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Purpose:We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC).Patients and Methods:In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence.Results:Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9–5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01).Conclusions:The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.

T-cell infiltration for patient LH-01-26 with BRAF V600 mutation in comparison to study population

Multispectral immunofluorescence. Number of cells positive for exhaustion (PD-L1, TIM3, LAG3, CTLA4) and activation markers (OX40) in baseline tumor specimens (A); Quantitation of signals per specimen (B). There are more PDL1 positive tumor cell in the pre-treatment metastatic site biopsy. While there are more activated, partially activated T cell-1 and exhausted T cell-1 in the primary tumor specimens (*p

Revised supplemental figures and tables

Consort Diagram.

Intratumoral NK cells (baseline) by change in tumor size (A) and change in CEA (B).

Correlate of baseline and on-treatment changes in intratumoral immune cells (flow cytometry) with clinical endpoints (Supplementary Table 2A), immune checkpoint expression by multispectral immunofluorescence in tumor microenvironment (Supplementary Table 2B) and correlation with clinical endpoints (Supplementary Table 2C), immune cells (flow cytometry) in peripheral blood samples at baseline and on treatment (Supplementary Table 2D) and correlation with clinical outcomes (Supplementary Table 2E).

Purpose:Small-bowel adenocarcinoma (SBA) is rare, and no standard of care exists for metastatic disease beyond first-line FOLFOX/CAPOX. SBA has higher rates of microsatellite instability (MSI-H) and T-lymphocyte infiltration than other gastrointestinal cancers. We hypothesize that pembrolizumab, a PD-1 inhibitor, will induce antitumor response.Patients and Methods:Patients with previously treated advanced SBA received pembrolizumab 200 mg i.v. every 3 weeks until disease progression (PD), toxicity, or 35 doses maximum. Primary endpoint was confirmed overall response rate (ORR) with secondary progression-free survival (PFS), overall survival (OS), and toxicity assessment endpoints. Outcomes were stratified by tumor location, microsatellite stability (MSS) or instability (MSI-H), and PD-L1 level.Results:Forty patients were treated for a median duration of four cycles (range, 1–35). All patients are off study treatment due to PD (75%), death (10%), 35 cycles completed (8%), refusal (3%), and adverse effects (AEs, 5%). Three confirmed partial responses [PRs; 8%; 95% confidence interval (CI), 2–20] did not meet predefined success criteria of ORR 30%. Median OS (7.1 months; 95% CI, 5.1–17.1) and median PFS (2.8 months; 95% CI, 2.7–4.2) were similar across primary tumor sites. One confirmed PR (3%) was seen in patients with low MSS/MSI tumors and correlated with high tumor mutation burden (TMB). Fifty percent of patients with MSI-H tumors achieved PR and remain alive without progression. Twenty-five patients (63%) had grade ≥3 AEs and 11 patients (28%) had grade 4/5 AEs.Conclusions:In the largest study of SBA to date, pembrolizumab did not induce the hypothesized response rate; however, we did identify responses in key biomarker-selected cohorts.

Changes in peripheral blood immune cell populations on treatment.

Adverse events with maximum grade seen

Change in CEA over time.

The aim of this paper is to summarize the current treatment landscape in metastatic colorectal cancer, as well as those on the horizon in the third-line and beyond settings. Herein, recent data regarding TAS-102, regorafenib, and novel anti-angiogenic agents are described. Data on chemotherapy re-challenge and EGFR re-challenge is reviewed. A summary of data on the use of BRAF-targeted therapies, HER-2-targeted therapies, rare fusions (NTRK, RET), MET amplification, and KRAS G12C is included, as well as a brief review on the current role of immune checkpoint inhibitors in metastatic colorectal cancer. Multiple new agents are on the horizon. There is increasing relevance of next generation sequencing to look for rare targets, and potentially to assess tumor mutational burden. ctDNA appears to be a valuable asset which may guide the use of therapies in the re-challenge setting.

