Progress Toward Identifying Exact Proxies for Predicting Response to Immunotherapies

Background: Emerging clinical evidence using immunotherapy in recent years has demonstrated its power to suppress tumor growth by releasing the brakes on the immune system. For example, blockade of immune checkpoints, such as PD-1, has revolutionized treatment options for patients with aggressive cancers such as head and neck squamous cell carcinoma (HNSCC). However, clinical responses to PD-1 inhibition vary widely among patients while majority of them do not show any anti-tumor response. Multiple FDA-approved drugs against the same immune checkpoints have resulted in globally distinct outcomes in the clinic. There is a huge unmet need to understand these disparities at the individual patient level and to maximize the clinical benefits of these agents. Methods: Here, we employed a patient-derived ex vivo model, CANScript™ (Majumder B et al. Nature Commun 2015 Feb 27;6:6169 and Goldman A et al. Nature Commun 2015 Feb 11;6:6139), which recreates the native 3D tumor microenvironment, autocrine-paracrine dynamic and response to therapy by incorporating fresh tumor tissue and autologous immune cells with immunotherapy agents. Utilizing late stage HNSCC (N=50) we interrogated phenotypic response to two FDA-approved PD-1 inhibitors, Pembrolizumab (KEYTRUDA) and Nivolumab (OPDIVO). To do this, we used a comprehensive panel of immunological assays to evaluate changes in the immune compartments by flowcytometry and immunohistochemistry (primarily CD8, CD45, FOXP3, CXCR4, CD68, PDL1, PD1), multiplex cytokine profiling (IL6, IL8, IFN-g, IL10, IL12, Perforin, GranzymeB), along with functional/phenotypic effects including tumor proliferation, histological changes and cell death. Results: The data demonstrated that CANScript™ preserves the tumor-immune contexture and native heterogeneity across different clinical stages and patients. Importantly, we observed that PD-1 blockade resulted in patient-specific therapeutic response, which was characterized by differential distribution and maintenance of infiltrating CD8+ and CD4+ lymphocytes, distinct patterning of cytokines linked to functional dysregulation, and changes in tumor proliferation and apoptosis. Interestingly, data suggest that both Pembrolizumab and Nivolumab act on the same immune network axis but trigger functionally diverse phenotypes in the tumor immune compartment and distinct antitumor effects within an individual patient tumor. Conclusion: Together, these findings demonstrate the utility of CANScript™ as an ex vivo platform to predict therapeutic response of immune checkpoint inhibitors at the individual patient level. It also highlights mechanistic variations that could impact clinical outcome of these agents having the same molecular target. Such information can re-shape our understanding of patient selection and rational combinations for novel immune checkpoint inhibitors. Citation Format: Padhma Radhakrishnan, Vasanthakumar Sekar, Nilesh Brijwani, Priyanka Chevour, Babu Balakrishnan, Dency D Pinto, Muthusami Oliyarasi, Debapriya G. Mehrotra, Manjusha Biswas, Sabitha K S, Kodaganur S. Gopinath, Arkasubhra Ghosh, M s Ganesh, Ashok M. Shenoy, Saravanan Thiyagarajan, Biswanath Majumder, Aaron Goldman. A patient derived ex vivo platform CANScript™ predicts distinct therapeutic outcomes to multiple PD-1 checkpoint inhibitors in single tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2017-3681

