Advanced Metastatic Colorectal Cancer Research Articles

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.

Structure-Activity Relationship Study of (E)-3-(6-Fluoro-1H-indol-3-Yl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (FC116) Against Metastatic Colorectal Cancers Resistant to Oxaliplatin.

Advanced metastatic colorectal cancer (mCRC) and the development of drug resistance to chemotherapy pose significant challenges in clinical settings. In previous studies, we have demonstrated the potent cytotoxic activity of (E)-3-(6-fluoro-1H-indol-3-yl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (FC116) and related 30 derivatives against mCRC by targeting microtubules. In this study, we aimed to evaluate the efficacy of the 31 compounds and explore the structure-activity relationship (SAR) against oxaliplatin-resistant mCRC. We found that most of the derivatives showed high sensitivity toward the oxaliplatin-resistant HCT-116/L cells. Particularly, FC116 exhibited a better GI50 value against the resistant mCRC cell line, HCT-116/L, compared to standard therapies. We also observed a safer therapeutic window for FC116 and a synergistic effect when it was used in combination with oxaliplatin. Mechanistically, FC116 induced the G2/M phase arrest by downregulating cyclin B1 expression through its interaction with microtubules in resistant colorectal cancer cells. Furthermore, in vivo experiments demonstrated that FC116 significantly suppressed tumor growth, achieving a 78% reduction at a dose of 3 mg/kg, which was superior to the 40% reduction achieved by oxaliplatin treatment. Overall, our findings suggest that the indole-chalcone compound FC116 represents a promising lead for chemotherapy in oxaliplatin-resistant mCRC.

Abstract LB206: The progression of chromosomal instability and aberrant DNA methylation during colorectal cancer development

Abstract Chromosomal instability (CIN) and aberrant DNA methylation play a pivotal role in the pathogenesis of colorectal cancer (CRC). Mapping both of these cancer hallmarks during the progression of the disease can help identify potential targets for pharmacological intervention and improve the management of CRC metastasis. In a set of 27 patients diagnosed with either high-grade colorectal adenoma or advanced metastatic CRC disease, high-throughput DNA methylation and/or DNA somatic copy number alterations (SCNAs) analyses were performed. Global genome methylation profiles and the most frequent hot-spot regions harboring SCNAs, identified in the patients' DNA, were verified in a validation set of eight CRC patients through whole-exome sequencing, genome-wide DNA methylation measurement, and gene expression profiling.The study is currently ongoing; however, a panel of 29 cancer-relevant genes, amplified in more than 50% of investigated adenoma tissues (such as PTK6, SRC, MALAT1, BRCA2), was identified. Chromosomal loci amplified in primary tumors were highly concordant with those in liver metastases. An increase in CIN was detected in all metastases, representing mostly gains of Chr7q, 8q, 13q, 20q, and the small arm of ChrX, while the lost chromosomal loci included Chr8p, 17p, and the entire Chr18. A gain unique to metastases was located on Chr6 (p25.1-p22.3), encoding the oncogene DEK. All metastases exhibited an increase in hypomethylated and a decrease in hypermethylated CpGs. The gene enrichment analysis of the differently methylated gene promoters showed the olfactory receptors pathway as the most frequently represented (FDR-adjusted P value=1.06E-8), with all the identified promoters (n=120) less methylated in metastases. Taken together, several genomic hot-spot regions with SCNAs, possibly important during the development of adenoma and also during the metastatic process, were identified. Furthermore, a decrease in methylation within promoters of odorant receptors was the most prominent epigenetic alteration occurring in our set of metastases. The study was funded by the Ministry of Health of the Czech Republic (NU22J-03-00028) and GACR 21-27902S. Citation Format: Katerina Saskova, Mattias Landfors, Josef Horak, Katerina Honkova, Kristyna Tomasova, Jana Drabova, Fernanda Schäfer Hackenhaar, Jitka Pavlikova, Ludmila Vodickova, Torbjörn Nilsson, Sanja Farkas, Veronika Vymetalkova, Pavel Vodicka, Sofie Degerman, Michal Kroupa. The progression of chromosomal instability and aberrant DNA methylation during colorectal cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB206.

Patient experience of medication administration and development of a Patient Experience and Preference Questionnaire (PEPQ) for patients with advanced or metastatic cancer.