Background This open-label, phase I/II study evaluated safety and efficacy for first-line liposomal irinotecan + oxaliplatin + 5-fluorouracil + leucovorin (NALIRIFOX). Methods Patients (aged ≥18 years) had locally advanced/metastatic pancreatic ductal adenocarcinoma (mPDAC), with an Eastern Cooperative Oncology Group performance status score of 0/1 and adequate organ function. Primary objectives were to determine the maximum tolerated dose (MTD) and to evaluate safety and tolerability. Treatment-emergent adverse events (TEAEs) were graded using National Cancer Institute Common Terminology Criteria for Adverse Events v4.03. Efficacy end-points included progression-free survival (PFS) and overall survival (OS); disease assessments used Response Evaluation Criteria in Solid Tumors 1.1. Results The MTD (liposomal irinotecan 50 mg/m2 [free-base equivalent], oxaliplatin 60 mg/m2, 5-fluorouracil 2400 mg/m2, leucovorin 400 mg/m2 every 2 weeks) was based on dose-limiting toxicities and cumulative safety data in four dose-exploration cohorts. The MTD was received by 32 of 56 patients, seven during dose exploration and 25 during dose expansion (median age 58.0 years [range, 39–76], 28 [87.5%] with metastatic disease at diagnosis [29 at study entry], and one receiving study treatment at data cutoff [26 February 2020]). Of these patients, 22 of 32 had grade ≥3 treatment-related TEAEs, most commonly neutropenia (31.3%), febrile neutropenia (12.5%) and hypokalaemia (12.5%); ten had serious treatment-related TEAEs; and three died from TEAEs considered unrelated to treatment. Median PFS and OS were 9.2 (95% CI: 7.69–11.96) and 12.6 (8.74–18.69) months, respectively. Conclusion First-line NALIRIFOX for patients with locally advanced/mPDAC was generally manageable and tolerable. A randomised, controlled phase III study is underway.

Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown.FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth.We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression.DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Irinotecan is a commonly used chemotherapeutic in solid tumor malignancies. Oratecan is an investigational product comprised of encequidar methanesulfonate, a novel minimally absorbed P-glycoprotein pump inhibitor, and irinotecan. This study sought to determine the maximum tolerated dose (MTD) of oratecan in patients with advanced malignancies.Using a "3 + 3″ dose-escalation design, patients were treated with oratecan on day 1 every 21 days. The irinotecan dose was escalated from 20 to 320 mg/mThirty-five patients were treated. The MTD was determined to be 280 mg/mThe MTD of oratecan was encequidar methanesulfonate 15 mg plus irinotecan 280 mg/m

Immune checkpoint inhibitors (ICIs), as a novel class of anticancer therapy, can be more efficacious and less toxic than chemotherapy, but their clinical success is confined to certain tumor types. Elucidating their targets, mechanisms and scope of action, and potential synergism with chemotherapy and/or targeted therapies are critical to widen their clinical indications. Treatment response to an ICI targeting programmed death-1 (anti-PD-1) is sought to be understood here by conducting a preplanned correlative analysis of a phase II clinical trial in patients with small bowel adenocarcinoma (SBA). The cytolytic capacity of circulating immune cells in cancer patients using a novel ex vivo cytotoxicity assay is evaluated, and the utility of circulating biomarkers is investigated to predict and monitor the treatment effect of anti-PD-1. Baseline expression of Bim and NKG7 and upregulation of CX3CR1 in circulating T cells are associated with the clinical benefit of anti-PD-1 in patients with SBA. Overall, these findings suggest that the frequency and cytolytic capacity of circulating, effector immune cells may differentiate clinical response to ICIs, providing a strong rationale to support immune monitoring using patient peripheral blood.

Background Timely diagnosis and management of iron deficiency anemia (IDA) in colorectal cancer (CRC) patients improves overall quality of life and survival. This study assessed the proportion of CRC patients who were formally diagnosed with IDA and factors that predict a formal diagnosis of IDA and receiving iron therapy. Methods We retrieved electronic medical records (EMRs) of CRC patients from a large comprehensive cancer center in the Northeastern part of the United States (n = 499). We abstracted sociodemographic characteristics, relevant laboratory results, IDA diagnosis, and iron supplementation from the EMRs. We assessed relationships between participant characteristics, a diagnosis of IDA and receiving iron therapy through adjusted logistic regressions. Results IDA was formally diagnosed in 26 (5.2%) individuals judged by EMR documentation. Only 153 (30.7%) participants had iron laboratory results available. Among the 153 patients with iron panel data available, 113 (73.9%) had iron deficiency. Seventy-six had absolute iron deficiency as shown by ferritin levels below 100 ng/mL and iron saturation less than 20% and 37 had functional iron deficiency as shown by ferritin levels between 100 and 500 ng/mL and iron saturation less than 20%. 12% of all patients had documentation of iron therapy receipt. A formal diagnosis of IDA was not associated with any of the covariates. Conclusions Iron deficiency anemia is under-diagnosed among CRC patients and most likely under-documented in clinical notes. Rates of iron repletion are low, suggesting that many patients with IDA are untreated. Future research should explore provider-level and other strategies for improving assessment and diagnosis of IDA among CRC patients.

