{"paper_id":"3ae2f221-7cc5-40fd-9307-0fef1827d9ee","body_text":"Absence of co-occurrence between HER2 amplification and dMMR/MSI in colorectal cancer: a multicentric study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Absence of co-occurrence between HER2 amplification and dMMR/MSI in colorectal cancer: a multicentric study Annalice Gandini, Federica Tosi, Luca Carmisciano, Federica Grillo, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9105189/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract 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. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Gastroenterology Health sciences/Oncology colorectal cancer dMMR/MSI HER2 precision medicine Figures Figure 1 Highlights • No HER2 amplification detected in 350 dMMR/MSI CRCs from two different centers. • Co-occurrence of HER2 amplification and dMMR/MSI appears anecdotal in CRC. • Routine HER2 testing in dMMR/MSI CRC may be unnecessary. Background The panorama of colorectal cancer (CRC) treatment has evolved in the last few decades thanks to the identification of molecular biomarkers able to predict response to specific therapies. The major determinant in CRC molecular classification is the identification of mismatch repair deficiency (dMMR)/microsatellite instability (MSI), which has both prognostic and therapeutic implications. dMMR/MSI is due to a deficit in the DNA mismatch repair system (MMR), which accounts for around 15% of all CRCs. Immunohistochemistry (IHC) is considered the most cost-effective methodology for testing as it allows the identification of the loss of expression of MMR proteins (MLH1, PMS2, MSH2, or MSH6), leading to the dMMR phenotype. Molecular biology defines the MSI phenotype, and it is usually recommended in equivocal IHC results in CRCs 1 . The identification of dMMR/MSI CRCs has now become mandatory at the time of diagnosis, regardless of the stage of the disease, due to two main reasons. First, it allows the identification of patients at risk of being affected by Lynch Syndrome (LS), an autosomal dominant inherited disorder caused by a germline pathogenic variant in the MMR genes 2 . Moreover, it predicts sensitivity to immunotherapy, both in early and advanced disease, which has revolutionized the therapeutic algorithm and the prognosis of these patients 3 . dMMR/MSI CRCs show a different clinical behavior when compared to proficient MMR (pMMR)/microsatellite stable (MSS) CRCs, and the impact on prognosis seems to vary between different stages of disease. While in advanced disease (5% of all CRCs), before the advent of immunotherapy, it identified a subpopulation with poorer prognosis 1 , in localized stages, it is associated with better prognosis when compared to pMMR/MSS, even if this better prognostic impact seems to be lost in stage III high-risk patients 4 . Due to the deficit in the DNA repair mechanism, and the subsequent accumulation of mutations, dMMR/MSI patients seem to be enriched in targetable genomic alterations, such as HER2 mutations, NTRK fusions 5 , as well as BRAF V600E mutation, which excludes the possibility of LS 6 . In this regard, although BRAF V600E represents an actionable alteration, microsatellite instability remains the dominant therapeutic driver, making immunotherapy the preferred upfront therapeutic choice. Accordingly, ongoing first-line trials such as SEAMARK are evaluating the combination of immunotherapy with anti-BRAF and anti-EGFR inhibitors 7 . However, the known clinical and molecular aspects of dMMR/MSI CRCs mostly derive from subgroup analyses of selected clinical trials, and data on large real-life cohorts are lacking. With the increasing identification of targetable alterations in CRC, such as KRAS G12C mutations and HER2 amplification, optimizing patient selection for molecular testing has become a relevant clinical issue. Current ESMO guidelines recommend extended next-generation sequencing (NGS) in advanced disease only when it does not generate additional costs, and not in localized CRC 8 . While all RAS and BRAF testing are routinely performed in the metastatic setting because of their predictive value for target therapies, HER2 amplification testing is not yet standard in Europe, despite emerging targeted options 9 . HER2 amplification occurs in approximately 2–3% of metastatic CRCs and has been associated with poor prognosis and resistance to anti-EGFR therapies 10–12 , while its prevalence in localized disease appears to be even lower 13,14 . Importantly, preliminary evidence suggests that HER2 amplification may be exceptionally rare in dMMR/MSI tumors, raising the hypothesis of a potential mutual exclusivity between these two molecular alterations 15 . If confirmed, this would have practical implications by identifying patient subsets in whom HER2 testing is unlikely to be informative, thereby avoiding low-yield analyses and improving the efficiency of precision medicine strategies. The aim of this study is to evaluate clinical and molecular characteristics of a large multicentric real-world cohort of unselected dMMR/MSI CRC patients, focusing on the co-occurrence of dMMR/MSI status and HER2 amplification as proof-of-concept. Results Study population Overall, 350 dMMR/MSI CRCs were identified, 293 from Genoa and 57 from Milan. Patients were mostly female (66%), with a median age of 75 years. Most patients had a localized CRC at the time of the diagnosis (stage I to III = 93%), and proximal tumors (86%), defined as right-sided or transverse colon cancer. More than half of CRCs were high grade - G3 and G4 (193/350 cases – 56%). Focusing on the IHC results in this cohort of dMMR/MSI CRC patients, loss of MLH-1/PMS2 was the most frequently detected (87%). Complete patients’ characteristics are reported in Table 1 . Table 1: Patient characteristics. Acronyms: dMMR= deficit of the Mismatch repair system. MSI= Microsatellite instability. IHC= Immunohistochemistry. SD= standard deviation. Characteristics N = 350 (%) Age – mean (SD) 74.93 (11.41) Sex Male Female 119 (34) 231 (66) Primary tumor location Right colon Transverse colon Left colon Rectum Multiple Unknown 266 (77.1) 31 (9) 40 (11.6) 6 (1.7) 2 (0.6) 5 Disease stage at diagnosis I II III IV 32 (9.1) 189 (54) 105 (30) 24 (6.9) Histotype Adenocarcinoma Medullary Mucinous Poorly differentiated 310 (88.6) 1 (0.3) 23 (6.6) 16 (4.6) Grade Low High Unknown 153 (44) 193 (56) 4 BRAF status Mutated Wild type Unknown 171 (48.9) 110 (31.4) 69 (19.7) RAS status Mutated Wild type Unknown 10 (2.9) 59 (16.9) 281 (80.3) Loss of MMR Proteins by IHC MLH-1 PMS-2 MSH-2 MSH-6 Other 304 (86.9) 24 (6.9) 22 (6.2) HER2 Positive Negative Unknown 0 349 1 Pathological and molecular variables Among the 350 patients, RAS status was available for 69 patients (20%) and, among these, 14% were mutated. Around 80% of the patients were tested for BRAF status, and 60% of these were mutated. None of the 350 patients tested for HER2 showed protein overexpression/gene amplification. Only one patient with a HER2 IHC 2 + was not tested through FISH and thus should be considered as “unknown”. Details on HER2 testing results are available in Table 2 . Assuming the prevalence of HER2 in the general CRC population is 2% 10 , and the prevalence of dMMR/MSI in non-metastatic CRC is around 15% 16 , then, under the assumption of independence, their joint prevalence would be approximately 0.3%. Under this same independence assumption, approximately 7 HER2 positive cases would be expected among 350 dMMR/MSI patients. In our cohort of 350 CRC dMMR/MSI patients, we identified no HER2 positive cases. We estimate a prevalence of HER2 in MSI cases between 0% and 0.9% with 95% confidence. The probability of observing a number of HER2 cases of 0 under the assumption of independence between HER2 and MSI is ~ 0.085%. Although we cannot formally establish mutual exclusivity, the absence of any HER2-positive cases in our MSI cases series indicates that their co-occurrence is essentially anecdotal. Table 2 Results of HER2 testing in Genoa and Milan, according to both scoring systems (GC and Heracles). *One patient with an IHC score of 2 + did not undergo FISH analysis due to technical issues. Genoa Milan GC scoring Heracles scoring Heracles scoring IHC results IHC results FISH performed FISH ampl. IHC results FISH performed FISH ampl. Score 0 197 Score 0 197 1 0 Score 0 41 0 0 Score 1+ 45 Score 1+ Score 2 + < 50% 45 35 0 35 0 0 Score 1+ Score 2 + < 50% 13 0 0 0 0 0 Score 2+ 49 Score 2 + > 50% 14 13* 0 Score 2 + > 50% 3 3 0 Score 3+ 2 Score 3 + < 50% Score 3 + > 50% 2 0 2 0 0 0 Score 3 + < 50% Score 3 + > 50% 0 0 0 0 0 0 Survival and genetic data After a median follow-up of 31.2 months (95%CI from 28.0 to 35.0), median overall survival (OS) was 76.9 months (95%CI: from 70.5, upper confidence limit not reached) across all stages. OS was strictly related to the stage at diagnosis, as expected, with 3-year OS rate ranging from 92.3% in stage I to 24.6% in stage IV (Fig. 1 ). Among all patients, 31% (n = 109) were referred for genetic counseling, and 17 patients were found to be Lynch Syndrome carriers (16% of those tested and 5% of the study cohort). Most patients presented with CRCs with loss of MLH1/PMS2 (304 patients representing 87% of the entire cohort); data on BRAF mutation with subsequent MLH1 promoter methylation status, if performed, according to the Lynch Syndrome diagnostic algorithm, are available for 259 patients. Two-thirds of these patients (211/259–81%) had CRCs that showed either BRAF mutation or were BRAF wild-type with MLH1 promoter methylation, in line with the literature 17 . On the other hand, among the 46 patients with loss of MMR proteins other than MLH1 (loss of MSH2/MSH6 or isolated loss of either PMS2 or MSH6), 30 underwent genetic counseling (65%), and testing, and 12 were LS carriers (about 40%). Discussion The therapeutic landscape of mCRC is rapidly evolving with increasing availability of targeted treatments thus eliciting a growing demand for molecular testing. European guidelines 8 indicate that HER2 amplifications occur in approximately 2% of mCRC and carry an ESCAT IIB actionability level. Several HER2-targeting regimens (i.e., trastuzumab-lapatinib 18 , trastuzumab-tucatinib 9 , trastuzumab deruxtecan 19,20 ) have reported objective response rates (ORR) of 30–40% in non-randomized studies, and are currently being evaluated in earlier lines of therapy 21 . However, a careful balance between the benefits of identifying actionable alterations and the associated costs is necessary — especially