in the first 150 cases prospectively collected. Nine-hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAF(V600E)IHC (BRAF(V600E)IHC-positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAF(V600E)IHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAF(V600E)IHC expression. Among the 12 IHC-equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF-mutated and nine BRAF-wildtype.Conclusion: BRAF(V600E)IHC can be used as a reliable surrogate of molecular testing after stringent in-house validation.

Mismatch repair (MMR) immunohistochemical (IHC) evaluation has entered pathology routine practice as the first-line screening method to identify patients with MMR deficient (MMRd)/microsatellite instability (MSI) colorectal cancer (CRC), and its misdiagnosis may significantly impact the personalization of CRC patient care. To determine the prevalence of MMR protein intratumor heterogeneity in real-world practice, we collected a series of 8282 CRCs tested for MMR proteins in the setting of Lynch syndrome universal screening. Four heterogenous cases were also investigated for tumor infiltrating lymphocytes count, MSI status, and consensus molecular subtypes by Nanostring nCounter® Platform. Overall, 1056 (12.8%) CRCs showed a MMR altered status, with 46 cases showing a heterogeneous MMR profile (0.56% of the total, and 4.36% of all MMRd cases). To conclude, the authors make some critical remarks regarding the approach to MMR heterogeneity in clinical practice and routine diagnostics.

Despite its clinical value, cascade genetic testing (CGT) in hereditary cancer syndromes remains underutilized for a number of reasons, including ineffective family communication of genetic risk information. Therefore, alternative strategies are being explored to improve CGT uptake rates; one such strategy is direct contact with at-risk relatives by healthcare professionals with proband consent. It is unclear how Italian laws and regulations pertaining to CGT—including the EU General Data Protection Regulation (GDPR)—should be understood and implemented in the context of such alternative strategies. The authors constructed a hypothetical case about CGT, reviewed laws and regulations on informed consent, privacy, and the right not to know, and analyzed how those laws and regulations might apply to different communicative strategies relevant to the case and aimed at supporting CGT. A constitutionally consistent reading of Italian law and of the GDPR, an integral part of the Italian privacy framework, suggests that multiple communicative approaches may be legally permissible in Italy to support the CGT process. This includes direct contact by healthcare professionals with proband consent, provided certain conditions are met. Understanding the effectiveness of such approaches in improving CGT uptake will require further research efforts.

Background Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling.Methods Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification.Results The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition.Conclusions Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Introduction Care for head and neck cancers is complex in particular for the rare ones. Knowledge is limited and histological heterogeneity adds complexity to the rarity. There is a wide consensus that to support clinical research on rare cancer, clinical registries should be developed within networks specializing in rare cancers. In the EU, a unique opportunity is provided by the European Reference Networks (ERN). The ERN EURACAN is dedicated to rare adults solid cancers, here we present the protocol of the EURACAN registry on rare head and neck cancers (ClinicalTrials.gov Identifier: NCT05483374). Study design Registry-based cohort study including only people with rare head and neck cancers. Objectives to help describe the natural history of rare head and neck cancers; to evaluate factors that influence prognosis; to assess treatment effectiveness; to measure indicators of quality of care. Methods Settings and participants It is an hospital based registry established in hospitals with expertise in head and neck cancers. Only adult patients with epithelial tumours of nasopharynx; nasal cavity and paranasal sinuses; salivary gland cancer in large and small salivary glands; and middle ear will be included in the registry. This registry won’t select a sample of patients. Each patient in the facility who meets the above mentioned inclusion criteria will be followed prospectively and longitudinally with follow-up at cancer progression and / or cancer relapse or patient death. It is a secondary use of data which will be collected from the clinical records. The data collected for the registry will not entail further examinations or admissions to the facility and/or additional appointments to those normally provided for the patient follow-up. Variables Data will be collected on patient characteristics (eg. patient demographics, lifestyle, medical history, health status); exposure data (eg. disease, procedures, treatments of interest) and outcomes (e.g. survival, progression, progression-free survival, etc.). In addition, data on potential confounders (e.g. comorbidity; functional status etc.) will be also collected. Statistical methods The data analyses will include descriptive statistics showing patterns of patients’ and cancers’ variables and indicators describing the quality of care. Multivariable Cox’s proportional hazards model and Hazard ratios (HR) for all-cause or cause specific mortality will be used to determine independent predictors of overall survival, recurrence etc. Variables to include in the multivariable regression model will be selected based on the results of univariable analysis. The role of confounding or effect modifiers will be evaluated using stratified analysis or sensitivity analysis. To assess treatment effectiveness, multivariable models with propensity score adjustment and progression-free survival will be performed. Adequate statistical (eg. marginal structural model) methods will be used if time-varying treatments/confounders and confounding by indication (selective prescribing) will be present. Results The registry initiated recruiting in May 2022. The estimated completion date is December 2030 upon agreement on the achievement of all the registry objectives. As of October 2022, the registry is recruiting. There will be a risk of limited representativeness due to the hospital-based nature of the registry and to the fact that hospital contributing to the registry are expert centres for these rare cancers. Clinical Follow-up could also be an issue but active search of the life status of the patients will be guaranteed.