Introduction: A better understanding of patient experience of intravenous (IV) or subcutaneous (SC) routes of administration is fundamental to providing optimal administration of medical therapies to oncology patients. The objective of this study was to examine patient experiences of IV and SC treatment with nivolumab and confirm the relevance of item concepts in the Patient Experience and Preference Questionnaire (PEPQ). The PEPQ is a clinical outcomes' assessment instrument developed to obtain patient-centric data and understand the experience with IV and SC treatment administration. Methods: Embedded qualitative interviews were conducted with a subset of participants from three treatment cohorts with metastatic non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), unresectable or advanced metastatic melanoma, hepatocellular carcinoma (HCC), or colorectal cancer (CRC) from the CA209-8KX clinical trial. Concept elicitation interviews were conducted within 14days of the initial treatment cycle and patient experiences with IV and SC treatment administration were assessed. Concepts from interviews were mapped to the PEPQ version 1.0 questions to assess relevance and convergence of concepts. Results: Interviews were conducted with 43 trial participants from clinical sites opting to participate from six countries (Argentina, France, the Netherlands, Poland, Spain, and New Zealand). The mean age of sub-study participants was 66 ± 11.3years (range 24-80years), and 67.4% (N = 29) were male. Sub-study participants with experience of SC most frequently reported symptoms or signs of injection-related redness (27.9%), itching (14.0%), and pain (of needle), and described the pain as pricking, stinging, or tingling (11.0% each). The amount of pain and time burden were widely endorsed as important factors for satisfaction and related to the route of medication administration. For 11 sub-study participants with experience with both IV and SC treatments, 10 (90.9%) preferred SC over IV treatment administration. Conclusion: This study summarizes the experience and satisfaction of receiving IV or SC treatment and confirms the relevance of the PEPQ in a subgroup of CA209-8KX clinical trial participants with metastatic NSCLC, RCC, melanoma, HCC, and CRC. Participant treatment experience and satisfaction with the route of medication mapped to the PEPQ question content support the relevance of PEPQ v2.0 in clinical trials as a self-report measure.

Evaluation of the IMPACT of a decision-support platform (Trapelo) on testing patterns for patients with advanced solid tumors.

513 Background: Given the evolving precision oncology landscape, it is challenging for oncologists to stay abreast of the latest biomarker testing guidelines. Texas Oncology (TxO), a network of community oncology providers, is evaluating Trapelo, a decision-support platform that assists in the ordering of appropriate biomarker tests. The objective of this study was to assess whether implementation of Trapelo’s testing pathways improves the ordering of timely and comprehensive biomarker testing (TCBT) among patients with advanced cancer. Methods: We conducted a retrospective analysis of patients with advanced non-small cell lung (NSCLC), metastatic breast (BC), or metastatic colorectal (CRC) cancer treated at TxO between July 24, 2018, and July 24, 2021, and had at least one oncology biomarker test ordered. The ‘Intervention’ group consisted of patients diagnosed after their physician’s onboarding to Trapelo; the ‘Control’ group consisted of patients diagnosed before onboarding. We also analyzed a subset of the intervention group: patients with biomarker tests actually ordered through Trapelo (Intervention - Trapelo.) Our primary endpoint, TCBT, was defined as the ordering of tests for all actionable biomarkers (i.e., with FDA approved first-line treatment) within 60 days following an advanced diagnosis. Because Trapelo implementation occurred over time, we used Two-Way Fixed Effects regression with fixed effects for time period and physician to assess the impact of the Intervention on TCBT. Results: In descriptive analysis, the rates of TCBT in the NSCLC cohort were 26, 28%, and 37% in the Control (n = 1023), Intervention (n = 554), and Intervention-Trapelo (n = 315) groups, respectively. In the CRC and BC cohorts, the rates were 32%, 40%, 55%, and 66%, 55%, and 45%, respectively. In the regression analysis, Intervention was associated with increases in TCBT rates of 7.5 ( p = 0.4) and 6.5 ( p = 0.4) percentage points in the CRC and NSCLC cohorts, respectively. The lack of statistical significance could be attributable to small sample sizes and clustered data by physicians. No differences were observed in the BC cohort. Conclusions: While trends observed in our study indicate that implementation of a decision support tool can improve rates of TCBT in a community oncology setting, studies with larger sample sizes are needed to assess the statistical significance of these findings. Despite these potential improvements, overall rates of TCBT, a critical aspect of optimal treatment in oncology, remained low. Further, over 43% of the NSCLC patients in the Intervention group did not have their orders placed through Trapelo, indicating an opportunity for improving post-onboarding adoption of Trapelo. Future analyses will investigate the impact of Trapelo utilization (as opposed to implementation/onboarding) on the receipt of TCBT.

HER-3 surface expression increases in advanced colorectal cancer representing a potential therapeutic target

HER-3 (also known as ErbB-3) is a human epidermal growth factor receptor tyrosine kinases family member, and its expression in CRC (colorectal cancer) tissues was previously associated with poor prognosis. In this study, HER-3 expression was analyzed by immunohistochemistry in two cohorts of early and advanced metastatic CRC patients. The first cohort included 180 patients diagnosed with CRC in absence of lymph nodes or distant metastases (Stage I and Stage II), while the second was obtained from 53 advanced metastatic CRC patients who developed synchronous (SM) and metachronous (MM) liver metastases. In the first early-stage CRC cohort, 86 out of 180 (47.8%) tumors showed membranous expression of HER-3, with a mean percentage of positive tumor cells of 25.7%; conversely, in advanced metastatic CRC primary tumors, HER-3 was detected in all specimens, with a mean percentage of positive tumor cells of 76.1%. Kaplan–Meier curves showed that in the advanced metastatic CRC group, patients with HER-3high tumors had a significantly lower Cancer-Specific Survival (CSS) rate compared to patients with HER-3low tumors (p = 0.021). Importantly, this worse CSS rate was observed only in the MM subgroup of patients with HER-3high tumors (p = 0.002). Multivariate analysis confirmed that high HER-3 expression represents a significant and strong risk factor for death in patients developing MM liver metastases (Hazard Ratio = 64.9; 95% Confidence Interval, 4.7–886.6; p = 0.002). In addition, using a specific anti-HER-3 antibody-drug conjugate, named EV20/MMAF, we showed that HER-3 + CRC cells can be efficiently targeted in vitro and in vivo. Overall, this study confirms that surface HER-3 is highly expressed in CRC and reveals that HER-3 expression increases in metastatic CRC patients compared to early stage. Importantly, the results suggest that HER-3 has a prognostic and therapeutic value in patients developing MM liver metastases.