in centers where financial and logistical constraints limit access to comprehensive NGS testing, and where the effort required to detect very rare alterations may not be justified by their low probability of occurrence. As combination strategies with immunotherapy and targeted therapies are currently being evaluated in the dMMR/MSI population as well 7 , we were interested in understanding whether HER2 amplification should be looked for in this population, by collecting data from two large tertiary referral centers for mCRC management. Only a few studies have evaluated this co-alteration 15,22,23 , and most included dMMR/MSI patients within much larger pMMR/MSS mCRC cohorts. In contrast, our series is the first and the largest one to focus exclusively on dMMR/MSI CRC across all disease stages. In the present cohort, no HER2-positive tumors were identified among 350 dMMR/MSI CRCs. Although a formal demonstration of mutual exclusivity would require explicit verification that no individuals with HER2 also exhibit dMMR/MSI, as well as larger sample size, the observed absence of co-occurrence is highly unlikely under the assumption of independence and suggests that HER2 positivity in dMMR/MSI tumors is exceedingly rare. From a clinical perspective, our results indicate that routine HER2 testing in dMMR/MSI CRC could be omitted or can be limited to clinical contexts of research setting. Larger datasets would be required to definitively assess mutual exclusivity. The inclusion of two high expertise CRC centers using two different HER2 scoring systems enabled a direct comparison between the two approaches. In Genoa, among the 49 patients classified as IHC 2 + according to the GC score, none resulted to have a gene amplification by FISH. If patients would be tested according to the HERACLES criteria, only 14 of these 49 would have undergone FISH testing, resulting in a reduction of costs and turnaround time. Based on our findings, we confirm that the HERACLES criteria appear to be more specific for CRC and should be therefore generally preferred in clinical practice 24 . Regarding genetic testing, the identification of only 5% of patients with Lynch syndrome in our series is markedly lower than the expected prevalence of this hereditary condition among dMMR/MSI CRCs (approximately 10–20%) 25,26 . The high proportion of CRCs with MLH1 loss and positive reflex testing (i.e., BRAF mutation or MLH1 promoter hypermethylation), together with the relatively high median age of our population, might be responsible for the large proportion of non-hereditary cases observed in our cohort. However, the underdiagnosis of hereditary syndromes remains a well-known issue in oncology, with important consequences in terms of delayed cancer detection and avoidable mortality. Indeed, identifying patients at increased genetic risk enables targeted surveillance protocols for second primary tumors, and cascade testing of relatives has been associated with a substantial reduction in cancer mortality—estimated at around 60% in some series 27 . Reflex testing, through the analysis of BRAF mutation analysis and MLH1 promoter hypermethylation in CRC patients with loss of MLH1 at IHC, effectively excludes LS. This approach may markedly reduce inappropriate referrals for genetic counseling and improve the detection of truly at-risk individuals. Its feasibility and clinical impact are currently being evaluated in the Italian ItaLynch study, coordinated by our group 28 . Preliminary results were presented at the ASCO Annual Meeting 2025 29 , although final results will be available in 2026. The main strength of our study lies in the large number of dMMR/MSI CRC patients with available HER2 analysis, all assessed in two major referral centers for pathology and compared using two different scoring systems, ensuring the reliability of the results. Among the limitations, we acknowledge its retrospective design and lack of data on systemic treatments, which prevented a proper evaluation of survival. In conclusion, this study represents the largest series focusing exclusively on dMMR/MSI and HER2 amplification in CRC. Our findings, together with available evidence, indicate that the co-occurrence of HER2 amplification in dMMR/MSI CRC is extremely rare and likely anecdotal, suggesting that HER2 testing could be omitted in dMMR/MSI CRC. Larger collaborative datasets are needed to confirm these results and refine clinical recommendations. Materials and methods We have included patients with dMMR/MSI CRC consecutively referred to the University Pathology Unit of IRCCS Azienda Ospedaliera Metropolitana Genoa, University of Genoa, and ASST Grande Ospedale Metropolitano Niguarda, Milan. MMR test was performed by immunohistochemistry (IHC); indeterminate/inadequate cases were sent to molecular MSI testing by PCR. BRAF and allRAS analyses were conducted in PCR. RAS status was analyzed only in selected patients (metastatic, recurrent, or at high risk of recurrence), while BRAF was systematically tested after February 2019, after the introduction of the reflex testing for the Lynch Syndrome 29 . Starting in 2016 in Genoa and 2019 in Milan, IHC was systematically performed to evaluate HER2 expression on all cases. The cases from Genoa were categorized following two different scoring systems for HER-2 evaluation: i) Gastric Cancer (GC) scoring system 30 , where a cut-off of 10% was used for complete or basolateral membranous reactivity with absent expression in score 0, faint/barely perceptible in score 1, weak to moderate in score 2 and moderate to strong expression in score 3; ii) the HERACLES study 24 scoring system designed specifically for HER-2 evaluation in colorectal cancer where both intensity (as seen above) and percentage of expression are considered (< 10%, 10–50%, ≥ 50%). The cases from Milan were all evaluated with the Heracles criteria. Table 3 shows the differences between the two scoring systems. Table 3 Comparison of the two different scoring systems for HER2 analysis (GC and Heracles). Acronyms: GC=Gastric Cancer. GC HER-2 Scoring System HERACLES Scoring system Intensity Percentage Score 0 No reactivity or membranous reactivity in < 10% of cells negative No staining (0) Negative Score 1+ Faint ⁄ barely perceptible membranous reactivity in > 10% of cells; cells are reactive only in part of their membrane negative Faint staining (1+) Any cellularity Negative Score 2+ Weak to moderate complete or basolateral membranous reactivity in > 10% of tumour cells Equivocal (requires FISH testing) Moderate (2+) < 50% Negative Moderate (2+) ≥ 50% Equivocal (requires FISH testing) Score 3+ Moderate to strong complete or basolateral membranous reactivity in > 10% of tumour cells positive Intense (3+) In ≤ 10% of cells Negative Intense (3+) In > 10% < 50% of cells Equivocal (requires FISH testing) Intense (3+) In ≥ 50% of cells Positive In brief, IHC results of 0 or 1 + were considered as negative, while 3 + were considered as positive. Results of IHC 2+ (in Genoa, according to GC scoring system, while in Milan, only in score 2 + cases with expression in > 50% of the tumor) were investigated by fluorescent in situ hybridization (FISH) for HER2 amplifications. The few cases scored as IHC 3 + but < 50% of expression according to the HERACLES scoring system were further evaluated by FISH. Data were summarized as frequencies and proportions or as median and range. To assess whether the probability of occurrence of HER2-positivity among patients with dMMR/MSI was consistent with the expected frequency under an assumption of independence, we performed a one-sided exact binomial test under the null hypothesis of a 2% HER2 prevalence in CRC 10 . Assuming independence between HER2 positivity and dMMR/MSI status (15% prevalence in non-metastatic CRC 1 ), the expected joint prevalence was 0.3%, corresponding to approximately 7 HER2-positive cases among 350 MSI patients. The probability of observing zero HER2-positive cases under this assumption was estimated using a binomial distribution. Overall survival (OS) was defined as the time from the diagnosis to death from any cause. Survival Curves were estimated by Kaplan-Meier method, and differences among subgroups were evaluated using the log-rank test. Median follow-up was estimated by reverse Kaplan-Meier method. Statistical analyses were conducted in R version 4.4.2 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria; 2024). The study was approved by the Regional Ethics Committee of Liguria (CER Liguria; approval numbers 102/2021 and 513/2020) and complied with the Declaration of Helsinki and good clinical practice guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects. Declarations Author contribution AG: conceptualization, data curation, formal analysis, investigation, methodology, project administration, writing – original draft. FT: data curation, investigation, methodology, writing – review and editing. LC: formal analysis, investigation, writing – review and editing. FG: data curation, methodology, writing – original draft. LM, MCA: data curation, methodology, writing – review and editing. SP, CP, SN, VM, APa, GM, AA, EB, KB, DP, SSi: data curation, writing – review and editing. SSc, ASB, APu: conceptualization, investigation, methodology, project administration, supervision, validation, writing – review and editing. Data availability statement The datasets generated and/or analysed during the current study are not publicly available due to patient confidentiality constraints but are available from the corresponding author on reasonable request. Funding None. Competing interests statement Authors declare the following conflict of interests, all not related to this manuscript: AG: speaker honoraria by Merck FT: advisory role for AstraZeneca, Daiichi-Sankyo, Bayer APa: consult and Advisory role honoraria by Merck Sharp & Dohme, Bristo-Myers Squibb, Merck Serono, Takeda, Servier, Bayer, Amgen, BeOne, Nordic Pharma, Pierre-Fabre, Natera, Incyte, Astra Zeneca. Research funding by Astra Zeneca, Bayer. Travel Expenses by Merck Sharp & Dohme, Pierre Fabre, Amgen, BeOne. GM: speaker honoraria by Amgen. AA: advisory role honoraria by Amgen and Italfarmaco SSc: Speaker: MSD, Gentili, Novartis, GSK; Travel/accomodation/expenses for congresses: Novartis, Gentili; Round Table: ALTEMS-GSK APu: reports consulting or advisory roles for GlaxoSmithKline, Takeda Pharmaceuticals U.S.A, Takeda Italia, Bayer, Daiichi Sankyo Italia, MSD Italia, BeOne, Amgen; Speaker Honoraria for Pierre Fabre, Servier, Amgen, Bristol Myers Squibb (BMS), Daiichi Sankyo, MSD, Merck Serono, BeOne, Takeda; Institutional funding from GlaxoSmithKline, Amgen; Travel, Accommodations, Expenses from AstraZeneca, Amgen, Merck Serono, BeOne, Takeda. All other authors declare no conflict of interests. References Venderbosch, S. et al. Mismatch Repair Status and BRAF Mutation Status in Metastatic Colorectal Cancer Patients: A Pooled Analysis of the CAIRO, CAIRO2, COIN, and FOCUS Studies. Clin. Cancer Res. 20, 5322–5330 (2014). Nakamori, S. et al. Clinicopathological characteristics of Lynch-like syndrome. Int. J. Clin. Oncol. https://doi.org/10.1007/s10147-024-02527-x (2024) doi:10.1007/s10147-024-02527-x. Cervantes, B., André, T. & Cohen, R. Deficient mismatch repair/microsatellite unstable colorectal cancer: therapeutic advances and questions. Ther. Adv. Med. Oncol. 