Mismatch repair (MMR) testing on all new cases of colorectal cancer (CRC) has customarily been preferably performed on surgical specimens, as more tissue is available; however, new clinical trials for the use of immune checkpoint inhibitors in the neoadjuvant setting require MMR testing on biopsy samples. This study aims at identifying advantages, disadvantages and any potential pitfalls in MMR evaluation on biopsy tissue and how to cope with them. The study is prospective-retrospective, recruiting 141 biopsies (86 proficient (p)MMR and 55 deficient (d)MMR) and 97 paired surgical specimens (48 pMMR; 49 dMMR). In biopsy specimens, a high number of indeterminate stains was observed, in particular for MLH1 (31 cases, 56.4%). The main reasons were a punctate nuclear expression of MLH1, relatively weak MLH1 nuclear expression compared to internal controls, or both (making MLH1 loss difficult to interpret), which was solved by reducing primary incubation times for MLH1. A mean of ≥ 5 biopsies had adequate immunostains, compared to ≤ 3 biopsies in inadequate cases. Conversely, surgical specimens rarely suffered from indeterminate reactions, while weaker staining intensity ( p < 0.007) for MLH1 and PMS2 and increased patchiness grade ( p < 0.0001) were seen. Central artefacts were almost exclusive to surgical specimens. MMR status classification was possible in 92/97 matched biopsy/resection specimen cases, and all of these were concordant (47 pMMR and 45 dMMR). Evaluation of MMR status on CRC biopsy samples is feasible, if pitfalls in interpretation are known, making laboratory-specific appropriate staining protocols fundamental for high-quality diagnoses.

2608 Background: Colorectal cancer (CRC) patients with mismatch repair system deficiency (dMMR/MSI-H) have shown impressive responses to immune checkpoint inhibitors in any lines of treatment. However, almost 30% of these patients are primary refractory and another 30% develop secondary resistance. Thus, many treatment combinations are being pursued to overcome resistance. One of the proposed strategies is the addition of radiotherapy to immunotherapy based on the potential abscopal effect and its consequent on-target and off-target activity. Methods: Between November 2019 and April 2021, we conducted a prospective interventional single-institution study to assess the feasibility, disease control and safety of the combination of pembrolizumab with stereotactic ablative radiotherapy (SABR) in a cohort of 14 consecutive dMMR CRC patients. SABR was administered in combination within the second or third cycle of Pembrolizumab 200 mg infused every 21 days. SABR was delivered on one or more targetable metastases with the ablative dose of 15 Gy in the first fraction followed by a second fraction of 5 Gy. Results: Fourteen patients were enrolled and 11 received radiotherapy in combination with Pembrolizumab as per protocol. Three patients who started Pembrolizumab never received radiotherapy due to deterioration of their general conditions, leading to death within two months. Two out of these three patients were BRAF V600E mutated. Five patients (35.7%) received Pembrolizumab as first line, 6 (43%) as second line and 3 (21%) as third or later lines. Six out of 14 patients (42.9%) harboured BRAF V600E mutation, 2 out of 14 (14.3%) RAS mutation and the others were RAS/BRAF wild type. Objective response was observed in 4 out of 14 patients (29%) and disease control was achieved in 7/14 patients (50%). Among these, six patients (42%) achieved a disease control lasting more than 15 months. This regimen caused substantial toxicity, mainly linked to SABR. Any grade TRAEs occurred in 50% of patients, with one patient with G1 event, three patients with G2 events, and two patients with G3 events (pneumonia, bowel perforation); no treatment-related deaths were observed. Conclusions: These data do not show hint of added efficacy of Pembrolizumab by SART in unresectable dMMR/MSI-H mCRC.