Despite lengthening survival, death rates from metastatic colorectal cancer (CRC) remain unacceptably high, with a bright spot being the demonstration of durable responses in patients with CRC who have mismatch repair-deficient (dMMR) and/or microsatellite instability-high (MSI-H) tumors and are treated with immune checkpoint inhibitor therapy. Nivolumab and pembrolizumab, as well as nivolumab in combination with low-dose ipilimumab-all checkpoint inhibitors-are currently approved by the U.S. Food and Drug Administration (FDA) for patients with MSI-H/dMMR metastatic CRC that progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. Nonetheless, there are a number of questions and considerations in the use of these checkpoint inhibitor therapies. Using a question-and-answer format, this review summarizes the scientific rationale for immune checkpoint inhibitor therapy in CRC, including the effects of tumor factors such as genetic aberrations and mutational load on the immune response, particularly in patients with MSI-H/dMMR disease. We discuss response patterns, response criteria, and immune-related adverse events using findings from published efficacy and safety data of immune checkpoint inhibitor therapy in metastatic CRC. We also discuss issues surrounding treatment sequencing, incorporating approved checkpoint inhibitors into the current treatment paradigm, and the multiple investigational strategies that may optimize immunotherapy for advanced CRC in the future, including novel combination therapies. IMPLICATIONS FOR PRACTICE: Colorectal cancer (CRC) is the third most common cancer in the U.S. Despite advances in chemotherapy, survival remains poor for patients with metastatic CRC. Certain immunotherapy agents have demonstrated long-lasting responses in previously treated patients with immune-responsive microsatellite instability-high/mismatch repair-deficient metastatic CRC, leading to U.S. Food and Drug Administration approval of the immune checkpoint inhibitors nivolumab (with or without low-dose ipilimumab) and pembrolizumab in this population. Combination therapy (e.g., nivolumab with low-dose ipilimumab) has demonstrated numerically higher response rates and improved long-term clinical benefit relative to anti-programmed death-1 monotherapy. Ongoing trials are evaluating immunotherapy in the broader CRC population and novel combinations to optimize immunotherapy for advanced CRC.

The Association of Long-term Treatment-related Side Effects With Cancer-specific and General Quality of Life Among Prostate Cancer Survivors

Neoantigens in the immuno-oncology space.

Pre-therapeutic Evaluation of Patient-Specific Responses to Immune-Checkpoint Inhibition in Colorectal Cancer

Autophagy has been identified as a catabolic mechanism in cells but its’ role in cancer remains controversial. Autophagy has been characterized either as tumor suppressor or inducer mechanism in many tumor types. Monoclonal antibodies against EGFR (cetuximab and panitumumab) represent a major step in the treatment of mCRC. Several studies propose that cetuximab and panitumumab trigger autophagy which reveals a potential resistance mechanism to these agents. The last years immunotherapy appears to be a novel promising strategy for the treatment of patients with solid tumors, including colorectal cancer. Checkpoint inhibitors, such as anti-PD1 (nivolumab and pembrolizumab) and anti-CTLA-4 (ipilimumab) antibodies have already been developed and applied in mCRC patients with MSI-H phenotype. The association between mtBRAF and autophagy or MSI status has already been characterized. In our study, we identify the autophagy initiation through anti-EGFR monoclonal antibodies and checkpoint inhibitors in colorectal carcinoma cell lines according to microsatellite status. The combination of autophagy inhibition, anti-EGFR antibodies and checkpoint inhibitors as well as autophagy targeting, MEK inhibition and anti-EGFR antibodies or checkpoint inhibitors appears to be the best treatment approach for microsatellite instability high and stable colorectal cancer cell lines, respectively. Both combinatorial approaches reduce cell viability through the induction of apoptotic cell death. The findings of this study point out the importance of different approach for the treatment of BRAF mutant metastatic colorectal cancers based on their microsatelite instability phenotype.

Challenges with the management of older patients with cancer during the COVID-19 pandemic

