Metastatic Colorectal Cancer Patients Research Articles

A consensus molecular subtypes classification strategy for clinical colorectal cancer tissues.

Consensus Molecular Subtype (CMS) classification of colorectal cancer (CRC) tissues is complicated by RNA degradation upon formalin-fixed paraffin-embedded (FFPE) preservation. Here, we present an FFPE-curated CMS classifier. The CMSFFPE classifier was developed using genes with a high transcript integrity in FFPE-derived RNA. We evaluated the classification accuracy in two FFPE-RNA datasets with matched fresh-frozen (FF) RNA data, and an FF-derived RNA set. An FFPE-RNA application cohort of metastatic CRC patients was established, partly treated with anti-EGFR therapy. Key characteristics per CMS were assessed. Cross-referenced with matched benchmark FF CMS calls, the CMSFFPE classifier strongly improved classification accuracy in two FFPE datasets compared with the original CMSClassifier (63.6% versus 40.9% and 83.3% versus 66.7%, respectively). We recovered CMS-specific recurrence-free survival patterns (CMS4 versus CMS2: hazard ratio 1.75, 95% CI 1.24-2.46). Key molecular and clinical associations of the CMSs were confirmed. In particular, we demonstrated the predictive value of CMS2 and CMS3 for anti-EGFR therapy response (CMS2&3: odds ratio 5.48, 95% CI 1.10-27.27). The CMSFFPE classifier is an optimized FFPE-curated research tool for CMS classification of clinical CRC samples.

Efficacy-effectiveness analysis on survival in a population-based real-world study of BRAF-mutated metastatic colorectal cancer patients treated with encorafenib-cetuximab.

Encorafenib-cetuximab has been approved for pretreated BRAFV600E-mutated metastatic colorectal cancer (mCRC) patients based on efficacy demonstrated in the randomized phase III BEACON trial. The aim of this real-world effectiveness study is to improve knowledge on the generalizability of trial results. This population-based real-world study includes all mCRC patients in the Netherlands treated with encorafenib-cetuximab since approval. Individual patient data and pathology reports were collected. Overall survival (OS) was compared to BEACON and subgroup analyses were conducted for patients who would have been eligible and ineligible for BEACON. 166 patients were included with a median follow-up time of 14.5 months. Median OS was 6.7 months (95% CI:6.0-8.3) and differed from BEACON (9.3 months; 95% CI:8.0-11.3, p-value 0.002). Thirty-six percent of real-world patients would have been ineligible for the BEACON trial. Trial ineligible subgroups with symptomatic brain metastases and WHO performance status ≥2 had the poorest median OS of 5.0 months (95% CI:4.0-NR) and 3.9 months (95% CI:2.4-NR). This real-world cohort of mCRC patients treated with encorafenib-cetuximab showed a clinically relevant efficacy-effectiveness gap for OS. The chance of survival benefit from encorafenib-cetuximab in patients with brain metastases and/or WHO performance status ≥2 is negligible as neither efficacy nor effectiveness has been demonstrated.

Common variation in a long non-coding RNA gene modulates variation of circulating TGF-β2 levels in metastatic colorectal cancer patients (Alliance)

BackgroundHerein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC). The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings.ResultsThe final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-β2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73.ConclusionsThis study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-β2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

Objective response after immune checkpoint inhibitors in a chemotherapy-refractory pMMR/MSS metastatic rectal cancer patient primed with experimental AlloStim® immunotherapy

BackgroundImmune Checkpoint Inhibitor (ICI) immunotherapy is most effective in immune effector cell infiltrated ‘hot’ tumor lesions, such as occurs in deficient mismatch repair, microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC). However, most all metastatic CRC tumors are mismatch repair proficient/microsatellite stable (pMMR/MSS) ‘cold’ lesions, without significant immune cell infiltration, and are unresponsive to ICI. AlloStim®, is an experimental, allogeneic immunomodulatory cell therapy designed to convert ‘cold’ metastatic tumor lesions to ‘hot’ inflamed lesions. After AlloStim® immunotherapy, this cold to hot inflammatory mechanism can make it difficult to distinguish between pseudoprogression and actual progression on restaging CT scans, as inflamed metastatic lesions can appear larger and occult disease can appear as new small lesions.MethodsTo explore whether radiological progression after AlloStim® immunotherapy is due to immune-flare or disease progression, we administered a short course of a combination ICI therapy to a pMMR/MSS chemotherapy-refractory metastatic colorectal cancer patient enrolled in the StimVax Phase IIb clinical study that presented with radiological progression after AlloStim® immunotherapy. Our rationale was that an accelerated response to ICI should occur if the lesions were inflamed, while if the enlarged lesions were due to disease progression there would not be a response.ResultsHere we report a rapid, significant reduction in tumor burden in response to ICI administration in an AlloStim® primed pMMR/MSS mCRC patient with retroperitoneal and lung metastases.ConclusionThis rare objective response to ICIs in a pMMR/MSS mCRC patient supports further evaluation of the combination of AlloStim® with ICI immunotherapy in MSS mCRC and other cold or ICI refractory tumors.Trial registrationNational Library of Medicine (NLM) at the National Institutes of Health (NIH). Registered 22 June 2020, https://clinicaltrials.gov/study/NCT04444622.