Colon cancer has a high incidence of metastasis, with an estimated 0.8–7.4% of colorectal adenocarcinoma (CRC) cases metastasizing to the ovary. The role of prophylactic bilateral oophorectomy in CRC is contested in the literature, particularly in premenopausal patients. Further, it is unclear if prophylactic removal of the contralateral ovary is indicated in cases of direct involvement of one ovary to reduce recurrence. Facing a lack of evidence for survival benefit, hormonal complications, and sterilization, some choose to pursue fertility sparing options. For female patients interested in additional pregnancies, the ovaries can be surgically relocated in a prophylactic procedure known as ovarian transposition; as even small doses of radiation to the ovary can effectively sterilize women in their 30 s. We present a case of a 29-year-old female who underwent ovarian transposition of the right ovary before initiating chemoradiation for primary left sided colon adenocarcinoma with direct invasion of the left ovary. Months later, she presented to the emergency department (ED) with abdominal pain suspicious for ovarian torsion. On restaging computerized tomography (CT), she was diagnosed with symptomatic right ovarian metastasis in the transposed ovary, requiring reoperation and oophorectomy. For this patient, and for others facing critical decisions about ovarian preservation in advanced colorectal cancer, the question remains how to balance fertility concerns with optimal minimization of metastasis and recurrence.

103 Background: Novel strategies to improve the efficacy of immune checkpoint inhibitors in microsatellite stable (MSS) mCRC are needed. CV301 is a vector-based vaccine that expresses carcinoembryonic antigen (CEA) and mucin 1 (MUC1), and in a phase II study in resected hepatic limited mCRC significantly improved OS compared with unvaccinated contemporary controls. Methods: In this multi-center randomized phase II study, patients with previously untreated resectable hepatic-limited mCRC were randomized to perioperative nivolumab + mFOLFOX +/- CV301 (Arm B) with a primary endpoint of 3-year OS. Treatment included mFOLFOX-nivo (+/- CV) x 4 cycles followed by resection, then 8 more cycles of mFOLFOX-nivo followed by maintenance nivo monthly for two years in both arms, and CV boosters concurrently with mFOLFOX, and then every 3 months for two years in arm B. Secondary endpoints of ORR (following induction pre-resection), PRR, and safety were determined. Correlative analyses included immune cell quantification using Immunoscore and T-cell clonality. Results: 17 patients were enrolled prior to premature closure for slow accrual (8 arm A, 9 arm B). At the time of data cutoff, 5 patients remained on treatment and no deaths had occurred. One patient was removed from study due to protocol non-compliance. The median age was 61, majority were male (59% vs 41%), and ECOG PS 0-1 (71% 0, 17% 1). All patients had complete surgical resection. Four patients (24%) experienced a SAE related to drug. The TRAE rate was 40.3%,. No AEs delayed/prevented surgical resection. The ORR in arm A was 50% (including 4 CR) and 87.5% in arm B (including 7 CR) (p=0.129, NS). There was no significant difference in pathologic response (p=0.9047). Correlative analyses demonstrated the Immunoscore CD3/CD8 predicted response to mFOLFOX + nivolumab, but did not correlate with response to CV301, though CV301 may induce a shift to predominantly cytotoxic CD8+ T cells. While there was no significant difference in T cell repertoire, clonality, fraction (TCFr) or richness, patients in arm B had significant decreases in blood TCFr and increase in tumor TCFr with treatment; those with CR had higher TCFr and clonality. Conclusions: The addition of CV301 to perioperative nivolumab and mFOLFOX was safe, did not delay or prevent surgical resection, and gave a higher response (p=ns due to sample size). Changes in T cells suggest a vaccine response. Clinical trial information: NCT03547999 . [Table: see text]