Colorectal cancer (CRC) ranks among the most common and lethal malignancies worldwide, accounting for 10% of all cancer cases. In Puerto Rico it is the third most prevalent cancer. In 2022, colorectal cancer ranked among the most diagnosed cancers in Puerto Rico, with a 2022 age-standardized incidence rate of 27.8 per 100,000—surpassing the U.S. rate of 27.0 per 100,000. The age-adjusted mortality rate in Puerto Rico was 13.0 per 100,000, compared to the U.S. rate of 7.9 per 100,000, highlighting a significant public health disparity. These differences may reflect variations in environmental exposures, dietary patterns, genetic susceptibility, and microbiome composition. However, role of microbial and immune factors in colorectal carcinogenesis among Puerto Rican Hispanics (PRHs) remains poorly understood. Pathogenic bacteria including Escherichia coli, Bacteroides fragilis, Fusobacterium nucleatum and Enterococcus faecalis are known to contribute to tumorigenesis through inflammation and immune modulation. Additionally, cytokines, play a critical role in CRC development by shaping the tumor microenvironment. This study aimed to characterize microbial composition, cytokine profiles, and immune signaling associated with CRC in PRHs. We analyzed plasma and tissue samples from the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR), including 15 CRC cases and 15 age- and gender-matched healthy controls. Cytokine levels were assessed using the ProcartaPlex Inflammation Panel (Luminex assay), while bacterial DNA was quantified by qPCR targeting uidA (E. coli), 16S rRNA ( B. fragilis), fus1 (F.nucleatum) and, groES (E.faecalis). Additionally, we conducted time and dose-response assays in metastatic CRC cell lines to assess the DNA damage produced by circulating bacterial DNA. Statistical analysis was performed using paired t-tests (GraphPad Prism) and ROC curve analysis using univariable and multivariable models (R studio) to evaluate diagnostic performance. CRC patients exhibited elevated plasma levels of CD62P, ICAM-1, IL-8, MCP-1, and IP-10, suggesting immune dysregulation and a tumor-promoting inflammatory milieu. Tissue levels of E. coli, F. nucleatum, and E. faecalis were significantly increased in CRC cases, supporting a role in dysbiosis and tumor progression. Among these, E. coli induced the highest DNA damage in metastatic CRC cells. A multivariable model combining MCP-1 and E.coli achieved the highest diagnostic accuracy (AUC: 0.96; 95% CI, 0.91-1.00). These findings underscore the intricate interaction between microbiota, immune signaling, and CRC development in PRHs. Population-specific research is essential for refining biomarker discovery and informing tailored prevention and diagnostic strategies that may also translate to broader U.S. populations. Citation Format: Byron K. Olivo-Natal, Sheila N. López-Acevedo, Camille N. Zenón-Meléndez, Marcia Cruz-Correa, Elba V. Caraballo-Rivera. Analysis of key microbial signatures and immune response interactions in colorectal cancer cases [abstract]. In: Proceedings of the 18th AACR Conference on the Science of Cancer Health Disparities; 2025 Sep 18-21; Baltimore, MD. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2025;34(9 Suppl):Abstract nr C114.

TPS2659 Background: Metastatic colorectal cancer (CRC) harbouring genetic defects in the mismatch-repair pathway (MMRd) presents with a high tumor mutational burden (TMB), and is highly sensitive to anti–programmed cell death protein 1 (PD-1) immune checkpoint inhibitors. We recently showed in preclinical models that the pharmacological treatment with temozolomide (TMZ) can induce the inactivation of MMR genes, and consequently the increase of TMB and immunogenic neoantigens, thus suggesting that TMZ could be used to prime MMR proficient (MMRp) tumors for response to checkpoint inhibitors. Accordingly, mCRC patients recruited in previous clinical trials where TMZ was administered, acquired alterations of MMR genes upon treatment and showed remarkable increase in TMB at disease progression (PD). We thus designed the ARETHUSA clinical trial to test whether a priming course with TMZ in patients can sensitize mCRC to the anti-PD1 inhibitor pembrolizumab. Methods: Arethusa (NCT03519412) is a 2-cohorts, phase II trial consisting of three different phases. In the SCREENING, 348 mCRC RAS-mutated patients will be tested for MMR status. MMRd patients will proceed directly to TRIAL for immediate pembrolizumab treatment (expected 14). MMR-proficient (MMRp) patients will be further tested for expression of O6-methylguanine-DNA methyltransferase (MGMT) by immunohistochemistry and by promoter methylation analysis. IHC-negative, promoter methylation-positive MMRp patients (expected 67) will enter in the PRIMING phase and will be treated with TMZ until PD. TMB will then be assessed on tumor biopsies at resistance. Those patients that will have > 20 mutations/megabase will proceed to TRIAL (expected 20) and will be treated with pembrolizumab. Overall response rate (primary outcome), Progression Free, and Overall Survival, and treatment related toxicities (secondary outcomes) in MMRp pembrolizumab-treated patients will be estimated., while the MMRd cohort will be used for comparison. Tissue biopsies, longitudinal blood and stool collection will be used for discovery of predictive molecular biomarkers and assessment of tumor evolution. Clinical trial information: NCT03519412.