Genomic landscape defines peritoneal metastatic pattern and related target of peritoneal metastasis in colorectal cancer.

The primary objective of this study is to develop a prediction model for peritoneal metastasis (PM) in colorectal cancer by integrating the genomic features of primary colorectal cancer, along with clinicopathological features. Concurrently, we aim to identify potential target implicated in the peritoneal dissemination of colorectal cancer through bioinformatics exploration and experimental validation. By analyzing the genomic landscape of primary colorectal cancer and clinicopathological features from 363 metastatic colorectal cancer patients, we identified 22 differently distributed variables, which were used for subsequent LASSO regression to construct a PM prediction model. The integrated model established by LASSO regression, which incorporated two clinicopathological variables and seven genomic variables, precisely discriminated PM cases (AUC 0.899; 95% CI 0.860-0.937) with good calibration (Hosmer-Lemeshow test p = .147). Model validation yielded AUCs of 0.898 (95% CI 0.896-0.899) and 0.704 (95% CI 0.622-0.787) internally and externally, respectively. Additionally, the peritoneal metastasis-related genomic signature (PGS), which was composed of the seven genes in the integrated model, has prognostic stratification capability for colorectal cancer. The divergent genomic landscape drives the driver genes of PM. Bioinformatic analysis concerning these driver genes indicated SERINC1 may be associated with PM. Subsequent experiments indicate that knocking down of SERINC1 functionally suppresses peritoneal dissemination, emphasizing its importance in CRCPM. In summary, the genomic landscape of primary cancer in colorectal cancer defines peritoneal metastatic pattern and reveals the potential target of SERINC1 for PM in colorectal cancer.

Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer

Colorectal cancer (CRC) is prone to metastasis and recurrence after surgery, which is one of the main causes for its poor treatment and prognosis. Therefore, it is essential to identify biomarkers associated with metastasis and recurrence in CRC. DNA methylation has a regulatory role in cancer metastasis, tumor immune microenvironment (TME), and prognosis and may be one of the most valuable biomarkers for predicting CRC metastasis and prognosis. We constructed a diagnostic model and nomogram that can effectively predict CRC metastasis based on the differential methylation CpG sites (DMCs) between metastatic and non-metastatic CRC patients. Then, we identified 17 DMCs associated with progression free survival (PFS) of CRC and constructed a prognostic model. The prognosis model based on 17 DMCs can predict the PFS of CRC with medium to high accuracy. The results of immunohistochemical analysis indicated that the protein expression levels of the genes involved in prognostic DMCs were different between normal and colorectal cancer tissues. According to the results of immune-related analysis, we found that the low-risk patients had better immunotherapy response. In addition, high risk scores were negatively correlated with high tumor mutation burden (TMB) levels, and patients with low TMB levels in the high-risk group had the worst PFS. Our work shows the clinical value of DNA methylation in predicting CRC metastasis and PFS, as well as their correlation with TME, immunotherapy, and TMB, which helps understand the changes of DNA methylation in CRC metastasis and improving the treatment and prognosis of CRC.

Differential expression of angiogenesis-related genes ‘VEGF’ and ‘angiopoietin-1’ in metastatic and EMAST-positive colorectal cancer patients

Abnormal angiogenesis leads to tumor progression and metastasis in colorectal cancer (CRC). This study aimed to elucidate the association between angiogenesis-related genes, including VEGF-A, ANGPT-1, and ANGPT-2 with both metastatic and microsatellite alterations at selected tetranucleotide repeats (EMAST) subtypes of CRC. We conducted a thorough assessment of the ANGPT-1, ANGPT-2, and VEGF-A gene expression utilizing publicly available RNA sequencing and microarray datasets. Then, the experimental validation was performed in 122 CRC patients, considering their disease metastasis and EMAST+/− profile by using reverse transcription polymerase chain reaction (RT-PCR). Subsequently, a competing endogenous RNA (ceRNA) network associated with these angiogenesis-related genes was constructed and analyzed. The expression level of VEGF-A and ANGPT-2 genes were significantly higher in tumor tissues as compared with normal adjacent tissues (P-value < 0.001). Nevertheless, ANGPT-1 had a significantly lower expression in tumor samples than in normal colon tissue (P-value < 0.01). We identified a significantly increased VEGF-A (P-value = 0.002) and decreased ANGPT-1 (P-value = 0.04) expression in EMAST+ colorectal tumors. Regarding metastasis, a significantly increased VEGF-A and ANGPT-2 expression (P-value = 0.001) and decreased ANGPT-1 expression (P-value < 0.05) were established in metastatic CRC patients. Remarkably, co-expression analysis also showed a strong correlation between ANGPT-2 and VEGF-A gene expressions. The ceRNA network was constructed by ANGPT-1, ANGPT-2, VEGF-A, and experimentally validated miRNAs (hsa-miR-190a-3p, hsa-miR-374c-5p, hsa-miR-452-5p, and hsa-miR-889-3p), lncRNAs (AFAP1-AS1, KCNQ1OT1 and MALAT1), and TFs (Sp1, E2F1, and STAT3). Network analysis revealed that colorectal cancer is amongst the 82 significant pathways. We demonstrated a significant differential expression of VEGF-A and ANGPT-1 in colorectal cancer patients exhibiting the EMAST+ phenotype. This finding provides novel insights into the molecular pathogenesis of colorectal cancer, specifically in EMAST subtypes. Yet, the generalization of in silico findings to EMAST+ colorectal cancer warrants future experimental investigations. In the end, this study proposes that the EMAST biomarker could serve as an additional perspective on CMS4 biology which is well-defined by activated angiogenesis and worse overall survival.