Prognostic and predictive impact of metastatic organ involvement on maintenance therapy in advanced metastatic colorectal cancer: Subgroup analysis of patients treated within the PanaMa trial (AIO KRK 0212).

Despite molecular selection, patients (pts) with RAS wildtype mCRC represent a heterogeneous population including diversity in metastatic spread. We investigated metastatic patterns for their prognostic and predictive impact on maintenance therapy with 5-fluorouracil/folinic acid ± panitumumab. The study population was stratified according to (1) number of involved metastatic sites (single vs multiple organ metastasis), liver-limited disease vs (2) liver metastasis plus one additional site, and (3) vs liver metastasis plus ≥two additional sites. Kaplan-Meier method and Cox regressions were used to correlate efficacy endpoints. Single organ metastasis was observed in 133 pts (53.6%) with 102 pts (41.1%) presenting with liver-limited disease, while multiple organ metastases were reported in 114 pts (46.0). Multiple compared to single organ metastases were associated with less favorable PFS (HR 1.48, 95% CI 1.13-1.93; P = .004) and OS (HR 1.37, 95% CI 0.98-1.93; P = .068) of maintenance therapy. While metastatic spread involving one additional extrahepatic site was not associated with clearly impaired survival compared to liver-limited disease, pts with liver metastasis plus ≥two additional sites demonstrated less favorable PFS (HR 1.92, 95% CI 1.30-2.83; P < .001), and OS (HR 2.38, 95% CI 1.51-3.76; P < .001) of maintenance therapy. Pmab-containing maintenance therapy appeared active in both pts with multiple (HR 0.58; 95% CI, 0.39-0.86; P = .006) as well as to a lesser numerical extent in pts with single organ metastasis (HR 0.83; 95% CI, 0.57-1.21; P = .332; Interaction P = .183). These data may support clinical decisions when EGFR-based maintenance therapy is considered.

612P Updated results from the multicenter phase II study of fruquintinib plus mFOLFOX6/FOLFIRI as first-line therapy in advanced metastatic colorectal cancer (mCRC)

612P Updated results from the multicenter phase II study of fruquintinib plus mFOLFOX6/FOLFIRI as first-line therapy in advanced metastatic colorectal cancer (mCRC)

Results of phase I-II bridging study for Nous-209, a neoantigen cancer immunotherapy, in combination with pembrolizumab as first line treatment in patients with advanced dMMR/MSI-h colorectal cancer.

e14665 Background: Microsatellite instability (MSI) is a molecular hallmark in which systemic deficiency of DNA mismatch-repair (dMMR) generates tumor-specific frameshift peptides (FSPs). Nous-209 is an off-the-shelf cancer vaccine targeting 209 FSPs selected from dMMR/MSI-H tumors (Leoni, G. et al., 2020). Treatment options for unresectable dMMR/MSI-H tumors have improved, but further efforts to increase long-term progression-free survival are needed. NOUS-209-01 Phase I demonstrated that Nous-209 and pembrolizumab is safe, well tolerated, and provides clinical benefit in advanced gastric and CRC dMMR/MSI-H tumors (Fakih et al., 2022). Moreover, Nous-209 induces durable specific anti-tumoral immune response, capable of successfully infiltrating the tumor (D’Alise et al., 2022). Here, we present data from NOUS-209-01 study Phase I-II expansion cohort at the recommended phase 2 dose (RP2D) of Nous-209 and pembrolizumab in adults with dMMR/MSI-H locally advanced unresectable or metastatic colorectal cancer (mCRC) eligible for 1st line immune checkpoint inhibition. Methods: NOUS-209-01 ( NCT04041310 ) Phase I-II bridge expansion cohort receives one GAd20-209-FSP prime intramuscularly at the 1st infusion of pembrolizumab, followed by 3 MVA-209-FSP boosts with the 2nd, 3rd and 4th pembrolizumab cycles. Patients can continue treatment with pembrolizumab for 18 months. Response assessment is evaluated every 9 (+/-1) weeks by CT imaging, according to RECIST v1.1 criteria. ctDNA assessments are at baseline, 11, 28, 49, 79 weeks and at objective progression on study. Results: Phase I-II bridge enrolled 8 patients with dMMR/MSI-H treatment-naïve mCRC. One patient had clinical progression before 1st scan and was excluded as non-evaluable and 7 were evaluable for tumor response. 5/7 (71%, CI: 95% 29.0, 96.3) patients achieved objective response: 1 (14%) complete response (CR) and 4 (57%) partial responses (PR). 2 patients (29%) had disease progression (PD) at the 1st scan. ctDNA assessments were available for 6 out of 7 patients (4 PR; 2 PD); ctDNA levels reduced &gt;50% at week 11 in the 4 PR patients, in contrast to a 28% increase in 1 PD. Interestingly 1 PD patient had ctDNA &gt; 50% reduction. The median follow-up at data cut-off was 7.5 months (range: 3.3-8.4), median progression free survival and median duration of response have not been reached. 6/7 (85.7%) patients had treatment emergent adverse events (TEAE), all mild or moderate grade; no TEAEs were serious or led to treatment discontinuation. Conclusions: The combination of Nous-209 and pembrolizumab is safe, well tolerated and shows encouraging clinical efficacy in patients with treatment-naive dMMR/MSI-H mCRC eligible for anti-PD-1 therapy. The study is ongoing and expanding to Phase II randomization with a new accelerated Nous-209 vaccination schedule. Clinical trial information: NCT04041310 .