A novel set of integrated procedures for quantification of fibroblast‐rich stroma and vascular characteristics has recently been presented allowing discovery of novel perivascular and stromal biomarkers in colorectal, renal cell, and ovarian cancer. In the present study, data obtained through these procedures from clinically well‐annotated collections of these three tumour types have been used to address two novel questions. First, data have been used to investigate if the three tumour types demonstrate significant differences regarding features such as vessel diameter, vessel density, and perivascular marker expression. Second, analyses of the cohorts have been used to explore the prognostic significance of a novel vascular metric, ‘vessel distance inter‐quartile range (IQR)’ that describes intra‐case heterogeneity regarding vessel distribution. The comparisons between the three tumour types demonstrated a set of significant differences. Vessel density of renal cell cancer was statistically significantly higher than in colorectal and ovarian cancer. Vessel diameter was statistically significantly higher in ovarian cancer. Concerning perivascular status, colorectal cancer displayed significantly higher levels of perivascular PDGFR‐β expression than the other two tumour types. Intra‐case heterogeneity of perivascular PDGFR‐β expression was also higher in colorectal cancer. Notably, these fibroblast‐dominated stroma phenotypes matched previously described experimental tumour stroma characteristics, which have been linked to differential sensitivity to anti‐VEGF drugs. High ‘vessel distance IQR’ was significantly associated with poor survival in both renal cell cancer and colorectal cancer. In renal cell cancer, this characteristic also acted as an independent prognostic marker according to multivariate analyses including standard clinico‐pathological characteristics. Explorative subset analyses indicated particularly strong prognostic significance of ‘vessel distance IQR’ in T stage 4 of this cancer type. Together, these analyses identified tumour‐type‐specific vascular‐stroma phenotypes of possible functional significance, and suggest ‘vessel distance IQR’ as a novel prognostic biomarker.

Simple SummaryImmune Checkpoint Inhibitors (ICIs) have demonstrated clinical efficacy in Microsatellite Instability High Colorectal Cancer (MSI-H CRC). However, in Microsatellite Stable (MSS) CRC, ICIs monotherapy provides limited clinical benefit. Therefore, efforts must be made to understand the highly heterogeneous CRC microenvironment and to find predictive biomarkers of response in order to adequately select CRC patients who may respond to ICIs-based therapies.Immune Checkpoint Inhibitors (ICIs) are well recognized as a major immune treatment modality for multiple types of solid cancers. However, for colorectal cancer (CRC), ICIs are only approved for the treatment of Mismatch-Repair-Deficient and Microsatellite Instability-High (dMMR/MSI-H) tumors. For the vast majority of CRC, that are not dMMR/MSI-H, ICIs alone provide limited to no clinical benefit. This discrepancy of response between CRC and other solid cancers suggests that CRC may be inherently resistant to ICIs alone. In translational research, efforts are underway to thoroughly characterize the immune microenvironment of CRC to better understand the mechanisms behind this resistance and to find new biomarkers of response. In the clinic, trials are being set up to study biomarkers along with treatments targeting newly discovered immune checkpoint molecules or treatments combining ICIs with other existing therapies to improve response in MSS CRC. In this review, we will focus on the characteristics of response and resistance to ICIs in CRC, and discuss promising biomarkers studied in recent clinical trials combining ICIs with other therapies.

PurposeImmune checkpoints, as pivotal regulators of immune escape in cancer, can motivate the emergence of immune checkpoint inhibitors (ICIs). The aim of this study is to identify the expression of the immune checkpoint genes (ICGs) in colorectal cancer (CRC) and to relate their individual as well as combined expression to prognosis and therapeutic effectiveness in CRC.MethodsRNA expression of 47 ICGs and clinical information of CRC patients were collected from two public databases to elucidate the expression levels and prognostic values of these ICGs in CRC. Then, the Shapiro–Wilk normality test was used to determine the normality of variables. Overall survival (OS) rates of each subset were found by Kaplan–Meier method, and the statistical significance was determined by the Log rank test (p < 0.05).ResultsThe expression of 13 and 9 ICGs was significantly associated with CRC prognosis in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. A series of ICGs was found to be significantly associated with TMB, neoantigens and MMR in CRC indicating that the combination of immunotherapy treatment biomarkers and ICGs may achieve accurate prognostic stratification of CRC, and potentially identify CRC cases that might respond to checkpoint inhibitors (CPIs). The subsets of high or low PD1/PD-L1/IDO1 expression stratified by CD48 were accurately associated with prognosis in CRC. In addition, in vitro experiments confirmed that VTCN1(B7-H4)-KD increases anti-PD-L1-mediated NK cell cytotoxicity on CRC tumor cells.ConclusionAlthough the expression of a single immune-checkpoint molecule does not predict the efficacy of immunotherapy in CRC, our findings infer that subsets defined by ICGs are associated with prognosis and imply the possibility that VTCN1 and CD48 serve as new immunotherapeutic targets.