Detailed analysis of metastatic colorectal cancer patients who developed cardiotoxicity on another fluoropyrimidine and switched to S-1 treatment (subgroup analysis of the CardioSwitch-study).

The CardioSwitch-study demonstrated that patients with solid tumors who develop cardiotoxicity on capecitabine or 5-fluorouracil (5-FU) treatment can be safely switched to S-1, an alternative fluoropyrimidine (FP). In light of the European Medicines Agency approval of S-1 in metastatic colorectal cancer (mCRC), this analysis provides more detailed safety and efficacy information, and data regarding metastasectomy and/or local ablative therapy (LAT), on the mCRC patients from the original study. This retrospective cohort study was conducted at 12 European centers. The primary endpoint was recurrence of cardiotoxicity after switch. For this analysis, safety data are reported for 78 mCRC patients from the CardioSwitch cohort (N = 200). Detailed efficacy and outcomes data were available for 66 mCRC patients. Data for the safety of S-1 in mCRC patients were similar to the original CardioSwitch cohort and that expected for FP-based treatment, with no new concerns. Recurrent cardiotoxicity (all grade 1) with S-1-based treatment occurred in 4/78 (5%) mCRC patients; all were able to complete FP treatment. Median progression-free survival from initiation of S-1-based treatment was 9.0 months and median overall survival 26.7 months. Metastasectomy and/or LAT was performed in 33/66 (50%) patients, and S-1 was successfully used in recommended neoadjuvant/conversion or adjuvant-like combination regimens and schedules as for standard FPs. S-1 is a safe and effective FP alternative when mCRC patients are forced to discontinue 5-FU or capecitabine due to cardiotoxicity and can be safely used in the standard recommended regimens, settings, and schedules.

Evaluation of the inflammation-based modified Glasgow Prognostic Score (mGPS) as a prognostic and predictive biomarker in patients with metastatic colorectal cancer receiving first-line chemotherapy: a post hoc analysis of the randomized phase III XELAVIRI trial (AIO KRK0110)

The inflammation-based modified Glasgow Prognostic Score (mGPS) combines serum levels of C-reactive protein and albumin and was shown to predict survival in advanced cancer. We aimed to elucidate the prognostic impact of mGPS on survival as well as its predictive value when combined with gender in unselected metastatic colorectal cancer (mCRC) patients receiving first-line chemotherapy in the randomized phase III XELAVIRI trial. In XELAVIRI, mCRC patients were treated with either fluoropyrimidine/bevacizumab followed by additional irinotecan at first progression (sequential treatment arm; Arm A) or upfront combination of fluoropyrimidine/bevacizumab/irinotecan (intensive treatment arm; Arm B). In the present post hoc analysis, survival was evaluated with respect to the assorted mGPS categories 0, 1 or 2. Interaction between mGPS and gender was analyzed. Out of 421 mCRC patients treated in XELAVIRI, 362 [119 women (32.9%) and 243 men (67.1%)] were assessable. For the entire study population a significant association between mGPS and overall survival (OS) was observed [mGPS= 0: median 28.9 months, 95% confidence interval (CI) 25.9-33.6 months; mGPS= 1: median 21.4 months, 95% CI 17.6-26.1 months; mGPS= 2: median 16.8 months, 95% CI 14.3-21.2 months; P < 0.00001]. Similar results were found when comparing progression-free survival between groups. The effect of mGPS on survival did not depend on the applied treatment regimen (P= 0.21). In female patients, a trend towards longer OS was observed in Arm A versus Arm B, with this effect being clearly more pronounced in the mGPS cohort 0 (41.6 versus 25.5 months; P= 0.056). By contrast, median OS was longer in male patients with an mGPS of 1-2 treated in Arm B versus Arm A (20.8 versus 17.4 months; P= 0.022). We demonstrate the role of mGPS as an independent predictor of OS regardless of the treatment regimen in mCRC patients receiving first-line treatment. mGPS may help identify gender-specific subgroups that benefit more or less from upfront intensive therapy.

Predictive significance of FGFR4 p.G388R polymorphism in metastatic colorectal cancer patients receiving trifluridine/tipiracil (TAS-102) treatment