16, 17588359231170473 (2024). Cohen, R. et al. Microsatellite Instability in Patients With Stage III Colon Cancer Receiving Fluoropyrimidine With or Without Oxaliplatin: An ACCENT Pooled Analysis of 12 Adjuvant Trials. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 39, 642–651 (2021). Hua, H. et al. Genomic and transcriptomic analysis of MSI-H colorectal cancer patients with targetable alterations identifies clinical implications for immunotherapy. Front. Immunol. 13, 974793 (2022). Seppälä, T. T. et al. Combination of microsatellite instability and BRAF mutation status for subtyping colorectal cancer. Br. J. Cancer 112, 1966–1975 (2015). Elez, E. et al. SEAMARK: phase II study of first-line encorafenib and cetuximab plus pembrolizumab for MSI-H/dMMR BRAF V600E-mutant mCRC. Future Oncol. 20, 653–663 (2024). Mosele, M. F. et al. Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024: a report from the ESMO Precision Medicine Working Group. Ann. Oncol. S092375342400111X (2024) doi:10.1016/j.annonc.2024.04.005. Strickler, J. H. et al. Tucatinib plus trastuzumab for chemotherapy-refractory, HER2-positive, RAS wild-type unresectable or metastatic colorectal cancer (MOUNTAINEER): a multicentre, open-label, phase 2 study. Lancet Oncol. 24, 496–508 (2023). Bekaii-Saab, T. S. et al. Impact of Anti-EGFR Therapies on HER2-Positive Metastatic Colorectal Cancer: A Systematic Literature Review and Meta-Analysis of Clinical Outcomes. The Oncologist 28, 885–893 (2023). Raghav, K. et al. Validation of HER2 Amplification as a Predictive Biomarker for Anti-Epidermal Growth Factor Receptor Antibody Therapy in Metastatic Colorectal Cancer. JCO Precis. Oncol. 3, 1–13 (2019). Pilati, C. et al. ERBB2 Comprehensive Profiling and Prognostication in Stage III Colon Cancer: Findings From PETACC8 and IDEA-France Cohorts. Gastroenterology 168, 714–724.e4 (2025). Richman, S. D. et al. HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials. J. Pathol. 238, 562–570 (2016). Ingold Heppner, B. et al. HER2/neu testing in primary colorectal carcinoma. Br. J. Cancer 111, 1977–1984 (2014). Lee, S. M. & Oh, H. RAS/RAF mutations and microsatellite instability status in primary colorectal cancers according to HER2 amplification. Sci. Rep. 14, 11432 (2024). Ambrosini, M. et al. Epidemiology, pathogenesis, biology and evolving management of MSI-H/dMMR cancers. Nat. Rev. Clin. Oncol. 22, 385–407 (2025). McRonald, F. E. et al. Identification of people with Lynch syndrome from those presenting with colorectal cancer in England: baseline analysis of the diagnostic pathway. Eur. J. Hum. Genet. 32, 529–538 (2024). Sartore-Bianchi, A. et al. Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial. Lancet Oncol. 17, 738–746 (2016). Raghav, K. et al. 737MO Trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-positive (HER2+) metastatic colorectal cancer (mCRC): Final analysis of DESTINY-CRC02, a randomized, phase II trial. Ann. Oncol. 36, S514–S515 (2025). Siena, S. et al. HER2-related biomarkers predict clinical outcomes with trastuzumab deruxtecan treatment in patients with HER2-expressing metastatic colorectal cancer: biomarker analyses of DESTINY-CRC01. Nat. Commun. 15, 10213 (2024). Strickler, J. H. et al. MOUNTAINEER-03 phase III study design: first-line mFOLFOX6 + tucatinib + trastuzumab for HER2 + metastatic colorectal cancer. Future Oncol. 21, 303–311 (2025). Qiu, M.-Z. et al. Relationship of HER2 Alteration and Microsatellite Instability Status in Colorectal Adenocarcinoma. The Oncologist 26, e1161–e1170 (2021). Luo, H. et al. HER2 Overexpression and Mismatch Repair Deficiency are Correlated with Malignancy in Colorectal Cancer. Cancer Manag. Res. Volume 13, 3443–3454 (2021). Valtorta, E. et al. Assessment of a HER2 scoring system for colorectal cancer: results from a validation study. Mod. Pathol. 28, 1481–1491 (2015). Abu-Ghazaleh, N., Kaushik, V., Gorelik, A., Jenkins, M. & Macrae, F. Worldwide prevalence of Lynch syndrome in patients with colorectal cancer: Systematic review and meta-analysis. Genet. Med. 24, 971–985 (2022). Latham, A. et al. Microsatellite Instability Is Associated With the Presence of Lynch Syndrome Pan-Cancer. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 37, 286–295 (2019). Järvinen, H. J. et al. Ten Years After Mutation Testing for Lynch Syndrome: Cancer Incidence and Outcome in Mutation-Positive and Mutation-Negative Family Members. J. Clin. Oncol. 27, 4793–4797 (2009). Puccini, A. et al. ItaLynch: an ongoing Italian study to evaluate the feasibility of mainstreaming the diagnosis of Lynch syndrome in colorectal cancer patients. ESMO Gastrointest. Oncol. 3, 100044 (2024). Puccini, A. et al. Streamlining the diagnostic pathway for Lynch syndrome in colorectal cancer patients: a 10-year experience in a single Italian Cancer Center. Eur. J. Cancer Prev. 33, 355–362 (2024). Hofmann, M. et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 52, 797–805 (2008). Additional Declarations Competing interest reported. Authors declare the following conflict of interests, all not related to this manuscript: AG: speaker honoraria by Merck FT: advisory role for AstraZeneca, Daiichi-Sankyo, Bayer APa: consult and Advisory role honoraria by Merck Sharp & Dohme, Bristo-Myers Squibb, Merck Serono, Takeda, Servier, Bayer, Amgen, BeOne, Nordic Pharma, Pierre-Fabre, Natera, Incyte, Astra Zeneca. Research funding by Astra Zeneca, Bayer. Travel Expenses by Merck Sharp & Dohme, Pierre Fabre, Amgen, BeOne. GM: speaker honoraria by Amgen. AA: advisory role honoraria by Amgen and Italfarmaco SSc: Speaker: MSD, Gentili, Novartis, GSK; Travel/accomodation/expenses for congresses: Novartis, Gentili; Round Table: ALTEMS-GSK APu: reports consulting or advisory roles for GlaxoSmithKline, Takeda Pharmaceuticals U.S.A, Takeda Italia, Bayer, Daiichi Sankyo Italia, MSD Italia, BeOne, Amgen; Speaker Honoraria for Pierre Fabre, Servier, Amgen, Bristol Myers Squibb (BMS), Daiichi Sankyo, MSD, Merck Serono, BeOne, Takeda; Institutional funding from GlaxoSmithKline, Amgen; Travel, Accommodations, Expenses from AstraZeneca, Amgen, Merck Serono, BeOne, Takeda. All other authors declare no conflict of interests. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 07 May, 2026 Reviews received at journal 01 May, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor invited by journal 20 Mar, 2026 Editor assigned by journal 19 Mar, 2026 Submission checks completed at journal 19 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9105189\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":626381456,\"identity\":\"f1f7982e-f961-4e06-9254-937e24555c5d\",\"order_by\":0,\"name\":\"Annalice Gandini\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Annalice\",\"middleName\":\"\",\"lastName\":\"Gandini\",\"suffix\":\"\"},{\"id\":626381457,\"identity\":\"9e2bac7c-aae4-45ce-a75f-b9961ab96e84\",\"order_by\":1,\"name\":\"Federica Tosi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Federica\",\"middleName\":\"\",\"lastName\":\"Tosi\",\"suffix\":\"\"},{\"id\":626381458,\"identity\":\"ee2cc99e-a4d3-41f3-8423-5efc0c16870c\",\"order_by\":2,\"name\":\"Luca Carmisciano\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Luca\",\"middleName\":\"\",\"lastName\":\"Carmisciano\",\"suffix\":\"\"},{\"id\":626381460,\"identity\":\"b75278b7-d227-4d6c-90fb-438453b8a8e7\",\"order_by\":3,\"name\":\"Federica Grillo\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Genoa\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Federica\",\"middleName\":\"\",\"lastName\":\"Grillo\",\"suffix\":\"\"},{\"id\":626381462,\"identity\":\"434b250e-2a0e-4641-aba5-cbcc2c3ecac4\",\"order_by\":4,\"name\":\"Luca Mastracci\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Genoa\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Luca\",\"middleName\":\"\",\"lastName\":\"Mastracci\",\"suffix\":\"\"},{\"id\":626381465,\"identity\":\"13ecd421-536e-4c89-b5eb-ac7c621c273d\",\"order_by\":5,\"name\":\"Silvia Puglisi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Silvia\",\"middleName\":\"\",\"lastName\":\"Puglisi\",\"suffix\":\"\"},{\"id\":626381466,\"identity\":\"44055b29-e5e9-468d-a058-4c3d0d85c26d\",\"order_by\":6,\"name\":\"Chiara Pirrone\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Santa Corona\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chiara\",\"middleName\":\"\",\"lastName\":\"Pirrone\",\"suffix\":\"\"},{\"id\":626381467,\"identity\":\"ef2f2886-a9ca-4792-a4c5-03234aa33a35\",\"order_by\":7,\"name\":\"Simone Nardin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Azienda Ospedaliero Universitaria Maggiore della Carita\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Simone\",\"middleName\":\"\",\"lastName\":\"Nardin\",\"suffix\":\"\"},{\"id\":626381468,\"identity\":\"7b42d515-71a1-4384-83b7-9b896b43edb2\",\"order_by\":8,\"name\":\"Valentino Martelli\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Valentino\",\"middleName\":\"\",\"lastName\":\"Martelli\",\"suffix\":\"\"},{\"id\":626381469,\"identity\":\"f981b7be-0afe-4e52-b0ed-70453e67a44c\",\"order_by\":9,\"name\":\"Alessandro Pastorino\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alessandro\",\"middleName\":\"\",\"lastName\":\"Pastorino\",\"suffix\":\"\"},{\"id\":626381470,\"identity\":\"1487c1bd-8fb5-429a-b456-cef00c5e894e\",\"order_by\":10,\"name\":\"Gianluca Mauri\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Gianluca\",\"middleName\":\"\",\"lastName\":\"Mauri\",\"suffix\":\"\"},{\"id\":626381471,\"identity\":\"4b69c74a-1988-4d25-bff9-43a047ca64ca\",\"order_by\":11,\"name\":\"Alessio Amatu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alessio\",\"middleName\":\"\",\"lastName\":\"Amatu\",\"suffix\":\"\"},{\"id\":626381472,\"identity\":\"cbd4cd81-2e02-493c-91d8-4e9998100f4c\",\"order_by\":12,\"name\":\"Erica