Zhu, Mojun

3594 Background: The efficacy of checkpoint inhibitor immunotherapy in MSI-H mCRC has not been replicated in MSS mCRC. Therefore, additional interventions are needed to convert immunologically “cold” MSS CRC to “hot” tumors resembling MSI-H tumors. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine containing 12 immunogenic epitopes derived from 7 shared tumor antigens which demonstrated early evidence of clinical activity in first-line MSS mCRC. Methods: Patients with MSS mCRC who have progressed on 2-3 lines of prior chemotherapy regimen received PolyPEPI1018 (1.2 mg, sc) and atezolizumab (1,200 mg, iv) Q3W until disease progression or unacceptable toxicity. The primary endpoint was safety. A Simon 2-stage design was applied. Data on objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and correlation studies will be presented. Results: 18 patients (66 % female) were enrolled for stage 1 of the study. Median age was 54 years (range 38–79), 50% had liver metastases and 67% received at least 3 lines of prior therapies. PD-L1 expression was <1% for 85% of patients. The combination was well-tolerated; most common side effect related to treatment was Grade (Gr) 1-2 local skin reaction (n=22). Gr 2 events (n=2) at least possibly related to treatment were nausea, pyrexia and increased blood alkaline phosphatase. There were no Gr 3-5 events or study discontinuation due to treatment AE. The ORR was 0% and the DCR was 61%. The mPFS was 2.7 months (95%CI 1.4-4.0) and the mOS was not reached (95%CI 8.75 – NE months) at the data cut-off date. Ex-vivo FluoroSpot identified 13/16 subjects with robust, vaccine-specific CD8+ and/or CD4+ T cell responses detected against multiple vaccine antigens. For patients with available biopsy pairs (n=8), both the average PD-L1 expression (IC%, p=0.029) and the density of CD8+ tumor-infiltrating lymphocytes (TILs, p=0.019) were significantly increased, post-treatment. Patients with increased PFS (> 12 weeks) had both higher levels of post-treatment PD-L1 (p=0.007) and TILs (p=0.016) than patients with PFS ≤ 12 weeks. Conclusions: PolyPEPI1018 in combination with atezolizumab was well-tolerated with no Grade 3 AEs observed. Despite no objective tumor responses could be detected, correlative data suggest contribution of PolyPEPI1018-induced immune responses to the modulation of tumor microenvironment and to improved disease control. The study did not proceed to stage 2; it is on-going for the collection of survival data. Clinical trial information: NCT05243862 .