Suvemcitug as second-line treatment of advanced or metastatic solid tumors and with FOLFIRI for pretreated metastatic colorectal cancer: phase Ia/Ib open label, dose-escalation trials

Suvemcitug (BD0801), a novel humanized rabbit monoclonal antibody against vascular endothelial growth factor, has demonstrated promising antitumor activities in preclinical studies. The phase Ia/b trials investigated the safety and tolerability and antitumor activities of suvemcitug for pretreated advanced solid tumors and in combination with FOLFIRI (leucovorin and fluorouracil plus irinotecan) in second-line treatment of metastatic colorectal cancer using a 3+ 3 dose-escalation design. Patients received escalating doses of suvemcitug (phase Ia: 2, 4, 5, 6, and 7.5 mg/kg; phase Ib: 1, 2, 3, 4, and 5 mg/kg plus FOLFIRI). The primary endpoint was safety and tolerability in both trials. All patients in the phase Ia trial had at least one adverse event (AE). Dose-limiting toxicities included grade 3 hyperbilirubinemia (one patient), hypertension and proteinuria (one patient), and proteinuria (one patient). The maximum tolerated dose was 5 mg/kg. The most common grade 3 and above AEs were proteinuria (9/25, 36%) and hypertension (8/25, 32%). Forty-eight patients (85.7%) in phase Ib had grade 3 and above AEs, including neutropenia (25/56, 44.6%), reduced leucocyte count (12/56, 21.4%), proteinuria (10/56, 17.9%), and elevated blood pressure (9/56, 16.1%). Only 1 patient in the phase Ia trial showed partial response, [objective response rate 4.0%, 95% confidence interval (CI) 0.1% to 20.4%] whereas 18/53 patients in the phase Ib trial exhibited partial response (objective response rate 34.0%, 95% CI 21.5% to 48.3%). The median progression-free survival was 7.2 months (95% CI 5.1-8.7 months). Suvemcitug has an acceptable toxicity profile and exhibits antitumor activities in pretreated patients with advanced solid tumors or metastatic colorectal cancer.

Abstract CT190: Oncolytic virus TG6002 safety and activity after intrahepatic artery administration in patients with liver-dominant metastatic colorectal cancer