Bonazzina\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Erica\",\"middleName\":\"\",\"lastName\":\"Bonazzina\",\"suffix\":\"\"},{\"id\":626381473,\"identity\":\"1635dd26-f6d0-47b5-8d55-3a7bd3362bf0\",\"order_by\":13,\"name\":\"Katia Bencardino\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Katia\",\"middleName\":\"\",\"lastName\":\"Bencardino\",\"suffix\":\"\"},{\"id\":626381474,\"identity\":\"df8f34fa-8f2e-4c91-874a-dd9e815a82c7\",\"order_by\":14,\"name\":\"Daniele Piscazzi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Daniele\",\"middleName\":\"\",\"lastName\":\"Piscazzi\",\"suffix\":\"\"},{\"id\":626381475,\"identity\":\"3fa8c32a-2153-4af5-9919-500f35bb4ce9\",\"order_by\":15,\"name\":\"Salvatore Siena\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Salvatore\",\"middleName\":\"\",\"lastName\":\"Siena\",\"suffix\":\"\"},{\"id\":626381476,\"identity\":\"d1ee872d-2d7d-4a07-bc52-bc4a370bfeec\",\"order_by\":16,\"name\":\"Maria Costanza Aquilano\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Maria\",\"middleName\":\"Costanza\",\"lastName\":\"Aquilano\",\"suffix\":\"\"},{\"id\":626381477,\"identity\":\"6b144147-ee6e-4360-83d2-91ea9344276b\",\"order_by\":17,\"name\":\"Stefania Sciallero\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Policlinico San Martino\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Stefania\",\"middleName\":\"\",\"lastName\":\"Sciallero\",\"suffix\":\"\"},{\"id\":626381478,\"identity\":\"f164aacf-18b5-4813-8776-c7fadafaa7f4\",\"order_by\":18,\"name\":\"Andrea Sartore-Bianchi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ospedale Niguarda Ca' Granda\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Andrea\",\"middleName\":\"\",\"lastName\":\"Sartore-Bianchi\",\"suffix\":\"\"},{\"id\":626381479,\"identity\":\"5315ee0b-85ad-4497-b15f-47d154fefea6\",\"order_by\":19,\"name\":\"Alberto Puccini\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDACdh4kTgKDhJwBkD7AwMCMWwszD1gpXIsxiVqAIHEDVBynDv5m3oOPK3/YMJhLJD/88HCHRfp2idyDh25UWDPwSyRg1SJxmC/Z8ExCGoPljDRjicQzErk7Z+QlHM45k84gOQO7FobDPGaSDQmHGQxu5LAxJLZJ5G64kWNwOLcNJIJdi/xhHvOfyFrSDcBa/h1msMehxQBoCyOylgSIlgagCA6/GAL9ItmQlsZj2fMM6Jc2CcMNZ94YHM45ls4jceYBVi1yx3sPfmywsZEzZ09++PFnW528wfEc4885NdZy/O04vA8FPAYYInjVgz1FUMUoGAWjYBSMWAAABjxcCSUt6jUAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"IRCCS Humanitas Research Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Alberto\",\"middleName\":\"\",\"lastName\":\"Puccini\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-12 13:09:51\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9105189/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9105189/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":107839194,\"identity\":\"037a2663-86bb-480e-8b7d-1c49ba4e3129\",\"added_by\":\"auto\",\"created_at\":\"2026-04-26 17:16:16\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":147637,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSurvival data.\\u003c/strong\\u003e Overall survival in all population and divided by stage. 3-year OS rate was 92.3%, 80.1%, 61.5%, and 24.6% in stage I, II, III, and IV, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9105189/v1/c61038dff63a10c9cbe6cfb3.png\"},{\"id\":107869860,\"identity\":\"27a0ee6f-291b-4655-a298-0bb71c71c16c\",\"added_by\":\"auto\",\"created_at\":\"2026-04-27 07:38:19\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":490216,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9105189/v1/17128e6e-7c27-48fa-bab6-3c367d844c8f.pdf\"}],\"financialInterests\":\"Competing interest reported. Authors declare the following conflict of interests, all not related to this manuscript: \\nAG: speaker honoraria by Merck \\nFT: advisory role for AstraZeneca, Daiichi-Sankyo, Bayer\\nAPa: consult and Advisory role honoraria by Merck Sharp \\u0026 Dohme, Bristo-Myers Squibb, Merck Serono, Takeda, Servier, Bayer, Amgen, BeOne, Nordic Pharma, Pierre-Fabre, Natera, Incyte, Astra Zeneca. Research funding by Astra Zeneca, Bayer. Travel Expenses by Merck Sharp \\u0026 Dohme, Pierre Fabre, Amgen, BeOne.\\nGM: speaker honoraria by Amgen.\\nAA: advisory role honoraria by Amgen and Italfarmaco\\nSSc: Speaker: MSD, Gentili, Novartis, GSK; Travel/accomodation/expenses for congresses: Novartis, Gentili; Round Table: ALTEMS-GSK\\nAPu: reports consulting or advisory roles for GlaxoSmithKline, Takeda Pharmaceuticals U.S.A, Takeda Italia, Bayer, Daiichi Sankyo Italia, MSD Italia, BeOne, Amgen; Speaker Honoraria for Pierre Fabre, Servier, Amgen, Bristol Myers Squibb (BMS), Daiichi Sankyo, MSD, Merck Serono, BeOne, Takeda; Institutional funding from GlaxoSmithKline, Amgen; Travel, Accommodations, Expenses from AstraZeneca, Amgen, Merck Serono, BeOne, Takeda. \\nAll other authors declare no conflict of interests.\",\"formattedTitle\":\"\\u003cp\\u003eAbsence of co-occurrence between \\u003cem\\u003eHER2\\u003c/em\\u003e amplification and dMMR/MSI in colorectal cancer: a multicentric study\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Highlights\",\"content\":\"\\u003cp\\u003e\\u0026bull; No \\u003cem\\u003eHER2\\u003c/em\\u003e amplification detected in 350 dMMR/MSI CRCs from two different centers.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026bull; Co-occurrence of \\u003cem\\u003eHER2\\u003c/em\\u003e amplification and dMMR/MSI appears anecdotal in CRC.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026bull; Routine HER2 testing in dMMR/MSI CRC may be unnecessary.\\u003c/p\\u003e\"},{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eThe panorama of colorectal cancer (CRC) treatment has evolved in the last few decades thanks to the identification of molecular biomarkers able to predict response to specific therapies. The major determinant in CRC molecular classification is the identification of mismatch repair deficiency (dMMR)/microsatellite instability (MSI), which has both prognostic and therapeutic implications. dMMR/MSI is due to a deficit in the DNA mismatch repair system (MMR), which accounts for around 15% of all CRCs. Immunohistochemistry (IHC) is considered the most cost-effective methodology for testing as it allows the identification of the loss of expression of MMR proteins (MLH1, PMS2, MSH2, or MSH6), leading to the dMMR phenotype. Molecular biology defines the MSI phenotype, and it is usually recommended in equivocal IHC results in CRCs \\u003csup\\u003e1\\u003c/sup\\u003e. The identification of dMMR/MSI CRCs has now become mandatory at the time of diagnosis, regardless of the stage of the disease, due to two main reasons. First, it allows the identification of patients at risk of being affected by Lynch Syndrome (LS), an autosomal dominant inherited disorder caused by a germline pathogenic variant in the MMR genes \\u003csup\\u003e2\\u003c/sup\\u003e. Moreover, it predicts sensitivity to immunotherapy, both in early and advanced disease, which has revolutionized the therapeutic algorithm and the prognosis of these patients \\u003csup\\u003e3\\u003c/sup\\u003e. dMMR/MSI CRCs show a different clinical behavior when compared to proficient MMR (pMMR)/microsatellite stable (MSS) CRCs, and the impact on prognosis seems to vary between different stages of disease. While in advanced disease (5% of all CRCs), before the advent of immunotherapy, it identified a subpopulation with poorer prognosis \\u003csup\\u003e1\\u003c/sup\\u003e, in localized stages, it is associated with better prognosis when compared to pMMR/MSS, even if this better prognostic impact seems to be lost in stage III high-risk patients \\u003csup\\u003e4\\u003c/sup\\u003e. Due to the deficit in the DNA repair mechanism, and the subsequent accumulation of mutations, dMMR/MSI patients seem to be enriched in targetable genomic alterations, such as \\u003cem\\u003eHER2\\u003c/em\\u003e mutations, \\u003cem\\u003eNTRK\\u003c/em\\u003e fusions \\u003csup\\u003e5\\u003c/sup\\u003e, as well as \\u003cem\\u003eBRAF V600E\\u003c/em\\u003e mutation, which excludes the possibility of LS \\u003csup\\u003e6\\u003c/sup\\u003e. In this regard, although \\u003cem\\u003eBRAF V600E\\u003c/em\\u003e represents an actionable alteration, microsatellite instability remains the dominant therapeutic driver, making immunotherapy the preferred upfront therapeutic choice. Accordingly, ongoing first-line trials such as SEAMARK are evaluating the combination of immunotherapy with anti-BRAF and anti-EGFR inhibitors \\u003csup\\u003e7\\u003c/sup\\u003e. However, the known clinical and molecular aspects of dMMR/MSI CRCs mostly derive from subgroup analyses of selected clinical trials, and data on large real-life cohorts are lacking. With the increasing identification of targetable alterations in CRC, such as \\u003cem\\u003eKRAS G12C\\u003c/em\\u003e mutations and \\u003cem\\u003eHER2\\u003c/em\\u003e amplification, optimizing patient selection for molecular testing has become a relevant clinical issue. Current ESMO guidelines recommend extended next-generation sequencing (NGS) in advanced disease only when it does not generate additional costs, and not in localized CRC \\u003csup\\u003e8\\u003c/sup\\u003e. While all\\u003cem\\u003eRAS\\u003c/em\\u003e and \\u003cem\\u003eBRAF\\u003c/em\\u003e testing are routinely performed in the metastatic setting because of their predictive value for target therapies, \\u003cem\\u003eHER2\\u003c/em\\u003e amplification testing is not yet standard in Europe, despite emerging targeted options \\u003csup\\u003e9\\u003c/sup\\u003e. \\u003cem\\u003eHER2\\u003c/em\\u003e amplification occurs in approximately 2\\u0026ndash;3% of metastatic CRCs and has been associated with poor prognosis and resistance to anti-EGFR therapies \\u003csup\\u003e10\\u0026ndash;12\\u003c/sup\\u003e, while its prevalence in localized disease appears to be even lower \\u003csup\\u003e13,14\\u003c/sup\\u003e. Importantly, preliminary evidence suggests that \\u003cem\\u003eHER2\\u003c/em\\u003e amplification may be exceptionally rare in dMMR/MSI tumors, raising the hypothesis of a potential mutual exclusivity between these two molecular alterations \\u003csup\\u003e15\\u003c/sup\\u003e. If confirmed, this would have practical implications by identifying patient subsets in whom \\u003cem\\u003eHER2\\u003c/em\\u003e testing is unlikely to be informative, thereby avoiding low-yield analyses and improving the efficiency of precision medicine strategies.