BackgroundTAS-102 (Lonsurf®) is an oral fluoropyrimidine consisting of a combination of trifluridine (a thymidine analog) and tipiracil (a thymidine phosphorylation inhibitor). The drug is effective in metastatic colorectal cancer (mCRC) patients refractory to fluorouracil, irinotecan and oxaliplatin. This study is a real-world analysis, investigating the interplay of genotype/phenotype in relation to TAS-102 sensitivity.MethodsForty-seven consecutive mCRC patients were treated with TAS-102 at the National Cancer Institute of Naples from March 2019 to March 2021, at a dosage of 35 mg/m2, twice a day, in cycles of 28 days (from day 1 to 5 and from day 8 to 12). Clinical-pathological parameters were described. Activity was evaluated with RECIST criteria (v1.1) and toxicity with NCI-CTC (v5.0). Survival was depicted through the Kaplan-Meyer curves. Genetic features of patients were evaluated with Next Generation Sequencing (NGS) through the Illumina NovaSeq 6000 platform and TruSigt™Oncology 500 kit.ResultsMedian age of patients was 65 years (range: 46–77). Forty-one patients had 2 or more metastatic sites and 38 patients underwent to more than 2 previous lines of therapies. ECOG (Eastern Cooperative Oncology Group) Performance Status (PS) was 2 in 19 patients. The median number of TAS-102 cycles was 4 (range: 2–12). The most frequent toxic event was neutropenia (G3/G4 in 16 patients). There were no severe (> 3) non-haematological toxicities or treatment-related deaths. Twenty-six patients experienced progressive disease (PD), 21 stable disease (SD). Three patients with long-lasting disease control (DC: complete, partial responses or stable disease) shared an FGFR4 (p.Gly388Arg) mutation. Patients experiencing DC had more frequently a low tumour growth rate (P = 0.0306) and an FGFR4 p.G388R variant (P < 0.0001). The FGFR4 Arg388 genotype was associated with better survival (median: 6.4 months) compared to the Gly388 genotype (median: 4 months); the HR was 0.25 (95% CI 0.12- 0.51; P = 0.0001 at Log-Rank test).ConclusionsThis phenotype/genotype investigation suggests that the FGFR4 p.G388R variant may serve as a new marker for identifying patients who are responsive to TAS-102. A mechanistic hypothesis is proposed to interpret these findings.Graphical

Efficacy of Regorafenib and Trifluridine/Tipiracil According to Extended RAS Evaluation in Advanced Metastatic Colorectal Cancer Patients: A Multicenter Retrospective Analysis.

There are few molecular markers driving treatment selection in later lines of treatment for advanced colorectal cancer patients. The vast majority of patients who progress after first- and second-line therapy undergo chemotherapy regardless of molecular data. We aimed to assess the prognostic and predictive effects of specific RAS mutations on overall survival of patients receiving regorafenib (rego), trifluridine/tipiracil (TFD/TPI), or both. This was a retrospective observational study based on data from a previous study of our research network, involving nine Italian institutions over a 10-year timeframe (2012-2022). Extended RAS analysis, involving KRAS exon 2-4 and NRAS exon 2-4, and BRAF were the main criteria for inclusion in this retrospective evaluation. Patients with BRAF mutation were excluded. Patients were classified according to treatment (rego- or TFD/TPI-treated) and RAS mutational status (wild-type [WT], KRAS codon 12 mutations, KRAS codon 13 mutations, KRAS rare mutations and NRAS mutations, KRAS G12C mutation and KRAS G12D mutation). Overall, 582 patients were included in the present analysis. Overall survival did not significantly differ in rego-treated patients according to RAS extended analysis, although a trend toward a better median survival in patients carrying G12D mutation (12.0 months), Codon 13 mutation (8.0 months), and Codon 12 mutation (7.0 months) has been observed, when compared with WT patients (6.0 months). Overall survival did not significantly differ in TFD/TPI-treated patients according to RAS extended analysis, although a trend toward a better median survival in WT patients had been observed (9.0 months) in comparison with the entire population (7.0 months). Patients receiving both drugs displayed a longer survival when compared with the population of patients receiving rego alone (p=0.005) as well as the population receiving TFD/TPI alone (p<0.001), suggesting a group enriched for favorable prognostic factors. However, when each group was analyzed separately, the addition of TFD/TPI therapy to the rego-treated group improved survival only in all-RAS WT patients (p=0.003). Differently, the addition of rego therapy to TFD/TPI-treated patients significantly improved OS in the Codon 12 group (p=0.0004), G12D group (p=0.003), and the rare mutations group (p=0.02), in addition to all-RAS WT patients (p=0.002). The rego-TFD/TPI sequence, compared with the reverse sequence, significantly improved OS only in the KRAS codon 12 group (p=0.003). Our data demonstrate that RAS mutations do not affect outcome in rego-treated patients as well as TFD/TPI-treated patients. Nevertheless, a trend toward a higher efficacy of rego in RAS-mutated (in particular codon 12, rare RAS mutations, and G12D) patients has been recorded. The rego-TFD/TPI sequence seems to be superior to the reverse sequence in patients carrying an RAS codon 12 mutation, although the impact of other factors as disease burden or performance status cannot be excluded.

Immunotherapy-induced colitis in metastatic colorectal cancer: a systematic review and meta-analysis

Colorectal cancer (CRC) presents significant mortality risks, underscoring the urgency of timely diagnosis and intervention. Advanced stages of CRC are managed through chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. Immunotherapy, while effective in bolstering the immune system against cancer cells, often carries toxic side effects, including colitis. This study aimed to evaluate the incidence of colitis in patients with metastatic CRC (mCRC) undergoing various immunotherapy treatments. Through a systematic search of Google Scholar and PubMed databases from inception until November 2023, nine relevant studies were identified. Subgroup analyses revealed a higher incidence of colitis, particularly in patients treated with anti-cytotoxic T-lymphocyte-associated molecule-4 (anti-CTLA-4) and combination therapies compared to monotherapy with programmed cell death receptor-1 (PD-1) or programmed cell death ligand receptor-1 (PDL-1) inhibitors. Notably, naive-treated metastatic CRC patients exhibited elevated colitis incidences compared to those previously treated. In conclusion, anti-CTLA-4 and combination therapies, such as nivolumab plus ipilimumab, were associated with increased colitis occurrences in metastatic CRC patients, highlighting the need for vigilant monitoring and management strategies, especially in immunotherapy-naive individuals.