Abstract The TG6002.03 trial is a dose-escalation phase 1 clinical trial of TG6002 infusion via the hepatic artery in patients with liver-dominant colorectal cancer metastases. TG6002 is an engineered Copenhagen strain oncolytic Vaccinia virus, deleted of thymidine kinase and ribonucleotide reductase to enhance tumor selective viral replication and expressing FCU1, an enzyme converting the non-cytotoxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). In this trial, patients with advanced unresectable liver-dominant metastatic colorectal cancer who had failed previous oxaliplatin and irinotecan-based chemotherapy were treated with up to 2 cycles of TG6002 infusion 6 weeks apart via the hepatic artery on day 1 combined with oral 5-FC on days 5 to 14 (where day 1 = TG6002 infusion). TG6002 infusion was performed over 30 minutes via selective catheterization of the hepatic artery proper. 5-FC oral dosing was 50mg/kg x4 daily. Blood was sampled for TG6002 pharmacokinetics and 5-FC and 5-FU measurements. Sampling of liver metastases was performed at screening and on day 4 or day 8 for virus detection and 5-FC and 5-FU quantification. In total, 15 patients (median age 61 years, range 37-78) were treated in 1 UK centre and 2 centres in France and received a dose of TG6002 of 1 × 106 (n=3), 1 × 107 (n=3), 1 × 108 (n=3), or 1 × 109 pfu (n=6). Fourteen of the 15 patients received a single cycle of treatment, including one patient who did not received 5-FC, and one patient received two cycles. TG6002 was transiently detected in plasma following administration, suggesting a strong tissue selectivity for viral replication. In the highest dose cohort, a virus rebound was observed on day 8, concordant with replication time of the virus. In serum samples, 5-FU was present on day 8 in all patients with a high variability ranging from 0.8 to 1072 ng/mL and was measurable over several days after initiation of therapy. Seven of the 9 patients evaluable showed the biodistribution of the virus in liver lesions by PCR testing on day 4 or day 8. Translational blood samples showed evidence for T-cell activation and immune checkpoint receptor-ligand expression. At 1 × 109 pfu, there was evidence for T-cell proliferation and activation against tumour-associated antigens by ELISpot and for immunogenic cell death. In terms of safety, a total of 34 TG6002-related adverse events were reported, of which 32 were grade 1-2 and 2 were grade 3. The maximum tolerated dose was not reached, and a single dose-limiting toxicity was observed consisting of a myocardial infarction in a context of recent Covid-19 infection in a 78-year-old patient. These results indicate that TG6002 infused via the hepatic artery in combination with oral 5-FC was well tolerated, effectively localized and replicated in the tumor tissues, expressed its therapeutic payload and showed anti-tumoral immunological activity. Citation Format: Adel Samson, Cristina Smolenschi, Philippe Cassier, Jai V. Patel, Chris Hammond, Marta Kurzawa, Sophie Sainte-Croix, Emma West, Alain Sadoun, Kaidre Bendjama. Oncolytic virus TG6002 safety and activity after intrahepatic artery administration in patients with liver-dominant metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT190.

Abstract 3199: Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells

Abstract The measurement of chimeric antigen receptor (CAR) expression is critical for the development, qualification and quantitation of CAR-T cells for clinical applications. There is a comprehensive range of tools commercially available for specifically measuring expression of the CAR on CD19-targeting CAR-T cells. However, with the rapidly expanding repertoire of CAR-T cells in development targeting a diverse range of tumour antigens, there is a paucity of readily implementable assays that specifically measure CAR expression, which is essential for translation into the clinic. We have developed several CARs targeting the cancer stem cell marker LGR5, one of which (CNA3103) is the lead candidate in an upcoming clinical trial in advanced metastatic colorectal cancer. Previously, the level of CNA3103 expression in this system has been determined indirectly by measuring expression of tEGFR on the CAR-T cell surface, taking advantage of co-expression of the CAR and tEGFR upon transduction with our lentiviral construct. tEGFR expression was found over many CAR-T cell manufacturing batches to report transduction efficiency reproducibly with high sensitivity. However, to specifically quantify expression of the CNA3103, an assay utilising an Fc-tagged recombinant human LGR5 protein, in combination with an anti-human IgG1 Fc-specific secondary antibody was developed. A number of optimisation steps including titration of multiple batches of rhLGR5-Fc, different anti-human IgG1 Fc-specific antibodies and a range of incubation parameters were tested. This resulted in development of an assay that yielded very similar reproducibility and sensitivity to that previously observed with the indirect tEGFR surrogate assay. Overall, the findings highlight the utility of using CAR-targeting recombinant proteins for evaluating CAR expression on CAR-T cells, providing a general flow cytometry-based staining strategy that may be adapted for assessing a diverse repertoire of CARs. Citation Format: Timona Tyllis, Caitlin Abbott, Dylan McPeake, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Emma Thompson, Lih Tan, Allison Cowin, Claudine Bonder, Timothy Sadlon, Simon Barry, Shaun McColl. Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3199.

Abstract 1819: Integrin inhibitor GLPG-0187 promotes T-cell killing of mismatch repair-deficient colorectal cancer cells by suppression of SMAD/TGF-β signaling

Abstract Colorectal cancer is the third leading cause of cancer-related death and the third most common cause of cancer. As the five-year survival with advanced metastatic colorectal cancer (mCRC) is 14%, new treatment strategies are needed. Immune checkpoint blockade which takes advantage of an individual's immune system in order to fight cancer, is a promising future direction of research, although it is only effective and approved for the treatment of mismatch repair (MMR)-deficient CRCs. Long-term outcomes even in MMR-deficient mCRC suggest that most patients are not cured but eventually develop therapy resistance. We hypothesized that targeting TGF- β signaling may enhance immune-mediated T-cell killing by MMR-deficient CRC cells. Using GLPG-0187, an inhibitor of multiple integrin receptors and TGF- β, we demonstrate minimal cytotoxicity against MMR-deficient HCT116 or p53-null HCT116 human CRC cells. However, GLPG-0187 promoted significant immune cell killing of the CRC cells by TALL-104 T lymphoblast cells. GLPG-0187 reduced pSMAD2 in HCT116 p53-null cells either in the absence or presence of exogenous TGF- β. We observed a reduction in CCL20, CXCL5, prolactin, and TRAIL-R3 while GDF-15 was increased in TALL-104 cells treated with a T-cell activating dose of GLPG-0187 (4 μM). Our results suggest that TGF- signaling inhibition by a general integrin receptor inhibitor may boost T-cell killing of MMR-deficient colorectal cancer cells, and suggest that a combination of anti-GDF-15 in combination with TGF- β blockade should be further investigated in the treatment of MMR-deficient mCRC. Our results support the development of a novel immune-based therapeutic strategy to treat colorectal cancer by targeting the TGF-β signaling pathway through integrin receptor blockade. Citation Format: Brooke Verschleiser, William MacDonald, Lindsey Carlsen, Kelsey E. Huntington, Lanlan Zhou, Wafik El-Deiry. Integrin inhibitor GLPG-0187 promotes T-cell killing of mismatch repair-deficient colorectal cancer cells by suppression of SMAD/TGF-β signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1819.