TPS257 Background: Refractory microsatellite stable colorectal cancer (MSS CRC) is immunologically cold and single-agent anti-PD-1/PD-L1 drugs are ineffective; novel immune-based approaches are needed. Evorpacept (E, ALX148) is an engineered protein (high-affinity CD47-blocker fused to an inactive IgG Fc region), which blocks the CD47/SIRPα innate immune inhibitory phagocytosis checkpoint expressed on CRC and phagocytes, respectively. The Fc region of E does not bind to Fcγ receptors, thereby limiting hematologic toxicity, and is intended to be given in combination. In CT26 CRC syngeneic models, E ± anti-PD-1 monoclonal antibody decreases tumor growth, reduces myeloid immunosuppression, increases dendritic cell activation, and increases T cell activation (Kauder, 2018); E enhances the antibody-dependent cellular phagocytosis activity of cetuximab (C) in vitro (Kauder, 2018); and E + pembrolizumab (P) was well-tolerated in the first-in-human trial (Lakhani, 2021). Methods: AGICC-ALX148 21CRC01 (NCT05167409) is a phase 2, single-arm, multicenter, investigator-initiated trial of E (15 mg/kg weekly), C (400 mg/m2 then 250 mg/m2 weekly), and P (200 mg every 3 weeks) in 21-day cycles for patients with unresectable MSS/proficient mismatch repair CRC refractory to oxaliplatin, irinotecan, and a fluoropyrimidine, regardless of tumor sidedness and RAS/BRAF status. Additional key eligibility criteria include ECOG performance status 0-1, evaluable disease per RECIST v1.1, adequate hematologic and end organ function, absence of prior checkpoint inhibitor use, and absence of significant autoimmune disease. Six patients will be enrolled in Stage 1 (safety run-in) and treated with ECP. The study will proceed to Stage 2 (dose expansion, N = 42, and all treated with ECP) if less than 33% of patients in Stage 1 experience a dose-limiting toxicity. Otherwise, additional patients will be enrolled in Stage 1 at lower dose level(s). The co-primary objectives are to determine 1) the recommended dose of E with CP, and 2) objective response rate by RECIST v1.1 (by one-sided exact test with α = 0.05, H0 p ≤ 3% [historical controls], HA p ≥ 15%; power is 87%). The study will close for futility if there are no responses (partial or complete) in the first 24 evaluable patients (by MinMax design with α = 0.025 [1-sided]; power is 87%). Secondary and exploratory aims include determination of progression-free survival, overall survival, safety, response assessment by iRECIST, and blood- and tumor-based immune modulation and baseline tumor expression (PD-L1, EGFR, and CD47) for association with tumor response. The study is open through the Academic GI Cancer Consortium and 5 patients have been enrolled at time of submission. Clinical trial information: NCT05167409 .

144 Background: TAS-102 (trifluridine/tipiracil) is a novel oral antimetabolite for late line metastatic colorectal cancer (CRC) approved in 2018. Many patients are treated early in their course with oxaliplatin (OX), particularly adjuvant, and may benefit from re-treatment. In this trial we combine the typical late line use of TAS with OX (BEV [bevacizumab] added at investigator discretion) with goal of improved response. Methods: Eligibility included measurable CRC previously treated with all approved drugs per TAS package insert (irinotecan, oxaliplatin, 5FU, anti-VEGF, anti-EGF) as appropriate, PS = 0-1, labs within usual range, neuropathy < grade 2, ability to take oral meds, appropriate contraception. If no contraindication to BEV, this could be added at patient. TAS was dosed at 35 mg/m2 days 1-5 with OX 85/m2 d1 every 14 days (and BEV 5 mg/kg, if given). All supportive care was allowed including growth factors. Results: 47 patients (pts, median age 55) were enrolled in a Simon mini-max design, including 45% female, 21% black, 11% Asian, 11% Hispanic and 5% mixed. 26 pts received BEV. For the first 40 pts, 385 cycles were given (mean = 7 cycles, median 8) with 18 pts (45%) requiring dose reductions (1 dose reduction = 9 pts, 2 = 6, 3 = 3), and 9 receiving (peg)/filgrastim. Toxicities leading to SAEs included gr 3 heme (2), heart failure, abd pain/n/v (6), sepsis (2), urinary (4); and related gr 3 included one gr 3 vomiting and one gr 3 neutropenia. Independently reviewed RECIST Response (N = 32) included PR 2(6%), SD 23 (72%), PD 7 (22%). Mean TTP was 4.5 m (median 4, range 1 – 18) with 9 (28%) pts more than 6 months. Conclusions: In patients with late-line CRC and candidates for TAS (trifluridine/tipiracil), treatment with TAS plus OX is both well tolerated and active. RR is higher than single agent and 78% (95% CI, 60-91%) of patients had stable disease or response, with 60% receiving 8 or more cycles. Randomized trials comparing to single agent TAS are warranted in this setting. Clinical trial information: NCT04294264 .