Abstract Background: It is a common practice to monitor trastuzumab-related cardiac toxicity (TRCT) using left ventricular ejection fraction (LVEF) in patients who receive trastuzumab-containing therapy for human epidermal growth factor receptor 2 (HER2)-positive cancers. However, this monitoring approach does not appear cost effective in patients with HER2-positive gastrointestinal (GI) cancers given poor overall survival (OS). Here, we aim to examine the incidence of clinically significant TRCT in this target population. Methods: This is a retrospective cohort study of patients with advanced HER2-positive GI cancers who received trastuzumab-containing therapies (trastuzumab +/- pertuzumab, chemotherapy +/- immunotherapy + trastuzumab, trastuzumab deruxtecan) between 2011 and 2023. HER2 status was determined by immunohistochemistry, fluorescent in situ hybridization, and/or next generation sequencing. LVEF was estimated by serial transthoracic echocardiograms (TTE). Continuous variables were summarized as median and interquartile range (IQR). Paired Wilcoxon signed rank test was used to compare serial LVEF. OS was estimated using Kaplan-Meier method. Results: A total of 243 patients were included in this study with a median age of 64 (IQR: 55-70) years. 139 (57%) patients had esophageal or GEJ adenocarcinoma; 45 (19%) patients had gastric cancer; 37 (15%) patients had colorectal cancer; and the rest had other GI cancers. Median follow-up time was 18.6 (IQR: 9.2-36) months. Median OS from cancer diagnosis and the first dose of trastuzumab-containing therapy were 24.1 (95%CI: 20.2-30.2) and 11.7 (95%CI: 10.4-15.6) months, respectively. Patients received a median of 13 cycles of trastuzumab-containing therapy for a median duration of 4.1 (IQR: 1.4-10.1) months. The median number of TTE each patient had throughout therapy was 2 (IQR: 1-4). Median baseline LVEF was 61% (IQR: 56-65%) which was significantly higher (p&lt;0.001) than the median lowest LVEF of 58% (IQR: 53-62%) during therapy, but not different (p=0.14) from the most recent median LVEF of 60% (IQR: 56-64%) after discontinuation of trastuzumab. Among 176 patients with serial LVEF data, 16 (9%) had &gt; 10% LVEF drop; 7 (44%) of these 16 patients had LVEF recovery. None of the cardiovascular risk factors prior to trastuzumab treatment including hypertension, diabetes, hyperlipidemia, peripheral arterial disease, coronary artery disease, and stroke were significantly associated with the development of TRCT. Conclusion: The incidence of clinically significant LVEF drop appears low in patients with advanced GI cancers as post-treatment LVEF recovery to baseline was observed at the population level. The value of using TTE to monitor TRCT in these patients with short OS should be further assessed in the perspective of cost effectiveness. Citation Format: Samual R. Kosydar, Matteo Castrichini, Mojun Zhu, Hao Xie. The value of routine trastuzumab-related cardiac toxicity monitoring in gastrointestinal cancers [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 6298.

Abstract Background: Benefit from single-agent immune checkpoint inhibitor has been observed in 5-10% of the patients with microsatellite stable (MSS) gastrointestinal (GI) cancers. However, a reliable predictive marker to immune checkpoint inhibitor has not been discovered in this patient population. We sought to assess the incidence of mismatch repair (MMR) gene alterations and their association with tumor mutational burden (TMB) in MSS GI cancers. Methods: This is a retrospective cohort study of patients with advanced GI cancer who had molecular profiling of the tumor using either Guardant360 blood-based or Tempus tissue next generation sequencing (NGS) platforms between 2020 and 2023. We assessed four MMR genes including MLH1, MSH2, MSH6 and PMS2 for alterations. Continuous variables were summarized as median and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were summarized as counts and proportions and compared using Fisher’s exact test. Results: A total of 2920 patients with Guardant360 were included with a median age of 65 (IQR: 56-73) years. 870 (30%) patients had pancreas cancer; 846 (29%) patients had colorectal cancer; 688 (24%) patients had biliary tract cancers; and the rest had other GI cancers. Among patients evaluable for microsatellite instability (MSI) status, 58 patients (2%) had MSI-high GI cancers, 31 (53%) of whom had MMR gene alterations. 2445 (98%) patients had MSS GI cancers, 130 (5.3%) of whom had MMR gene alterations. Patients with MSS GI cancers bearing MMR gene alterations had a median age of 70 (IQR: 62-75) years, significantly older than those without MMR gene alterations (p&lt;0.001). In addition, patients with MMR gene alterations had significantly higher median blood TMB of 11 (IQR: 7.4-16) mut/Mb compared to 7.7 (IQR: 4.8-11) mut/Mb in patients without MMR gene alterations. In MSS GI cancers, MMR gene alterations are more commonly associated with the presence of alterations in APC (p=0.03), TP53 (p=0.01), CTNNB1 (p&lt;0.001) and SMAD4 (p=0.01). 1453 patients with GI cancer and Tempus tissue NGS data were included as a validation cohort. They had comparable demographics with the Guardant360 cohort. 14 (1.6%) of 896 patients with MSS GI cancers had MMR gene alterations, significantly fewer (p&lt;0.001) than those found in the Guardant360 cohort. Patients with MMR gene alterations and wild type POLE had significantly higher (p=0.001) median TMB of 9.8 (IQR: 4.8-14) mut/Mb compared to 4.7 (IQR: 3.2-6.3) mut/Mb in patients without MMR gene alterations. Conclusions: MMR gene alterations can be identified by clinical NGS platforms in a small proportion of patients with MSS GI cancers. They are associated with elevated TMB, which may suggest a hypermutated profile serving as a basis for potential role of immune checkpoint inhibitor in MSS GI cancers. Citation Format: Conor D. O'Donnell, Mojun Zhu, Hao Xie. The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers [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 6469.