Abstract 1072: Combination of 5-Fluorouracil or ONC212 plus KRAS G12D inhibitor MRTX1133 against colorectal and pancreatic cancer cells results in immune-stimulatory cytokine patterns and unexpected synergies independent of G12D mutation

Abstract BACKGROUND: Advanced metastatic colorectal cancer (mCRC) has a 14% 5-year survival rate with little progress made for microsatellite stable tumors. KRAS mutations occur in about 40-50% of mCRC and are associated with more aggressive drug resistance and lack of response to anti-EGFR therapies. Recent advances have led to small molecules targeting KRAS G12C that have been undergoing clinical testing in lung, mCRC, and other tumor types. ONC212 is a fluorinated imipridone with strong anti-cancer activity in nM range and preclinical efficacy against pancreatic and other malignancies. Through extensive structure-based drug design, MRTX1133 was identified as a noncovalent, potent, and selective inhibitor of KRASG12D. MRTX1133 suppresses KRASG12D signaling in cells and in vivo, MRTX1133 binds to the switch II pocket and inhibits the protein-protein interactions necessary for activation of the KRAS pathway. MATERIALS &amp; METHODS: We investigated cell viability and drug synergies of 5-FU or ONC212 plus KRAS G12D inhibitor MRTX1133 in 6 human CRC and 4 human pancreatic cancer cell lines at 72 hours. Experiments evaluated drug effects on pERK using western blot at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. RESULTS: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM for 5-FU 0.2-0.8 microM for ONC212, &amp;gt;100 nM to &amp;gt;5,000 nM for MRTX1133 (G12D N=4: LS513 &amp;gt;100, HPAF-II &amp;gt;1,000, SNUC2B &amp;gt;5,000, PANC-1 &amp;gt;5,000). For non-G12D, the range of IC50 for MRTX1133 was &amp;gt;1,000 to &amp;gt;5,000 nM for CRC lines with G12V, G13D, or WT KRAS (N=7). Synergies between MRTX1133 and either 5-FU or ONC212 were observed across cell lines regardless of the presence of KRAS G12D mutation with combination indices of &amp;lt;0.5 indicating strong synergy. The observed synergy was greater with the combination of MRTX1133 and ONC212 compared to the synergy with 5-FU. Among the cytokine alterations, IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased with all treatments. CONCLUSIONS: Our ongoing studies reveal the potential activity of MRTX1133 against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation and synergies were observed with 5-FU and ONC212 regardless of KRAS G12D mutation. Immune stimulatory cytokine profiles were observed with 5-FU, MRTX1133 and combination. The results suggest that KRAS G12D, KRAS G13D and WT KRAS should not be excluded from clinical trials especially with combination therapies involving MRTX1133 and standard-of-care 5-FU. Citation Format: Vida Tajiknia, Wafik El-Deiry, Maximilian Pinho Schwermann, Lanlan Zhou, Kelsey Huntington. Combination of 5-Fluorouracil or ONC212 plus KRAS G12D inhibitor MRTX1133 against colorectal and pancreatic cancer cells results in immune-stimulatory cytokine patterns and unexpected synergies independent of G12D mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1072.

Healthcare experiences of people with advanced colorectal cancer: A qualitative study.

Qualitative research examining healthcare experiences and needs of people with advanced (metastatic or recurrent) colorectal cancer CRC-A is limited. This study aimed to fill this gap in CRC-A survivors treated with surgical or palliative chemotherapy, through a qualitative study. Australian adults treated for CRC-A were recruited 0.5-2 years post-surgery or post-diagnosis of CRC-A (for palliative chemotherapy groups). Semi-structured telephone interviews, analysed via framework analysis, explored healthcare experiences. Demographic, clinical, and quality of life data characterised the sample and informed framework analyses. Data was compared against the Institute of Medicine's framework for quality healthcare. Interviews from 38 participants (22 female) of median age 59 years (range 27-84) revealed six overarching themes relating to the safety, effectiveness, timeliness, patient-centredness, efficiency, and equity of CRC-A care: 1) Early experiences influence later perceptions; 2) Trusting the system, trusting the professionals; 3) The benefits of multidisciplinary care co-ordination; 4) Feeling lost in follow-up; 5) Whose role is it anyway? Gaps in responsibility for survivorship care; and 6) Useful or useless? Perceptions of psychosocial support. Healthcare systems for CRC-A can be improved through delivery of repeated information, upskilling general practitioners and/or implementing written survivorship care plans or survivorship clinics, to ensure quality healthcare.