Immunotherapy with immune checkpoint inhibitors (ICIs) is highly effective in microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC); however, specific predictive biomarkers are lacking. Data and samples from 85 patients with MSI-H mCRC treated with ICIs were gathered. Tumor infiltrating lymphocytes (TILs) and tumor mutational burden (TMB) were analyzed in an exploratory cohort of "super" responders and "clearly" refractory patients; TILs were then evaluated in the whole cohort of patients. Primary objectives were the correlation between the number of TILs and TMB and their role as biomarkers of ICI efficacy. Main endpoints included response rate (RR), progression-free survival (PFS), and overall survival (OS). In the exploratory cohort, an increasing number of TILs correlated to higher TMB (Pearson's test, p = .0429). In the whole cohort, median number of TILs was 3.6 in responders compared with 1.8 in nonresponders (Mann-Whitney test, p = .0448). RR was 70.6% in patients with high number of TILs (TILs-H) compared with 42.9% in patients with low number of TILs (odds ratio = 3.20, p = .0291). Survival outcomes differed significantly in favor of TILs-H (PFS: hazard ratio [HR] = 0.42, p = .0278; OS: HR = 0.41, p = .0463). A significant correlation between higher TMB and increased number of TILs was shown. A significantly higher activity and better PFS and OS with ICI in MSI-H mCRC were reported in cases with high number of TILs, thus supporting further studies of TIL count as predictive biomarker of ICI efficacy. Microsatellite instability is the result of mismatch repair protein deficiency, caused by germline mutations or somatic modifications in mismatch repair genes. In metastatic colorectal cancer (mCRC), immunotherapy (with immune checkpoint inhibitors [ICIs]) demonstrated remarkable clinical benefit in microsatellite instability-high (MSI-H) patients. ICI primary resistance has been observed in approximately 25% of patients with MSI-H mCRC, underlining the need for predictive biomarkers. In this study, tumor mutational burden (TMB) and tumor infiltrating lymphocyte (TIL) analyses were performed in an exploratory cohort of patients with MSI-H mCRC treated with ICIs, demonstrating a significant correlation between higher TMB and increased number of TILs. Results also demonstrated a significant correlation between high number of TILs and clinical responses and survival benefit in a large data set of patients with MSI-H mCRC treated with ICI. TMB and TILs could represent predictive biomarkers of ICI efficacy in MSI-H mCRC and should be incorporated in future trials testing checkpoint inhibitors in colorectal cancer.

6621 Background: Clinical outcomes for patients (pts) treated in non-clinical trial settings have not been compared to clinical trial participant outcomes. Moreover, the degree to which reference regimens (Ref Reg) are utilized in actual clinical settings is not known. We created ETPs based on trial reports and guidelines and measured clinician compliance with regimens, degree of variance from Ref Reg, and outcomes in heterogenous community oncology practice settings. Methods: ETPs including Ref Reg were created for 1st and 2nd line therapy in metastatic colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and prostate cancer (PRC) based on review of published and presented Phase III trials and supplemented by NCCN guidelines when phase III data were not available. A range of acceptable regimens and a preferred regimen for each disease were identified, but clinicians were free to utilize any regimen of their choice after pt prospective registration with IRB approved informed consent on the ETP. Results: Five community oncology clinics with median (range) 13 (8–26) medical oncologists enrolled 169 pts as of 12/1/07: 64 CRC, 85 NSCLC, and 20 PRC. Pt demographics: 43% female, 14% African American, 50% Medicare primary payer, ECOG PS 0 31%, PS 1, 47% and > 2, 17%. Of 160 evaluable pts 67(41.9%) were treated identically to Ref Reg, 57 (35.6%) received a standard regimen with dose variation, 20 (12.5%) were enrolled on a research protocol and 11% received a non-ETP regimen. From time to event analysis, the estimated median (95%CI) duration of first line regimen ETP was 210 (98–322) days for CRC, 111 (86–135) days for NSCLC and 225 (181–268) days for PRC. There was no statistically significant difference in the duration of 1st line therapy for Ref Reg compared to dose variation/non-standard regimens. The ETP preferred regimen (± dose variation) was used in 30/54 CRC, 47/70 NSCLC, and 10/16 PRC pts. Accruals to all three ETPs are ongoing. Conclusions: ETPs can be created and utilized successfully in community oncology practice. Most patients received a Ref Reg although a priori dose modifications were common. Initial analysis indicates duration of 1st line therapy is similar in patients receiving clinical trial derived Ref Reg or alternative doses or regimens. No significant financial relationships to disclose.