TPS228 Background: Colorectal cancer (CRC) is the third most frequently diagnosed cancer and the second leading cause of cancer-related mortality worldwide. PD-1/PD-L1 inhibition, in combination with other modalities, has demonstrated significant benefit in patients (pts) with microsatellite instability-high or mismatch repair–deficient CRC. However, these agents have limited, if any, clinical benefit in pts with microsatellite stable (MSS) or mismatch repair–proficient (pMMR) CRC. RP2 is an enhanced potency oncolytic herpes simplex virus type 1 which expresses the fusogenic gibbon ape leukemia virus glycoprotein with the R sequence deleted (GALV-GP-R–), granulocyte-macrophage colony-stimulating factor (GM-CSF), and an anti–CTLA-4 antibody-like molecule; RP3 additionally expresses 4-1BB and CD40 activating ligands but does not express GM-CSF. Both agents have demonstrated preliminary safety and antitumor activity in pts with solid tumors. This study will evaluate the safety and activity of RP2 and RP3 in combination with atezolizumab (Atezo) plus bevacizumab (Bev) in pts with advanced MSS/pMMR CRC (NCT05733611). Methods: Pts with a histologic diagnosis of unresectable and/or metastatic CRC, with documented MSS or pMMR status, and previously treated with up to 3 standard-of-care systemic regimens will be enrolled in the RP2 + Atezo + Bev or RP3 + Atezo + Bev treatment groups (30 pts per group). Further key inclusion criteria include Eastern Cooperative Oncology Group performance status 0 to 1 and adequate hepatic, renal, and hematologic function. RP2/RP3 will be injected into tumors by direct or image-guided injection. Pts will receive 8 total doses of up to 10 mL of RP2 or RP3, with a first dose concentration of 1 × 106 plaque-forming units (PFU)/mL, followed by 3 doses of 1 × 107 PFU/mL every 2 weeks, and then 4 doses of 1 × 107 PFU/mL every 3 weeks. Pts may receive a second course of up to 8 injections of RP2/RP3 if study criteria are met. Bev will be administered starting on week 1; Atezo will be administered starting week 7. The primary endpoint is objective response rate; secondary endpoints are safety, overall survival, progression-free survival, duration of response, and complete response rate. Antitumor activity will be evaluated using Response Evaluation Criteria in Solid Tumors version 1.1 as modified for this study. Safety will be assessed by physical examination, clinical laboratory evaluations, vital signs, and monitoring for adverse events (AEs; including serious AEs). Clinical trial information: NCT05733611 .