Efficacy and safety of toripalimab with fruquintinib in the third-line treatment of refractory advanced metastatic colorectal cancer: results of a single-arm, single-center, prospective, phase II clinical study.

The most effective treatment with immune checkpoint inhibitors (ICIs) is limited to the microsatellite instability high (MSI-H) subgroup of advanced colorectal cancer. ICIs are completely ineffective in microsatellite stabilized (MSS) patients with advanced colorectal cancer. Fruquintinib, a tyrosine kinase inhibitor (TKI) domestically made in China that specifically inhibits vascular endothelial growth factor receptors, is used to treat refractory metastatic colorectal cancer (mCRC). Researches showed that anti-angiogenic therapy combined with immunotherapy induces a long-lasting antitumor immune response. Here, we aimed to evaluate antitumor efficacy and safety of fruquintinib with anti-programmed death-1 (PD-1) antibody toripalimab in Chinese patients with non-MSI-H/mismatch repair proficient (pMMR) mCRC. This was a single-arm, single-center, prospective, phase II clinical trial. A total of 19 MSS patients with refractory or advanced mCRC were enrolled They received fruquintinib (5 mg, orally, once daily for 3 weeks followed by 1 week off in 4-week cycles) and toripalimab (240 mg, intravenously administered on day 1 once every 3 weeks) until disease progression or unacceptable toxicity. The objective response rate (ORR), progression-free survival (PFS), overall survival (OS), 1-year PFS rate, disease control rate (DCR), and toxicity were reviewed and evaluated. The Cox regression model was used to analyze the influence on OS and PFS. Among the 19 patients, the median age was 52 years (range, 30-71 years); 4 patients (21.05%) achieved partial response, 10 patients (52.63%) experienced stable disease, and 4 patients (21.05%) experienced progressive disease. The ORR was 21.05%. The median PFS and OS were 5.98 months and 11.10 months, respectively. Patients with peritoneal metastasis received greater benefit from combination therapy, with a longer PFS (P=0.043) in the univariate analysis. The most common treatment-related adverse reactions were fatigue (57.89%), hepatic dysfunction (42.11%) and hypertension (36.84%). No serious adverse effects or adverse effect-related deaths were reported. Our study provides evidence supporting fruquintinib combined with an anti-PD-1 monoclonal antibody have the better effect than fruquintinib alone in the third-line setting for Chinese patients with MSS advanced colorectal cancer. Primary lesion excision and peritoneal metastasis were independent prognostic factors of PFS. Further well-designed, prospective, large-scale studies are needed to validate this outcome.

Correlation between SMADs and Colorectal Cancer Expression, Prognosis, and Immune Infiltrates.

In recent years, the incidence and mortality of colorectal cancer (CRC) are increasing, and the 5-year survival rate of advanced metastatic CRC is poor. Small mothers against decapentaplegic (SMAD) superfamily are intracellular signal transduction proteins associated with the development and prognosis of a variety of tumors. At present, no study has systematically analysed the relationship between SMADs and CRC. Here, R3.6.3 was used to analyse the expression of SMADs in pan-cancer and CRC. Protein expression of SMADs were analysed by Human Protein Atlas (HPA). Gene expression profiling interactive analysis (GEPIA) was used to evaluate the correlation between SMADs and tumor stage in CRC. The effect of R language and GEPIA on prognosis was analysed. Mutation rates of SMADs in CRC were determined by cBioPortal, and potentially related genes were predicted using GeneMANIA. R analysis was used to correlate immune cell infiltration in CRC. Both SMAD1 and SMAD2 were found to be weakly expressed in CRC and correlated with the immune invasion level. SMAD1 was correlated with patient prognosis, and SMAD2 was correlated with tumor stage. SMAD3, SMAD4, and SMAD7 were all expressed at low levels in CRC and associated with a variety of immune cells. SMAD3 and SMAD4 proteins were also expressed at low levels, and SMAD4 had the highest mutation rate. SMAD5 and SMAD6 were overexpressed in CRC, and SMAD6 was also associated with patient overall survival (OS) and CD8+ T cells, macrophages, and neutrophils. Our results reveal innovative and strong evidence that SMADs can be used as biomarkers for the treatment and prognosis of CRC.

Efficacy and safety of fruquintinib plus mFOLFOX6/FOLFIRI as first-line therapy in advanced metastatic colorectal cancer (mCRC).