\\u003c/p\\u003e \\u003cp\\u003eThe aim of this study is to evaluate clinical and molecular characteristics of a large multicentric real-world cohort of unselected dMMR/MSI CRC patients, focusing on the co-occurrence of dMMR/MSI status and \\u003cem\\u003eHER2\\u003c/em\\u003e amplification as proof-of-concept.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003eStudy population\\u003c/h2\\u003e\\n \\u003cp\\u003eOverall, 350 dMMR/MSI CRCs were identified, 293 from Genoa and 57 from Milan. Patients were mostly female (66%), with a median age of 75 years. Most patients had a localized CRC at the time of the diagnosis (stage I to III\\u0026thinsp;=\\u0026thinsp;93%), and proximal tumors (86%), defined as right-sided or transverse colon cancer. More than half of CRCs were high grade - G3 and G4 (193/350 cases \\u0026ndash; 56%). Focusing on the IHC results in this cohort of dMMR/MSI CRC patients, loss of MLH-1/PMS2 was the most frequently detected (87%). Complete patients\\u0026rsquo; characteristics are reported in Table \\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTable 1:\\u003c/strong\\u003e Patient characteristics. Acronyms: dMMR= deficit of the Mismatch repair system. MSI= Microsatellite instability. IHC= Immunohistochemistry. SD= standard deviation.\\u003c/p\\u003e\\n \\u003cdiv align=\\\"left\\\"\\u003e\\n \\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCharacteristics\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eN = 350 (%)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAge \\u0026ndash; mean (SD)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e74.93 (11.41)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSex\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eMale\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Female\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e119 (34)\\u003c/p\\u003e\\n \\u003cp\\u003e231 (66)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ePrimary tumor location\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Right colon\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Transverse colon\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Left colon\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Rectum\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Multiple\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Unknown\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e266 (77.1)\\u003c/p\\u003e\\n \\u003cp\\u003e31 (9)\\u003c/p\\u003e\\n \\u003cp\\u003e40 (11.6)\\u003c/p\\u003e\\n \\u003cp\\u003e6 (1.7)\\u003c/p\\u003e\\n \\u003cp\\u003e2 (0.6)\\u003c/p\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eDisease stage at diagnosis\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;I\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;II\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;III\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;IV\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e32 (9.1)\\u003c/p\\u003e\\n \\u003cp\\u003e189 (54)\\u003c/p\\u003e\\n \\u003cp\\u003e105 (30)\\u003c/p\\u003e\\n \\u003cp\\u003e24 (6.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHistotype\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eAdenocarcinoma\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Medullary\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Mucinous\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Poorly differentiated\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e310 (88.6)\\u003c/p\\u003e\\n \\u003cp\\u003e1 (0.3)\\u003c/p\\u003e\\n \\u003cp\\u003e23 (6.6)\\u003c/p\\u003e\\n \\u003cp\\u003e16 (4.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGrade\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Low\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;High\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Unknown\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e153 (44)\\u003c/p\\u003e\\n \\u003cp\\u003e193 (56)\\u003c/p\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eBRAF\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;status\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Mutated\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Wild type\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Unknown\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e171 (48.9)\\u003c/p\\u003e\\n \\u003cp\\u003e110 (31.4)\\u003c/p\\u003e\\n \\u003cp\\u003e69 (19.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eRAS\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;status\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eMutated\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Wild type\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Unknown\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e10 (2.9)\\u003c/p\\u003e\\n \\u003cp\\u003e59 (16.9)\\u003c/p\\u003e\\n \\u003cp\\u003e281 (80.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eLoss of MMR Proteins by IHC\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;MLH-1 PMS-2\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;MSH-2 MSH-6\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003cstrong\\u003eOther\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e304 (86.9)\\u003c/p\\u003e\\n \\u003cp\\u003e24 (6.9)\\u003c/p\\u003e\\n \\u003cp\\u003e22 (6.2)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eHER2\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Positive\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Negative\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Unknown\\u003cstrong\\u003e\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003cp\\u003e0\\u003c/p\\u003e\\n \\u003cp\\u003e349\\u003c/p\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003ePathological and molecular variables\\u003c/h3\\u003e\\n\\u003cp\\u003eAmong the 350 patients, \\u003cem\\u003eRAS\\u003c/em\\u003e status was available for 69 patients (20%) and, among these, 14% were mutated. Around 80% of the patients were tested for \\u003cem\\u003eBRAF\\u003c/em\\u003e status, and 60% of these were mutated. None of the 350 patients tested for \\u003cem\\u003eHER2\\u003c/em\\u003e showed protein overexpression/gene amplification. Only one patient with a HER2 IHC 2\\u0026thinsp;+\\u0026thinsp;was not tested through FISH and thus should be considered as \\u0026ldquo;unknown\\u0026rdquo;. Details on HER2 testing results are available in Table \\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eAssuming the prevalence of HER2 in the general CRC population is 2% \\u003csup\\u003e10\\u003c/sup\\u003e, and the prevalence of dMMR/MSI in non-metastatic CRC is around 15% \\u003csup\\u003e16\\u003c/sup\\u003e, then, under the assumption of independence, their joint prevalence would be approximately 0.3%. Under this same independence assumption, approximately 7 HER2 positive cases would be expected among 350 dMMR/MSI patients. In our cohort of 350 CRC dMMR/MSI patients, we identified no HER2 positive cases. We estimate a prevalence of HER2 in MSI cases between 0% and 0.9% with 95% confidence. The probability of observing a number of HER2 cases of 0 under the assumption of independence between HER2 and MSI is ~\\u0026thinsp;0.085%. Although we cannot formally establish mutual exclusivity, the absence of any HER2-positive cases in our MSI cases series indicates that their co-occurrence is essentially anecdotal.\\u003c/p\\u003e\\n\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eResults of HER2 testing in Genoa and Milan, according to both scoring systems (GC and Heracles). *One patient with an IHC score of 2\\u0026thinsp;+\\u0026thinsp;did not undergo FISH analysis due to technical issues.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"6\\\" nameend=\\\"c6\\\" namest=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eGenoa\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c10\\\" namest=\\\"c7\\\"\\u003e\\n \\u003cp\\u003eMilan\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c2\\\" namest=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eGC scoring\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c4\\\" namest=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eHeracles scoring\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\n \\u003cp\\u003eHeracles scoring\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c10\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c2\\\" namest=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eIHC results\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c4\\\" namest=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eIHC results\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003eFISH performed\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eFISH ampl.\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\n \\u003cp\\u003eIHC results\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\n \\u003cp\\u003eFISH performed\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c10\\\"\\u003e\\n \\u003cp\\u003eFISH ampl.\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e197\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e197\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e41\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 1+\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e45\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 1+\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 2\\u0026thinsp;+\\u0026thinsp;\\u0026lt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e45\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e35\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e35\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 1+\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 2\\u0026thinsp;+\\u0026thinsp;\\u0026lt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e13\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 2+\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e49\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 2\\u0026thinsp;+\\u0026thinsp;\\u0026gt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e14\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e13*\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 2\\u0026thinsp;+\\u0026thinsp;\\u0026gt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e3\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e3\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 3+\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 3\\u0026thinsp;+\\u0026thinsp;\\u0026lt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 3\\u0026thinsp;+\\u0026thinsp;\\u0026gt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 3\\u0026thinsp;+\\u0026thinsp;\\u0026lt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eScore 