Hypertension as predictive factor for bevacizumab-containing first-line therapy in metastatic breast and colorectal cancer in BRECOL (GEICAM/2011-04) study.

Retrospective data suggest an association between bevacizumab efficacy and the incidence of arterial hypertension (AHT). Additionally, epigenetic mechanisms have been related to AHT. This prospective observational study conducted by GEICAM Spanish Breast Cancer Research Group included metastatic breast (MBC) or colorectal (mCRC) cancer patients treated with bevacizumab-containing chemotherapy as first-line treatment. Blood pressure (BP) levels were measured (conventional and 24-h Holter monitoring) at baseline and up to cycle 3. Primary endpoint assessed BP levels increase as predictive factor for progression-free survival (PFS). Germline DNA methylation profile was explored in pre-treatment blood samples; principal component analysis was used to define an epigenetic predictive score for increased BP levels. From Oct-2012 to Jul-2016, 143 (78 MBC and 65 mCRC) patients were included. The incidence of AHT according to guidelines was neither predictive of PFS nor of best overall tumor response (BOR). No statistically significant association was observed with systolic BP nor diastolic BP increment for PFS or BOR. Grade 3 and 4 adverse events were observed in 37 and 5% of patients, respectively. We identified 27 sites which baseline methylation status was significantly associated to BP levels increase secondary to bevacizumab-containing chemotherapy. Neither the frequency of AHT nor the increase of BP levels were predictive of efficacy in MBC and mCRC patients treated with bevacizumab-containing chemotherapy. ClinicalTrials.gov Identifier: NCT01733628.

Allosteric SHP2 inhibition enhances regorafenib's effectiveness in colorectal cancer treatment

Colorectal cancer (CRC) is the third most common cancer globally. Regorafenib, a multi-target kinase inhibitor, has been approved for treating metastatic colorectal cancer patients who have undergone at least two prior standard anti-cancer therapies. However, regorafenib efficacy as a single agent remains suboptimal. A promising target at the crossroads of multiple signaling pathways is the Src homology 2 domain-containing protein tyrosine phosphatase (SHP2). However, a combination approach using SHP2 inhibitors (SHP099) and anti-angiogenic drugs (Regorafenib) has not been reported in current research. In this study, we conducted in vitro experiments combining SHP099 and regorafenib and established an MC-38 colon cancer allograft mouse model. Our results revealed that co-treatment with SHP099 and regorafenib significantly inhibited cell viability and altered the biological characteristics of tumor cells compared with treatment alone in vitro. Furthermore, the combination strategy demonstrated superior therapeutic efficacy compared to monotherapy with either drug. This was evidenced by reduced tumor size, decreased proliferation, increased apoptosis, normalized tumor microvasculature, and improved antitumor immune response in vivo. These findings suggest that the combination of an SHP2 inhibitor and regorafenib is a promising therapeutic approach for patients with colorectal cancer.

Abstract 2498: Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue

Abstract Introduction Tumor profiling including RAS, BRAF, HER2, and MSI/MMR status, is required to determine the treatment for patients with metastatic colorectal cancer (mCRC) at the time of diagnosis. While comprehensive tumor profiling using tissue-based next-generation sequencing (NGS) is increasingly adopted for initial profiling in mCRC, the role of circulating tumor DNA (ctDNA) NGS as initial testing in patients with sufficient tumor tissue is not clearly understood. We assessed the clinical value of ctDNA sequencing compared to tumor NGS in patients with newly diagnosed mCRC who have sufficient tumor specimens. Methods We prospectively enrolled consecutive patients with newly diagnosed mCRC at the National Cancer Center Korea. As per the institutional protocol for mCRC, initial tumor profiling was performed on primary tumor tissue using an in-house NGS panel (NCC PCP ver.3), which included 525 genes. For ctDNA sequencing, patients were evaluated using the AlphaLiquid®100 comprehensive cancer panel (IMBdx, Inc.), which included 118 genes, before the initiation of chemotherapy. Additionally, immunohistochemical (IHC) testing for HER2 and polymerase chain reaction (PCR)/IHC testing for MSI and/or MMR were performed to assess the accuracy of HER2 and MSI/MMR status. Results A total of 188 patients were enrolled. In 139 eligible patients, 275 potentially actionable mutations were found in 12 selected CRC-related genes (APC, TP53, KRAS, NRAS, BRAF, FBXW7, PIK3CA, ERBB2, SMAD4, NF1, EGFR, MET). Of these, 32% were found both in ctDNA and tissue; 54% were found in ctDNA only; 12% in tissue only, and 2% were discordant. For RAS/BRAF mutations, which are required for anti-EGFR treatment decisions, the concordance rate between ctDNA and tissue NGS was 83.1%, and the concordance was higher in patients with higher ctDNA concentrations. For 9 patients with potentially actionable copy number variations (CNV) in EGFR, HER2, MET, and FGFR1, 3 cases were found by both assays; 4 were found by ctDNA only, and 2 were found by tissue only. ctDNA NGS correctly predicted MSI/MMR status in 2 out of 4 patients with MSI-H/dMMR; in the 2 other patients, the MSI and MMR statuses were different, suggesting potential false positivity. In addition, the fold changes in ctDNA dynamics during treatment significantly correlated with changes in tumor size and CEA levels, as well as with droplet digital PCR copy number fold changes. In a patient with MET amplification, ctDNA NGS identified MET Y1230H as a potential acquired resistance mutation after crizotinib treatment, which responded to cabozantinib but not to capmatinib. Conclusions Initial tumor profiling using ctDNA NGS yielded outcomes comparable to those of tumor tissue NGS in guiding treatment for patients with newly diagnosed mCRC, thereby suggesting its utility as an initial profiling method in mCRC. Citation Format: Yongjun Cha, Bun Kim, Dong Woon Lee, Hyun Yang Yeo, Chang Won Hong, Kyung Su Han, Byung Chang Kim, Hee Jin Chang. Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2498.