152 Background: Fruquintinib is a highly selective, and potent oral inhibitor of VEGF receptor 1, 2, 3 and has been recommended as a third-line treatment for mCRC patients (pts) in China because of its low toxicity, favorable ORR and survival benefit. FOLFOX/FOLFIRI is the preferred chemotherapy regimens of first-line for mCRC pts according to the NCCN guideline. Our study aimed to investigate the efficacy and safety of fruquintinib plus mFOLFOX6/FOLFIRI as first-line therapy in advanced mCRC pts. Methods: In this prospective, open-label, multi-center, single-arm phase 2 study (NCT05004441), pts who are diagnosed with unresectable mCRC, without prior systemic threated are recruited. RAS/BRAF status should be detected to exclude BRAF mutations before enrollment. Pts receive fruquintinib (3mg, QD, PO, Q4W) in combination with mFOLFOX6/FOLFIRI (Q2W) for up to 8 cycles. Pts with SD or above are followed by maintenance therapy (fruquintinib 3mg, QD, PO, Q4W, capecitabine 850mg/m2, BID, PO, D1-7, D15-21, Q4W) until disease progression or intolerable toxicity. The primary endpoint is objective response rate (ORR), and secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Results: Based on a data cutoff of September 2, 2022, 19 pts have been enrolled and treated, of which 13 pts have efficacy evaluation data. Media age is 60 (range: 50-73) years and 62% pts are male. 7 (54%) pts harboring RAS mutations while the remaining pts are wild-type (wt). 54% treated with mFOLFOX6. At a median follow-up of 5.9mo, all 13 pts are still on treatment. In best overall response assessment, the ORR is 77%, with 10 (77%) partial response (PR). Stable disease (SD) is 3 (23%) with a DCR of 100%. Median PFS has not yet reached. Safety profile exhibited that the regimen is tolerable and mainly grade 1/2. Grade 3 treatment-related adverse events (TRAEs) are neutrophil count decreased (23%), GGT increased (15%), leukopenia, AST increased and hypertension accounted for 1% respectively. No pts have serious adverse events. Conclusions: Combination treatment with fruquintinib plus mFOLFOX6/FOLFIRI as the first-line therapy followed by fruquintinib plus capecitabine maintenance in advanced mCRC pts shows promising efficacy and manageable safety profile. Longer follow up data with PFS, and OS will be presented. Clinical trial information: NCT05004441 .

Trial in progress: A phase 2 study of CTX-009 in adult patients with metastatic colorectal cancer who have received two or three prior systemic chemotherapy regimens.

TPS281 Background: CTX-009 is a recombinant bispecific antibody that binds both delta-like ligand-4 (DLL4) and vascular endothelial growth factor A (VEGF-A). The antibody simultaneously inhibits the DLL4-Notch 1 and VEGF A-VEGF receptor signaling pathways. Patients with unresectable locally advanced and unresectable metastatic colorectal cancer (CRC) are sequentially treated with various combinations or single agent courses of cytotoxic chemotherapy agents including 5-fluorouracil and its orally administered family members (capecitabine, TAS-102) oxaliplatin, and irinotecan, as well with targeted therapies such as bevacizumab or aflibercept (anti-VEGF), cetuximab or panitumumab (anti-EGFR), or multitargeted tyrosine kinase inhibitors (regorafenib). In subsets of patients who have had multigene panel testing done and whose tumors have been identified as having mutations that drive their disease such as defective mismatch repair, HER-2 overexpression, or NTRK fusions specific treatments with respectively PD-1 or PD-L1 inhibitors, traztuzumab or larotrectinib or entrectinib can also be used effectively. Despite the approval of multiple new agents in the past two decades, the goals of therapy are generally palliative although some patients will have long disease-free intervals when they respond to therapy. In the cases of patients whose disease has progressed, or who have not tolerated 2 or 3 prior lines of therapy, the expectation for tumor response with the available treatments are less than 5% and the median overall survival is approximately 6 months (for 3rd line therapy) or less (for 4th line therapy). In a Phase 1 study with CTX-009, responses have been seen in patients with colorectal cancers who were treated in the 4th line setting. Since CTX-009 targets tumor induced neoangiogenesis, and monoclonal antibodies that target vascular endothelial growth factor (VEGF) has been shown to extend the lives of patients with advanced colorectal cancer, testing of CTX-009 in the setting of advanced treatment refractory colorectal cancer is justified. Methods: CTX-009 is being evaluated in an open label study in patients with previously treated metastatic CRC. This study is designed as an adaptive Simon Two Stage study, with approximately 37 patients to be enrolled into Stage 1. If criteria are met to move to Stage 2, an additional 47 patients will be enrolled, for a total of approximately 84 patients. To move to stage 2 of the study, 3 or more Complete or Partial Responses must be confirmed via RECIST 1.1. Patients will be treated in 28-day cycles, with CTX-009 administration on Day 1 and 15. The primary objective of the study is to assess the efficacy of CTX-009, as measured by Overall Response Rate (ORR). The study is currently activating sites with first patient in expected in 2022. Clinical trial information: NCT05513742 .

Research on the Efficacy of Anlotinib in Treating Advanced Metastatic Colorectal Cancer

Research on the Efficacy of Anlotinib in Treating Advanced Metastatic Colorectal Cancer