3\\u0026thinsp;+\\u0026thinsp;\\u0026gt;\\u0026thinsp;50%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c10\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003ch3\\u003eSurvival and genetic data\\u003c/h3\\u003e\\n\\u003cp\\u003eAfter a median follow-up of 31.2 months (95%CI from 28.0 to 35.0), median overall survival (OS) was 76.9 months (95%CI: from 70.5, upper confidence limit not reached) across all stages. OS was strictly related to the stage at diagnosis, as expected, with 3-year OS rate ranging from 92.3% in stage I to 24.6% in stage IV (Fig. \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eAmong all patients, 31% (n\\u0026thinsp;=\\u0026thinsp;109) were referred for genetic counseling, and 17 patients were found to be Lynch Syndrome carriers (16% of those tested and 5% of the study cohort). Most patients presented with CRCs with loss of MLH1/PMS2 (304 patients representing 87% of the entire cohort); data on \\u003cem\\u003eBRAF\\u003c/em\\u003e mutation with subsequent \\u003cem\\u003eMLH1\\u003c/em\\u003e promoter methylation status, if performed, according to the Lynch Syndrome diagnostic algorithm, are available for 259 patients. Two-thirds of these patients (211/259\\u0026ndash;81%) had CRCs that showed either \\u003cem\\u003eBRAF\\u003c/em\\u003e mutation or were \\u003cem\\u003eBRAF\\u003c/em\\u003e wild-type with \\u003cem\\u003eMLH1\\u003c/em\\u003e promoter methylation, in line with the literature \\u003csup\\u003e17\\u003c/sup\\u003e. On the other hand, among the 46 patients with loss of MMR proteins other than MLH1 (loss of MSH2/MSH6 or isolated loss of either PMS2 or MSH6), 30 underwent genetic counseling (65%), and testing, and 12 were LS carriers (about 40%).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe therapeutic landscape of mCRC is rapidly evolving with increasing availability of targeted treatments thus eliciting a growing demand for molecular testing. European guidelines \\u003csup\\u003e8\\u003c/sup\\u003e indicate that \\u003cem\\u003eHER2\\u003c/em\\u003e amplifications occur in approximately 2% of mCRC and carry an ESCAT IIB actionability level. Several HER2-targeting regimens (i.e., trastuzumab-lapatinib \\u003csup\\u003e18\\u003c/sup\\u003e, trastuzumab-tucatinib \\u003csup\\u003e9\\u003c/sup\\u003e, trastuzumab deruxtecan \\u003csup\\u003e19,20\\u003c/sup\\u003e) have reported objective response rates (ORR) of 30\\u0026ndash;40% in non-randomized studies, and are currently being evaluated in earlier lines of therapy \\u003csup\\u003e21\\u003c/sup\\u003e. However, a careful balance between the benefits of identifying actionable alterations and the associated costs is necessary \\u0026mdash; especially in centers where financial and logistical constraints limit access to comprehensive NGS testing, and where the effort required to detect very rare alterations may not be justified by their low probability of occurrence.\\u003c/p\\u003e \\u003cp\\u003eAs combination strategies with immunotherapy and targeted therapies are currently being evaluated in the dMMR/MSI population as well \\u003csup\\u003e7\\u003c/sup\\u003e, we were interested in understanding whether \\u003cem\\u003eHER2\\u003c/em\\u003e amplification should be looked for in this population, by collecting data from two large tertiary referral centers for mCRC management. Only a few studies have evaluated this co-alteration \\u003csup\\u003e15,22,23\\u003c/sup\\u003e, and most included dMMR/MSI patients within much larger pMMR/MSS mCRC cohorts. In contrast, our series is the first and the largest one to focus exclusively on dMMR/MSI CRC across all disease stages.\\u003c/p\\u003e \\u003cp\\u003eIn the present cohort, no HER2-positive tumors were identified among 350 dMMR/MSI CRCs. Although a formal demonstration of mutual exclusivity would require explicit verification that no individuals with HER2 also exhibit dMMR/MSI, as well as larger sample size, the observed absence of co-occurrence is highly unlikely under the assumption of independence and suggests that HER2 positivity in dMMR/MSI tumors is exceedingly rare. From a clinical perspective, our results indicate that routine HER2 testing in dMMR/MSI CRC could be omitted or can be limited to clinical contexts of research setting. Larger datasets would be required to definitively assess mutual exclusivity.\\u003c/p\\u003e \\u003cp\\u003eThe inclusion of two high expertise CRC centers using two different HER2 scoring systems enabled a direct comparison between the two approaches. In Genoa, among the 49 patients classified as IHC 2\\u0026thinsp;+\\u0026thinsp;according to the GC score, none resulted to have a gene amplification by FISH. If patients would be tested according to the HERACLES criteria, only 14 of these 49 would have undergone FISH testing, resulting in a reduction of costs and turnaround time. Based on our findings, we confirm that the HERACLES criteria appear to be more specific for CRC and should be therefore generally preferred in clinical practice \\u003csup\\u003e24\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eRegarding genetic testing, the identification of only 5% of patients with Lynch syndrome in our series is markedly lower than the expected prevalence of this hereditary condition among dMMR/MSI CRCs (approximately 10\\u0026ndash;20%) \\u003csup\\u003e25,26\\u003c/sup\\u003e. The high proportion of CRCs with MLH1 loss and positive reflex testing (i.e., \\u003cem\\u003eBRAF\\u003c/em\\u003e mutation or MLH1 promoter hypermethylation), together with the relatively high median age of our population, might be responsible for the large proportion of non-hereditary cases observed in our cohort. However, the underdiagnosis of hereditary syndromes remains a well-known issue in oncology, with important consequences in terms of delayed cancer detection and avoidable mortality. Indeed, identifying patients at increased genetic risk enables targeted surveillance protocols for second primary tumors, and cascade testing of relatives has been associated with a substantial reduction in cancer mortality\\u0026mdash;estimated at around 60% in some series \\u003csup\\u003e27\\u003c/sup\\u003e. Reflex testing, through the analysis of \\u003cem\\u003eBRAF\\u003c/em\\u003e mutation analysis and MLH1 promoter hypermethylation in CRC patients with loss of MLH1 at IHC, effectively excludes LS. This approach may markedly reduce inappropriate referrals for genetic counseling and improve the detection of truly at-risk individuals. Its feasibility and clinical impact are currently being evaluated in the Italian ItaLynch study, coordinated by our group \\u003csup\\u003e28\\u003c/sup\\u003e. Preliminary results were presented at the ASCO Annual Meeting 2025 \\u003csup\\u003e29\\u003c/sup\\u003e, although final results will be available in 2026.\\u003c/p\\u003e \\u003cp\\u003eThe main strength of our study lies in the large number of dMMR/MSI CRC patients with available HER2 analysis, all assessed in two major referral centers for pathology and compared using two different scoring systems, ensuring the reliability of the results. Among the limitations, we acknowledge its retrospective design and lack of data on systemic treatments, which prevented a proper evaluation of survival.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, this study represents the largest series focusing exclusively on dMMR/MSI and \\u003cem\\u003eHER2\\u003c/em\\u003e amplification in CRC. Our findings, together with available evidence, indicate that the co-occurrence of \\u003cem\\u003eHER2\\u003c/em\\u003e amplification in dMMR/MSI CRC is extremely rare and likely anecdotal, suggesting that HER2 testing could be omitted in dMMR/MSI CRC. Larger collaborative datasets are needed to confirm these results and refine clinical recommendations.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003eWe have included patients with dMMR/MSI CRC consecutively referred to the University Pathology Unit of IRCCS Azienda Ospedaliera Metropolitana Genoa, University of Genoa, and ASST Grande Ospedale Metropolitano Niguarda, Milan. MMR test was performed by immunohistochemistry (IHC); indeterminate/inadequate cases were sent to molecular MSI testing by PCR. \\u003cem\\u003eBRAF\\u003c/em\\u003e and \\u003cem\\u003eallRAS\\u003c/em\\u003e analyses were conducted in PCR. \\u003cem\\u003eRAS\\u003c/em\\u003e status was analyzed only in selected patients (metastatic, recurrent, or at high risk of recurrence), while \\u003cem\\u003eBRAF\\u003c/em\\u003e was systematically tested after February 2019, after the introduction of the reflex testing for the Lynch Syndrome \\u003csup\\u003e29\\u003c/sup\\u003e. Starting in 2016 in Genoa and 2019 in Milan, IHC was systematically performed to evaluate HER2 expression on all cases. The cases from Genoa were categorized following two different scoring systems for HER-2 evaluation: i) Gastric Cancer (GC) scoring system \\u003csup\\u003e30\\u003c/sup\\u003e, where a cut-off of 10% was used for complete or basolateral membranous reactivity with absent expression in score 0, faint/barely perceptible in score 1, weak to moderate in score 2 and moderate to strong expression in score 3; ii) the HERACLES study \\u003csup\\u003e24\\u003c/sup\\u003e scoring system designed specifically for HER-2 evaluation in colorectal cancer where both intensity (as seen above) and percentage of expression are considered (\\u0026lt;\\u0026thinsp;10%, 10\\u0026ndash;50%, \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026ge;\\u003c/span\\u003e\\u0026thinsp;50%). The cases from Milan were all evaluated with the Heracles criteria. Table \\u003cspan refid=\\\"Tab5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e shows the differences between the two scoring systems.\\u003c/p\\u003e\\n\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eComparison of the two different scoring systems for HER2 analysis (GC and Heracles). Acronyms: GC=Gastric Cancer.