Abstract 2000: Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response

Abstract Rationale: Fluoropyrimidine drugs (FPs; e.g., 5-FU) are widely used to treat colorectal cancer (CRC), the 3rd leading cause of cancer-related mortality. However, acquired resistance to 5-FU is a principal cause of treatment failure in the metastatic setting. Further, previous studies selected for cells resistant to single agent 5-FU however 5-FU is invariably used clinically in combination with the reduced folate leucovorin (LV). Our studies focus on resistance mechanisms upon treatment with 5-FU+LV in folate-restricted media and test if CF10, a novel polymeric FP being developed in our laboratory displays activity in CRC cells resistant to 5-FU+LV. Experimental: We selected CRC cells (HCT-116, LS174T, HCT15, MC38) resistant to 5-FU+LV by selecting for survival upon culture in folate-restricted media with stepwise increased concentrations of 5-FU+LV over 5 months. Since elevated thymidylate synthase (TS) levels were observed in multiple studies selecting CRC cells for 5-FU resistance, we performed Western blot on cell lysates from parental and (5-FU+LV)-R cells. Since Myc and p53 expression are regulated in part via translational inhibition by TS binding to these mRNAs we also compared Myc and p53 levels in parental and (5-FU+LV)-R cells by Western blot. Further, since TS, Myc, and p53 protein stability are all regulated in part via O-GlcNAc conjugation we tested if OGT levels were changed in (5-FU+LV)-R cells. Resistance factors for CF10 were also determined in (5-FU+LV)-R cells. Results: A surprising result was that basal TS levels were reduced in (5-FU+LV)-R cells relative to the parental cells. We also observed that TS levels were increased in both parental and (5-FU+LV)-R cells by 5-FU+LV treatment however the effect was much greater in (5-FU+LV)-R cells. We observed strongly decreased p53 and Myc levels in the (5-FU+LV)-R cells. CF10 treatment further decreased p53 and Myc levels in parental cells, but increased p53 and further decreased Myc in (5-FU+LV)-R cells. In contrast, 5-FU alone treatment in (5-FU+LV)-R cells increased Myc and decreased p53 levels and further decreased in combination with LV. CF10 retained strong activity in (5-FU+LV)-R cells. Conclusions: Overall, we have validated that cancer cells can be cultured under conditions with human-like folate levels and selected for resistance to 5-FU+LV under folate-restricted conditions. The causes of 5-FU+LV resistance are multi-factorial and include TS overexpression induced by 5-FU+LV treatment that may be mediated by O-GlcNAc conjugation. We demonstrated that CF10 is more potent than 5-FU, and importantly its potency advantage is enhanced in cells selected for 5-FU+LV resistance. Our studies support CF10 translation for treatment of metastatic CRC in patients with progressive disease following treatment with combination therapy that includes 5-FU+LV. Citation Format: Charles C. Okechukwu, Xue Ma, William H. Gmeiner. Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2000.

Abstract 927: Real-world (RW) biomarker characteristics and clinical outcomes in pre-treated non-MSI-H/dMMR metastatic colorectal cancer (mCRC) patients