175 Background: Alterations to mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, and PMS2, can lead to microsatellite instability-high (MSI-H) tumors. MMR mutations can be inherited in Lynch syndrome (LS) but can also be a result of de novo alterations resulting in MSI-H malignancies. Treatment with immune checkpoint inhibitors (ICIs) have been shown to improve survival in such patients (pts) compared to systemic chemotherapy. However, there is a paucity of information in the literature with respect to outcomes of pts with germline vs somatic MMR mutations in MSI-H colorectal cancer (CRC). Furthermore, the presence of underlying BRAF mutation confers resistance to chemotherapy in pts with CRC, however data regarding outcomes with immunotherapy in such pts with MSI-H CRC is lacking. Methods: Pt records from the Mayo Clinic (AZ, MN, FL) between 2008-2023 denoted as MSI-H CRC, were included for retrospective review. Pt demographics, treatment courses, and genomic profiles were collected. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Outcome differences between sub-groups were accessed with the log rank test and univariate analyses were assessed with Cox-regression. Results: A total of 81 pt records were identified (n = 18 LS, n = 63 non-LS). Stage IV disease was found in 28% of LS pts vs 59% of non-LS pts at diagnosis. Pembrolizumab was the most common ICI selected for metastatic CRC pts for both LS and non-LS pts (65% vs 94%) with median treatment duration for each group 11.7 vs 8.8 months. There were no differences in OS or PFS for any stage between LS vs non-LS. Median OS for all patients was 82 months. For all stage IV patients, median OS was 44 months, and median PFS was 19 months. Interestingly, for stage IV pts treated with immunotherapy, tumors that harbored BRAF V600E mutation (n = 21) vs BRAF wild type (n = 20) had a significantly lower median OS 19 vs 113 months (HR = 2.69, p = 0.043) as well as median PFS 12 vs 95 months (HR = 2.48, p = 0.041). Conclusions: In this study, we found that patient treated with ICIs have similar outcomes in the presence of germline vs. somatic MMR mutations in MSI-H CRC. The presence of an associated BRAF V600E mutation, which occurs in non-LS MSI-H CRC conferred worse outcomes.

170 Background: KRAS-mutant colorectal cancer (CRC) has a worse prognosis and greater resistance to therapy, attracting tremendous efforts to develop both allele-specific and pan-KRAS targeting agents including KRAS G12C and G12D inhibitors. CRC has demonstrated both primary and acquired resistance to KRAS G12C inhibitors, which can be better evaluated by circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) methods without tissue sampling bias. Here, we utilized clinical ctDNA data to delineate the landscape of candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Methods: Samples from advanced CRC patients with Guardant360 data in 2021 were categorized into a KRAS G12D group and comparator groups of KRAS G12C and KRAS not detected (ND) according to KRAS mutation status. Demographics, MSI-H status, and blood tumor mutational burden (bTMB) were summarized and compared between groups using t-test for continuous variables and Fisher’s exact test for categorical variables. Co-occurrence patterns of alterations between KRAS and other genes were assessed. Primary resistance mutations were inferred by acquired resistance findings to KRAS G12C inhibitors reported by Awad et al. N Engl J Med. 2021 and Tanaka et al. Cancer Discov. 2021. Results: Among approximately 2500 samples, 16.8% had KRAS G12D, the most common KRAS mutation in CRC. The mean allele frequency of KRAS G12D (11.4%) is comparable to that of KRAS G12C (10.9%). Mean age (61.6 years), MSI-H frequency (3.8%), and mean bTMB (14.2 Mut/Mb) of KRAS G12D group is comparable to those of KRAS ND group. This contrasts with KRAS G12C with significantly lower mean age (60.2 years, p=0.01), lower MSI-H frequency (0.56%, p=0.03), and higher mean bTMB (16.3 Mut/Mb, p=0.02) compared to KRAS ND. Both KRAS G12D and G12C significantly co-occur with APC, PIK3CA, and SMAD4 alterations. FBXW7, AR, and KEAP1 alterations were unique co-occurrences to KRAS G12D, while KIT was unique to KRAS G12C. Candidate alterations associated with primary resistance in KRAS G12D CRC included simultaneously present mutations of KRAS Q61H (14.1%), G12V (6.4%), G12C (5.9%), G13D (3.6%), BRAF V600E (2.7%) and others, amplification of MET (2%), MYC (1.6%), and KRAS (1.4%), and FGFR3-TACC3 translocation (Table). Conclusions: KRAS G12D is associated with unique co-occurring molecular alterations compared to KRAS G12C in CRC. ctDNA-based NGS platforms can survey candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Further validation in preclinical models is needed. [Table: see text]

Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. Methods: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. Results: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90–232 days). Conclusions: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

AbstractPatient‐derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non‐resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold‐standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)