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ccolgroup cols=\\\"6\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"3\\\" morerows=\\\"1\\\" nameend=\\\"c3\\\" namest=\\\"c1\\\" rowspan=\\\"2\\\" style=\\\"width: 55.2681%;\\\"\\u003e\\n \\u003cp\\u003eGC HER-2 Scoring System\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c6\\\" namest=\\\"c4\\\" style=\\\"width: 44.5471%;\\\"\\u003e\\n \\u003cp\\u003eHERACLES Scoring system\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3086%;\\\"\\u003e\\n \\u003cp\\u003eIntensity\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003ePercentage\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eScore 0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eNo reactivity or membranous reactivity in \\u0026lt;\\u0026thinsp;10% of cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" style=\\\"width: 14.6026%;\\\"\\u003e\\n \\u003cp\\u003enegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eNo staining (0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eNegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eScore 1+\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eFaint \\u0026frasl; barely perceptible membranous reactivity in \\u0026gt;\\u0026thinsp;10% of cells; cells are reactive only in part of their membrane\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" style=\\\"width: 14.6026%;\\\"\\u003e\\n \\u003cp\\u003enegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eFaint staining (1+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003eAny cellularity\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eNegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eScore 2+\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eWeak to moderate complete or basolateral membranous reactivity in \\u0026gt;\\u0026thinsp;10% of tumour cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\" style=\\\"width: 14.6026%;\\\"\\u003e\\n \\u003cp\\u003eEquivocal\\u003c/p\\u003e\\n \\u003cp\\u003e(requires FISH testing)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eModerate (2+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;50%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eNegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eModerate (2+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026ge;\\u003c/span\\u003e\\u0026thinsp;50%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eEquivocal\\u003c/p\\u003e\\n \\u003cp\\u003e(requires FISH testing)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eScore 3+\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eModerate to strong complete or basolateral membranous reactivity in \\u0026gt;\\u0026thinsp;10% of tumour cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\" style=\\\"width: 14.6026%;\\\"\\u003e\\n \\u003cp\\u003epositive\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eIntense (3+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003eIn \\u0026le;\\u0026thinsp;10% of cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eNegative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eIntense (3+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003eIn \\u0026gt;\\u0026thinsp;10% \\u0026lt; 50% of cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003eEquivocal\\u003c/p\\u003e\\n \\u003cp\\u003e(requires FISH testing)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" style=\\\"width: 13.3087%;\\\"\\u003e\\n \\u003cp\\u003eIntense (3+)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\n \\u003cp\\u003eIn \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026ge;\\u003c/span\\u003e\\u0026thinsp;50% of cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\n \\u003cp\\u003ePositive\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eIn brief, IHC results of 0 or 1\\u0026thinsp;+\\u0026thinsp;were considered as negative, while 3\\u0026thinsp;+\\u0026thinsp;were considered as positive. Results of IHC 2+ (in Genoa, according to GC scoring system, while in Milan, only in score 2\\u0026thinsp;+\\u0026thinsp;cases with expression in \\u0026gt;\\u0026thinsp;50% of the tumor) were investigated by fluorescent in situ hybridization (FISH) for \\u003cem\\u003eHER2\\u003c/em\\u003e amplifications. The few cases scored as IHC 3\\u0026thinsp;+\\u0026thinsp;but \\u0026lt;\\u0026thinsp;50% of expression according to the HERACLES scoring system were further evaluated by FISH.\\u003c/p\\u003e\\n\\u003cp\\u003eData were summarized as frequencies and proportions or as median and range. To assess whether the probability of occurrence of HER2-positivity among patients with dMMR/MSI was consistent with the expected frequency under an assumption of independence, we performed a one-sided exact binomial test under the null hypothesis of a 2% HER2 prevalence in CRC \\u003csup\\u003e10\\u003c/sup\\u003e. Assuming independence between HER2 positivity and dMMR/MSI status (15% prevalence in non-metastatic CRC \\u003csup\\u003e1\\u003c/sup\\u003e), the expected joint prevalence was 0.3%, corresponding to approximately 7 HER2-positive cases among 350 MSI patients. The probability of observing zero HER2-positive cases under this assumption was estimated using a binomial distribution. Overall survival (OS) was defined as the time from the diagnosis to death from any cause. Survival Curves were estimated by Kaplan-Meier method, and differences among subgroups were evaluated using the log-rank test. Median follow-up was estimated by reverse Kaplan-Meier method. Statistical analyses were conducted in R version 4.4.2 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria; 2024). The study was approved by the Regional Ethics Committee of Liguria (CER Liguria; approval numbers 102/2021 and 513/2020) and complied with the Declaration of Helsinki and good clinical practice guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAG: conceptualization, data curation, formal analysis, investigation, methodology, project administration, writing \\u0026ndash; original draft.\\u003c/p\\u003e\\n\\u003cp\\u003eFT: data curation, investigation, methodology, writing \\u0026ndash; review and editing.\\u003c/p\\u003e\\n\\u003cp\\u003eLC: formal analysis, investigation, writing \\u0026ndash; review and editing.\\u003c/p\\u003e\\n\\u003cp\\u003eFG: data curation, methodology, writing \\u0026ndash; original draft.\\u003c/p\\u003e\\n\\u003cp\\u003eLM, MCA: data curation, methodology, writing \\u0026ndash; review and editing.\\u003c/p\\u003e\\n\\u003cp\\u003eSP, CP, SN, VM, APa, GM, AA, EB, KB, DP, SSi: data curation, writing \\u0026ndash; review and editing.\\u003c/p\\u003e\\n\\u003cp\\u003eSSc, ASB, APu: conceptualization, investigation, methodology, project administration, supervision, validation, writing \\u0026ndash; review and editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated and/or analysed during the current study are not publicly available due to patient confidentiality constraints but are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNone.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors declare the following conflict of interests, all not related to this manuscript:\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAG: speaker honoraria by Merck\\u003c/p\\u003e\\n\\u003cp\\u003eFT: advisory role for AstraZeneca, Daiichi-Sankyo, Bayer\\u003c/p\\u003e\\n\\u003cp\\u003eAPa: consult and Advisory role honoraria by Merck Sharp \\u0026amp; Dohme, Bristo-Myers Squibb, Merck Serono, Takeda, Servier, Bayer, Amgen, BeOne, Nordic Pharma, Pierre-Fabre, Natera, Incyte, Astra Zeneca. Research funding by Astra Zeneca, Bayer. Travel Expenses by Merck Sharp \\u0026amp; Dohme, Pierre Fabre, Amgen, BeOne.\\u003c/p\\u003e\\n\\u003cp\\u003eGM: speaker honoraria by Amgen.\\u003c/p\\u003e\\n\\u003cp\\u003eAA: advisory role honoraria by Amgen and Italfarmaco\\u003c/p\\u003e\\n\\u003cp\\u003eSSc: Speaker: MSD, Gentili, Novartis, GSK; Travel/accomodation/expenses for congresses: Novartis, Gentili; Round Table: ALTEMS-GSK\\u003c/p\\u003e\\n\\u003cp\\u003eAPu: reports consulting or advisory roles for GlaxoSmithKline, Takeda Pharmaceuticals U.S.A, Takeda Italia, Bayer, Daiichi Sankyo Italia, MSD Italia, BeOne, Amgen; Speaker Honoraria for Pierre Fabre, Servier, Amgen, Bristol Myers Squibb (BMS), Daiichi Sankyo, MSD, Merck Serono, BeOne, Takeda; Institutional funding from GlaxoSmithKline, Amgen; Travel, Accommodations, Expenses from AstraZeneca, Amgen, Merck Serono, BeOne, Takeda.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAll other authors declare no conflict of interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eVenderbosch, S. \\u003cem\\u003eet al.\\u003c/em\\u003e Mismatch Repair Status and \\u003cem\\u003eBRAF\\u003c/em\\u003e Mutation Status in Metastatic Colorectal Cancer Patients: A Pooled Analysis of the CAIRO, CAIRO2, COIN, and FOCUS Studies. \\u003cem\\u003eClin. Cancer Res.\\u003c/em\\u003e 20, 5322\\u0026ndash;5330 (2014).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eNakamori, S. \\u003cem\\u003eet al.\\u003c/em\\u003e Clinicopathological characteristics of Lynch-like syndrome. \\u003cem\\u003eInt. J. Clin. Oncol.\\u003c/em\\u003e https://doi.org/10.1007/s10147-024-02527-x (2024) doi:10.1007/s10147-024-02527-x.\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eCervantes, B., Andr\\u0026eacute;, T. \\u0026amp; Cohen, R. Deficient mismatch repair/microsatellite unstable colorectal cancer: therapeutic advances and questions. \\u003cem\\u003eTher. Adv. Med. Oncol.\\u003c/em\\u003e 16, 17588359231170473 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eCohen, R. \\u003cem\\u003eet al.\\u003c/em\\u003e Microsatellite Instability in Patients With Stage III Colon Cancer Receiving Fluoropyrimidine With or Without Oxaliplatin: An ACCENT Pooled Analysis of 12 Adjuvant Trials. \\u003cem\\u003eJ. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.\\u003c/em\\u003e 39, 642\\u0026ndash;651 (2021).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eHua, H. \\u003cem\\u003eet al.\\u003c/em\\u003e Genomic and transcriptomic analysis of MSI-H colorectal cancer patients with targetable alterations identifies clinical implications for immunotherapy. \\u003cem\\u003eFront. Immunol.\\u003c/em\\u003e 13, 974793 (2022).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eSepp\\u0026auml;l\\u0026auml;, T. T. \\u003cem\\u003eet al.\\u003c/em\\u003e Combination of microsatellite instability and BRAF mutation status for subtyping colorectal cancer. \\u003cem\\u003eBr. J. Cancer\\u003c/em\\u003e 112, 1966\\u0026ndash;1975 (2015).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eElez, E. \\u003cem\\u003eet al.\\u003c/em\\u003e SEAMARK: phase II study of first-line encorafenib and cetuximab plus pembrolizumab for MSI-H/dMMR \\u003cem\\u003eBRAF\\u003c/em\\u003e V600E-mutant mCRC. \\u003cem\\u003eFuture Oncol.\\u003c/em\\u003e 20, 653\\u0026ndash;663 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eMosele, M. F. \\u003cem\\u003eet al.\\u003c/em\\u003e Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024: a report from the ESMO Precision Medicine Working Group. \\u003cem\\u003eAnn. Oncol.