33 Background: Receptor tyrosine kinase fusion (RTK+) has been identified in colorectal carcinoma (CRC). Tumor agnostic treatment has been approved in NTRK fusion ( NTRK+) and RET fusion ( RET+) positive solid tumor. However, overall response rate (ORR) was the lowest among entrectinib-treated NTRK+ CRC (20%, 2/10) and selpercatinib treated RET+ CRC (20%, 2/10) among all NTRK+ and RET+ solid tumors respectively. Methods: We surveyed all 58 human RTKs to identify RTK fusions in CRC and RET and NTRK fusions in other solid tumors using AACR GENIE cbioportal public version 13.0. We abstracted and compared the tumor mutation burden (TMB) of these fusions from the cbioportal database. Patient and tumor characteristics were also abstracted. Results: Among the 14812 unique CRC patients identified in the AACR GENIE cbioportal, a total of 153 unique RTK fusions were identified (44 with known fusion partners, 109 with intragenic rearrangement) in 27 out of the total 58 human RTKs. The most common RTK fusions in CRC were in FGFR1 (N = 23), EGFR (N = 21), ERBB2, NTRK1, RET, FGFR2 (N = 10 each), FLT1 and FLT3 (N = 9 each), FLT4 and ALK (N = 7 each). Considering only samples with identified fusion partners, the mean TMB was 66.6 +/-15.8 (mt/MB) for NTRK+ CRC (N = 9), and 35 +/-11.5 for RET+ CRC (N = 4), which were significantly higher when compared to mean number of 6.2 +/- 5.4 of TMB for all other RTK+ CRC (ALK, EGFR, FGFR1/2, ERBB4, FLT1/3/4, LMKT2, MET, NTRK2, PDGFR-b, ROS1) (N = 30, P&lt; 0.05). Furthermore, NTRK+ CRC harbored significantly higher TMB than RET+ CRC (P = 0.003). The mean TMB was 4.0 +/- 1.9 for RET+ NSCLC (N = 65), 2.6 +/- 1.6 for RET+ Thyroid cancer (N = 52), which were both significantly lower than RET+ CRC. Mean TMB was 3.1 +/- 1.7 for NTRK+ glioma (N = 21), 3.3 +/- 1.7 for NTRK+ thyroid cancer (N = 21), 6.5+/-5.1 for NTRK+ Melanoma (N = 8), 5.9 +/- 3.4 for NTRK+ NSCLC (N = 7), 5.1 +/- 1.2 for NTRK+ breast cancer (N = 7) all of them were significantly lower than the mean TMB of NTRK+ CRC. 1482 (10.0%) patients with CRC had their MSI status reported. 3 out of 3 NTRK+ CRC patients with known MSI status were all MSI-H (100%). 12 out of 12 non-NTRK/non-RET RTK+ CRC patients were MSI-S. Conclusions: In this comprehensive survey of human RTK fusions in CRC, NRTK+ and RET+ CRC harbor significantly higher TMB compared to other RTK+ CRC or to other NTRK+/RET+ solid tumors. Limited data showed NTRK+ CRC were all MSI-H. NTRK+ and RET+ CRC are distinct molecular subtypes of CRC with unique pathogenesis from all other NTRK+ or RET+ solid tumors. We hypothesize that NTRK+/RET+ CRC may benefit from pembrolizumab that has been approved for histologic agnostic high TMB tumors rather than RET or NTRK tyrosine kinase inhibitors (low ORR in CRC) that also have histological agnostic indications.