Abstract Introduction: Given sparse RW evidence, we assessed biomarker characteristics &amp; clinical outcomes in community oncology practices among pre-treated non-MSI-H/dMMR mCRC patients. Methods: This retrospective cohort study used structured &amp; chart review data from iKnowMed, The US Oncology Network electronic health record data &amp; included non-MSI-H/dMMR adult mCRC patients pre-treated with standard of care (SoC) chemo who initiated subsequent systemic anticancer treatment (SACT), best supportive care (BSC) or neither from 1/1/2016 - 12/31/2021. Index date for SACT users was the regimen start date post SoC chemo &amp; for patients on BSC &amp; no SACT/BSC, it was last chemo administration date. Patients were followed from index date until 8/31/2022 for overall survival (OS) &amp; real-world progression-free survival (rwPFS). KM analyses assessed outcomes, overall &amp; stratified by treatment, RAS status &amp; metastasis sites. Results: Of mCRC patients with &amp;gt;2 visits, 70.9% received MSI/MMR testing. In 292 eligible chart review patients, 69.5% received SACT in pre-treated setting (regorafenib 14.8%, trifluridine/tipiracil (TAS-102) 12.3%, other 72.9%), 28.8% received BSC &amp; 1.7% neither. 76.7% reported KRAS/NRAS testing with 60.7% wild-type &amp; 39.3% mutant. Common metastasis sites were liver-only (44.2%), liver &amp; lung (10.3%) &amp; lung-only (8.9%). Median (range) duration of follow-up was 5.2 (2.7, 10.0) months for regorafenib, 5.3 (2.5, 8.6) for TAS-102 &amp; 10.7 (5.3, 18.0) for the other treatments group. Median OS was 7.4 (95% CI: 5.8, 8.8) months &amp; median rwPFS was 3.5 (95% CI: 3.2, 4.3) months (outcomes data in Table). Conclusions: 29.1% pre-treated, mCRC patients did not receive guideline recommended MSI/MMR testing, highlighting the need to improve testing. Outcomes varied by treatment, RAS status, &amp; metastatic sites. The modest RW clinical benefit for SoC regorafenib &amp; TAS-102, was consistent with trials &amp; other RW studies; all highlighting an unmet need. Cohorts Median (95% CI) OS (months) Median (95% CI) rwPFS1 (months) Overall 7.4 (5.8, 8.8) 3.5 (3.2, 4.3) Regorafenib 5.6 (3.1, 9.7) 2.4 (1.8, 3.0) TAS-102 5.9 (3.4, 8.6) 3.0 (2.1, 3.4) Other SACT2 12.8 (9.8, 17.2) 4.9 (3.7, 5.8) BSC 2.4 (1.8, 3.2) - RAS mutation Wild-type 8.6 (6.6, 11.5) 4.0 (3.3, 4.9) Mutant 5.4 (3.4, 9.5) 3.2 (2.1, 4.6) Metastatic sites Liver only 8.0 (5.7, 11.7) 3.9 (3.0, 4.9) Lung only 17.0 (7.8, 35.6) 7.9 (3.3, 10.5) Liver &amp; lung only 5.4 (2.4, 7.2) 3.5 (2.1, 5.4) Other 6.7 (4.1, 9.4) 3.2 (2.7, 3.9) Abbreviation: CI: confidence interval, TAS-102: trifluridine/tipiracil, OS: overall survival, rwPFS: real-world progression-free survival, SACT: systemic anticancer treatment 1Overall rwPFS was calculated among patients receiving subsequent SACT only (n = 203) 2Top 2 “other SACT” included irinotecan- or oxaliplatin-based treatment Citation Format: Mayur M. Amonkar, Sneha Sura, Kaushal Desai, Rishi Jain, Zhimei Liu, Nicole Niehoff, Thomas W. Wilson, Gregory Patton, David Cosgrove. Real-world (RW) biomarker characteristics and clinical outcomes in pre-treated non-MSI-H/dMMR metastatic colorectal cancer (mCRC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 927.

Abstract 56: Preclinical in vivo characterization underpinning LGR5-targeting CAR-T cells as a cancer immunotherapy

Abstract Incidences of early-onset colorectal cancer (CRC) have been accelerating worldwide. To begin to address the lack of therapeutic options for CRC, we have developed a CAR-T cell therapy specific for leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5). LGR5 is known to potentiate Wnt/Β-catenin signaling and mark cells-of-origin in intestinal cancers. Extensive screening has demonstrated elevated expression of LGR5 in CRC, which is correlated with reduced patient survival. In CRC, LGR5 has been intimately linked with metastasis, cell survival and perturbation of chemotherapeutic interventions. We have developed a 9-day preclinical manufacturing method to generate LGR5-targeting CAR-T cells with a minimally differentiated T cell phenotype, which was concomitant with significant cytotoxic and activation marker expression. In human CRC xenograft mouse models, LGR5-targeting CAR-T cells eradicated tumors at doses as low as 800,000 CAR+ cells, and were capable of significantly inhibiting advanced tumor growth following delayed administration. Robust CAR-T cell-mediated immunological protection was observed in rechallenge experiments, in which secondary tumor growth was undetectable in mice which had tumors rejected following previous CAR-T cell administration. LGR5 protein and mRNA expression was also detected in a diverse range of cancer families, including ovarian, brain, liver and stomach, expanding upon the clinical indications with which LGR5-targeting CAR-T cells may be harnessed. These results contribute to a preclinical body of work that culminated in a Phase I/IIa clinical trial in metastatic colorectal cancer patients, which was approved for initiation by the FDA and commenced in 2023 (NCT05759728). Citation Format: Jade Foeng, Timona Tyllis, Caitlin Abbott, Dylan McPeake, Emma Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Claudine Bonder, Iain Comerford, Tim Sadlon, Simon Barry, Shaun McColl. Preclinical in vivo characterization underpinning LGR5-targeting CAR-T cells as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 56.