\\u003c/em\\u003e S092375342400111X (2024) doi:10.1016/j.annonc.2024.04.005.\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eStrickler, J. H. \\u003cem\\u003eet al.\\u003c/em\\u003e Tucatinib plus trastuzumab for chemotherapy-refractory, HER2-positive, RAS wild-type unresectable or metastatic colorectal cancer (MOUNTAINEER): a multicentre, open-label, phase 2 study. \\u003cem\\u003eLancet Oncol.\\u003c/em\\u003e 24, 496\\u0026ndash;508 (2023).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eBekaii-Saab, T. S. \\u003cem\\u003eet al.\\u003c/em\\u003e Impact of Anti-EGFR Therapies on HER2-Positive Metastatic Colorectal Cancer: A Systematic Literature Review and Meta-Analysis of Clinical Outcomes. \\u003cem\\u003eThe Oncologist\\u003c/em\\u003e 28, 885\\u0026ndash;893 (2023).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eRaghav, K. \\u003cem\\u003eet al.\\u003c/em\\u003e Validation of HER2 Amplification as a Predictive Biomarker for Anti-Epidermal Growth Factor Receptor Antibody Therapy in Metastatic Colorectal Cancer. \\u003cem\\u003eJCO Precis. Oncol.\\u003c/em\\u003e 3, 1\\u0026ndash;13 (2019).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003ePilati, C. \\u003cem\\u003eet al.\\u003c/em\\u003e ERBB2 Comprehensive Profiling and Prognostication in Stage III Colon Cancer: Findings From PETACC8 and IDEA-France Cohorts. \\u003cem\\u003eGastroenterology\\u003c/em\\u003e 168, 714\\u0026ndash;724.e4 (2025).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eRichman, S. D. \\u003cem\\u003eet al.\\u003c/em\\u003e HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials. \\u003cem\\u003eJ. Pathol.\\u003c/em\\u003e 238, 562\\u0026ndash;570 (2016).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eIngold Heppner, B. \\u003cem\\u003eet al.\\u003c/em\\u003e HER2/neu testing in primary colorectal carcinoma. \\u003cem\\u003eBr. J. Cancer\\u003c/em\\u003e 111, 1977\\u0026ndash;1984 (2014).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eLee, S. M. \\u0026amp; Oh, H. RAS/RAF mutations and microsatellite instability status in primary colorectal cancers according to HER2 amplification. \\u003cem\\u003eSci. Rep.\\u003c/em\\u003e 14, 11432 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eAmbrosini, M. \\u003cem\\u003eet al.\\u003c/em\\u003e Epidemiology, pathogenesis, biology and evolving management of MSI-H/dMMR cancers. \\u003cem\\u003eNat. Rev. Clin. Oncol.\\u003c/em\\u003e 22, 385\\u0026ndash;407 (2025).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eMcRonald, F. E. \\u003cem\\u003eet al.\\u003c/em\\u003e Identification of people with Lynch syndrome from those presenting with colorectal cancer in England: baseline analysis of the diagnostic pathway. \\u003cem\\u003eEur. J. Hum. Genet.\\u003c/em\\u003e 32, 529\\u0026ndash;538 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eSartore-Bianchi, A. \\u003cem\\u003eet al.\\u003c/em\\u003e Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial. \\u003cem\\u003eLancet Oncol.\\u003c/em\\u003e 17, 738\\u0026ndash;746 (2016).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eRaghav, K. \\u003cem\\u003eet al.\\u003c/em\\u003e 737MO Trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-positive (HER2+) metastatic colorectal cancer (mCRC): Final analysis of DESTINY-CRC02, a randomized, phase II trial. \\u003cem\\u003eAnn. Oncol.\\u003c/em\\u003e 36, S514\\u0026ndash;S515 (2025).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eSiena, S. \\u003cem\\u003eet al.\\u003c/em\\u003e HER2-related biomarkers predict clinical outcomes with trastuzumab deruxtecan treatment in patients with HER2-expressing metastatic colorectal cancer: biomarker analyses of DESTINY-CRC01. \\u003cem\\u003eNat. Commun.\\u003c/em\\u003e 15, 10213 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eStrickler, J. H. \\u003cem\\u003eet al.\\u003c/em\\u003e MOUNTAINEER-03 phase III study design: first-line mFOLFOX6\\u0026thinsp;+\\u0026thinsp;tucatinib\\u0026thinsp;+\\u0026thinsp;trastuzumab for HER2\\u0026thinsp;+\\u0026thinsp;metastatic colorectal cancer. \\u003cem\\u003eFuture Oncol.\\u003c/em\\u003e 21, 303\\u0026ndash;311 (2025).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eQiu, M.-Z. \\u003cem\\u003eet al.\\u003c/em\\u003e Relationship of HER2 Alteration and Microsatellite Instability Status in Colorectal Adenocarcinoma. \\u003cem\\u003eThe Oncologist\\u003c/em\\u003e 26, e1161\\u0026ndash;e1170 (2021).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eLuo, H. \\u003cem\\u003eet al.\\u003c/em\\u003e HER2 Overexpression and Mismatch Repair Deficiency are Correlated with Malignancy in Colorectal Cancer. \\u003cem\\u003eCancer Manag. Res.\\u003c/em\\u003e Volume 13, 3443\\u0026ndash;3454 (2021).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eValtorta, E. \\u003cem\\u003eet al.\\u003c/em\\u003e Assessment of a HER2 scoring system for colorectal cancer: results from a validation study. \\u003cem\\u003eMod. Pathol.\\u003c/em\\u003e 28, 1481\\u0026ndash;1491 (2015).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eAbu-Ghazaleh, N., Kaushik, V., Gorelik, A., Jenkins, M. \\u0026amp; Macrae, F. Worldwide prevalence of Lynch syndrome in patients with colorectal cancer: Systematic review and meta-analysis. \\u003cem\\u003eGenet. Med.\\u003c/em\\u003e 24, 971\\u0026ndash;985 (2022).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eLatham, A. \\u003cem\\u003eet al.\\u003c/em\\u003e Microsatellite Instability Is Associated With the Presence of Lynch Syndrome Pan-Cancer. \\u003cem\\u003eJ. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.\\u003c/em\\u003e 37, 286\\u0026ndash;295 (2019).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eJ\\u0026auml;rvinen, H. J. \\u003cem\\u003eet al.\\u003c/em\\u003e Ten Years After Mutation Testing for Lynch Syndrome: Cancer Incidence and Outcome in Mutation-Positive and Mutation-Negative Family Members. \\u003cem\\u003eJ. Clin. Oncol.\\u003c/em\\u003e 27, 4793\\u0026ndash;4797 (2009).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003ePuccini, A. \\u003cem\\u003eet al.\\u003c/em\\u003e ItaLynch: an ongoing Italian study to evaluate the feasibility of mainstreaming the diagnosis of Lynch syndrome in colorectal cancer patients. \\u003cem\\u003eESMO Gastrointest. Oncol.\\u003c/em\\u003e 3, 100044 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003ePuccini, A. \\u003cem\\u003eet al.\\u003c/em\\u003e Streamlining the diagnostic pathway for Lynch syndrome in colorectal cancer patients: a 10-year experience in a single Italian Cancer Center. \\u003cem\\u003eEur. J. Cancer Prev.\\u003c/em\\u003e 33, 355\\u0026ndash;362 (2024).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cspan\\u003eHofmann, M. \\u003cem\\u003eet al.\\u003c/em\\u003e Assessment of a HER2 scoring system for gastric cancer: results from a validation study. \\u003cem\\u003eHistopathology\\u003c/em\\u003e 52, 797\\u0026ndash;805 (2008).\\u003c/span\\u003e\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"colorectal cancer, dMMR/MSI, HER2, precision medicine\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9105189/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9105189/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eMismatch repair deficiency (dMMR)/Microsatellite instability (MSI) plays a central role in colorectal cancer (CRC) as a predictive and prognostic biomarker and as a \\u0026ldquo;red flag\\u0026rdquo; for diagnosis of Lynch Syndrome (LS). \\u003cem\\u003eHER2\\u003c/em\\u003e is emerging as a therapeutic target and clinical trials are ongoing. Several reports suggested that \\u003cem\\u003eHER2\\u003c/em\\u003e amplification could be mutually exclusive with dMMR/MSI, which is mostly associated with \\u003cem\\u003eHER2\\u003c/em\\u003e 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. \\u003cem\\u003eHER2\\u003c/em\\u003e amplification was evaluated with immunohistochemistry (IHC) and FISH for IHC 2\\u0026thinsp;+\\u0026thinsp;patients. Median age was 75 years, 84% were stage II and stage III. \\u003cem\\u003eBRAF\\u003c/em\\u003e and \\u003cem\\u003eRAS\\u003c/em\\u003e mutation occurred in 60% and 14% of the patients with available molecular data, respectively. No patients harbored \\u003cem\\u003eHER2\\u003c/em\\u003e amplification, suggesting that the co-occurrence of HER2 amplification and dMMR/MSI is essentially anecdotal, making therefore HER2 testing in this subgroup less compelling.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Absence of co-occurrence between HER2 amplification and dMMR/MSI in colorectal cancer: a multicentric study\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-04-26 17:16:08\",\"doi\":\"10.21203/rs.3.rs-9105189/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-05-11T06:27:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-07T13:48:39+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-01T17:05:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"241251310978632705995341584436492700387\",\"date\":\"2026-04-20T13:55:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"149399533316516466913737901199193584328\",\"date\":\"2026-04-19T19:28:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"61905924645918732515078512623655101293\",\"date\":\"2026-04-17T14:10:46+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-04-17T14:07:54+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-03-20T10:23:49+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-03-20T03:37:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-03-19T08:01:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2026-03-19T07:47:33+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"250ce80f-a321-44e7-a071-dd4f9ff61bd6\",\"owner\":[],\"postedDate\":\"April 26th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-05-11T06:27:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-07T13:48:39+00:00\",\"index\":103,\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-01T17:05:23+00:00\",\"index\":102,\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":66965824,\"name\":\"Health sciences/Biomarkers\"},{\"id\":66965825,\"name\":\"Biological sciences/Cancer\"},{\"id\":66965826,\"name\":\"Health sciences/Gastroenterology\"},{\"id\":66965827,\"name\":\"Health sciences/Oncology\"}],\"tags\":[],\"updatedAt\":\"2026-05-14T05:25:40+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-04-26 17:16:08\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9105189\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9105189\",\"identity\":\"rs-9105189\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}