Abstract 5: Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors

Abstract Among gastrointestinal tumors, colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are major unmet medical needs, with a high mortality rate mainly related to the onset of metastases, hindering the therapeutic efficacy of conventional treatments. Adoptive Cell Therapy (ACT) with TCR-edited T cells is a promising therapeutic strategy for patients with late-stage tumors, but its applicability is limited by the paucity of TCRs targeting relevant tumor antigens and the presence of an immunosuppressive tumor microenvironment. Moreover, the safety of the therapeutic cellular products, generated by genome editing tools, is fundamental for an effective and rapid clinical translation. With the aim of improving the safety profile of T-cell based therapeutic products manufactured by genome editing, we evaluated the efficacy and safety of cytosine base editor (CBE). We disrupted the endogenous TCR to redirect T cells specificity toward tumor cells, and TIGIT, a major inhibitory molecule in gastrointestinal tumors to enhancing T cell ability to counteract immunosuppression. Beyond the high efficiency of CBE in simultaneously disrupting the target genes, we observed only marginal sgRNA-dependent and -independent off-target events as measured by targeted sequencing of the top predicted off-target loci and by ultra-deep whole exome sequencing, respectively. In addition, unlike CRISPR/Cas9-treated cells, we could not detect translocations in CBE edited T cells. To select the antigens to target by engineered T cells, we investigated published datasets and identified 19 relevant tumor-associated antigens. By clonal tracking of the TCR repertoire of T cells stimulated with autologous APCs loaded with the selected antigens, we isolated 5 different TCRs specificities, including mesothelin. Importantly, these antigens are expressed in our cohort of primary and metastatic PDAC and CRC patients. TCR-edited T cells displayed anti-tumor activity against PDAC cell lines, CRC and PDAC patient-derived organoids while sparing HLA-unrelated and antigen-negative controls. To boost the efficacy of anti-tumor T cells, we coupled TIGIT disruption with TCR editing. By challenging TCR-edited TIGITKO T cells in vitro and in an orthotopic model of CRC liver metastases, we observed that TIGIT disruption can ameliorate the ability of tumor-specific T cells to control tumor growth. Our findings suggest that base editors can generate cellular products with a better safety profile and a promising therapeutic efficacy in gastrointestinal tumors. Citation Format: Martina Spiga, Alessia Potenza, Stefano Beretta, Alessia Airaghi, Chiara Iozzi, Martina Fiumara, Samuele Ferrari, Neda Mohammadi, Oronza Botrugno, Maximilian Reichert, Maria Pia Protti, Stefano Crippa, Massimo Falconi, Luigi Naldini, Eliana Ruggiero, Chiara Bonini. Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5.

Abstract 4728: Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer

Abstract Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody (mAb), results in beneficial, yet limited, clinical improvement in metastatic colorectal cancer (mCRC) patients with wild-type (WT) KRAS gene. Numerous studies have shown that circRNAs are aberrantly expressed in tumors and play crucial roles in tumor growth and metastasis in various human malignancies, including colorectal cancer (CRC). One such circRNA, circ-EGFR, has been identified as having associations with tumor progression and chemotherapy response in diverse cancer types. However, exploring the functional role and underlying mechanisms of circ-EGFR in the context of the response to EGFR-targeted therapy may offer new therapeutic avenues for mCRC. Methods: The expression level of circ-EGFR (hsa_circ_0080229) was evaluated in cetuximab-sensitive and resistant RAS and BRAF WT CRC cell lines by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The biological function of circ-EGFR was investigated by loss- and gain-of-function assays in cultured cells and an animal model. The potential targets of circ-EGFR were identified through in-silico analysis, and the circ-EGFR/miR-942-3p/GAS1 axis pathway was verified using the luciferase reporter assay and RT-qPCR. The association of circ-EGFR expression with clinical prognosis in mCRC patients with different cetuximab responses was analyzed by Kaplan-Meier survival analysis. Results: The expression of circ-EGFR was remarkedly higher in sensitive (DiFi and SW48) compared with resistant cell lines (CaCO2 and SNU-C1) after exposure of cells to cetuximab (sensitive vs. resistant: p &amp;lt; 0.001). Biologically, genetic depletion of circ-EGFR significantly enhanced cell proliferation, migration [DiFi: sh-circ-EGFR vs. shNC: Foldchange (FC) = 1.97, p &amp;lt; 0.001; SW48: FC = 1.96, p &amp;lt; 0.001], and invasion (DiFi: sh-circ-EGFR vs. shNC: FC = 2.23, p &amp;lt; 0.001; SW48: FC = 1.78, p &amp;lt; 0.001) after 24-48 hours of cetuximab treatment in DiFi and SW48 cell lines, and its overexpression suppressed tumor growth in vivo under cetuximab treatment. Mechanically, we identified circ-EGFR as a sponge for miR-942-3p to regulate the expression of Growth Arrest Specific 1 (GAS1), which promoted sensitivity to cetuximab treatment in CRC. Notably, our findings identified circ-EGFR as a promising therapeutic indicator in clinical applications. Conclusion: Our study illustrated that circ-EGFR plays a vital role in affecting cetuximab efficacy by regulating the miR-942-3p/GAS1 axis in CRC. This holds promise for improving patient selection and management in this malignancy. Citation Format: Silei Sui, Caiming Xu, Yuan Li, Wenyi Wei, Joan Maurel, Man Li, Ajay Goel. Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4728.