178 Background: Anti-EGFR therapy has the potential to increase a localized anti-tumor immune response. We recently published the results of a phase Ib/II trial of the anti-EGFR antibody cetuximab in combination with the anti-PD1 antibody pembrolizumab in pts with advanced, RASwt CRC (PMID: 34645646). Despite its partial local immunologic efficacy, this combination of cetuximab and pembrolizumab was inactive. Here we present the results of comprehensive molecular characterization of pts with tumors amenable to DNA and RNA sequencing. Methods: Forty-two pts with RASwt mCRC were treated with cetuximab plus pembrolizumab. Archival or fresh tumor samples were obtained at baseline and, in select patients, on-treatment. Tumor samples underwent targeted DNA sequencing and whole transcriptome sequencing. Gene set enrichment analysis (GSEA), metabolic dysregulation assessment, and immune deconvolution were performed. Genomic data were linked to clinical outcomes. Results: Eighteen pts had tissue available for dual DNA/RNA extraction and sequencing. Of these, 10 had available matched on-treatment tumor samples. The most common mutations detected in protein coding regions were TP53 (14 pts), ERBB4 (5 pts), CDKN2A and APC (6 pts each). There were no statistically significant differences in progression-free survival (PFS) in pts with and without resistance-associated mutations (i.e., RAS, MET, ERBB4). Further, there was no significant difference associated with the consensus molecular subtype (CMS). However, when we compared patients with stable/increased tumor change percentage to those with decreased tumor change percentage, we found downstream transcriptional differences associated with altered metabolism, and in particular lipid and amino acid metabolism. Conclusions: We identified a significant number of patients with mutations predicting resistance to either or both cetuximab and pembrolizumab; however, none were associated with survival. However, we did identify metabolic pathways of interest, which may be associated with response to therapy. It is important to note, our analysis is limited by the small number of specimens (Trial registration NCT02713373). Clinical trial information: NCT02713373 .

Purpose: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC). Patients and Methods: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence. Results: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9–5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01). Conclusions: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.

The immune tumor microenvironment (TME) of colorectal cancer (CRC) is a crucial contributor to disease biology, making it an important target for therapeutic intervention. The diversity of immune cell populations within various subsets of CRC has led to the discovery that immune characterization of the TME has both prognostic and predictive value for patients. The convergence of improved molecular and cellular characterization of CRC along with the widespread use of immunotherapy in solid tumors has led to a revolution in the approach to clinical care. Monoclonal antibodies (mAbs) which target key immune checkpoints, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), have demonstrated remarkable clinical activity in microsatellite instability-high (MSI-H) CRCs and are now used in routine practice. The observation that MSI-H cancers are highly infiltrated with immune cells and carry a high neoantigen load led to the successful targeting of these cancers with immunotherapy. More recently, the Food and Drug Administration (FDA) approved a PD-1 inhibitor for microsatellite stable (MSS) cancers with high tumor mutation burden. However, the anti-tumor activity of immunotherapy is rare in the majority of CRC. While immune cell characterization does provide prognostic value in these patients, these observations have not yet led to therapeutic interventions. By delineating factors that predict efficacy, resistance, and therapeutic targets, ongoing research will inform the development of effective combination strategies for the vast majority of MSS CRC and immunotherapy-resistant MSI-H cancers.

Background: Liver metastases develop in 20-50% of colorectal cancer (CRC) patients, being typically resistant to immune checkpoint inhibitors (ICIs) and having a poor prognosis. Our preliminary data showed synergy between IFNα and TLR3 ligands in selectively enhancing the intratumoral production of CD8+ T cell (CTL) attracting chemokines (but not Treg attractants) in ex vivo-treated CRC explants and preferential impact of this combination on liver-metastatic CRC tumor tissues (rather than surrounding liver tissues). Based on these data and previous reports showing the prognostic value of intratumoral CTLs in the CRC outcomes, we hypothesized that systemic infusion of the combination of IFNα2b with rintatolimod (selective TLR3 ligand for i.v. use) can reprogram local balance between the CTL- and regulatory T cell (Treg)-attracting chemokines and the resulting patterns of immune infiltrate in liver lesions. Methods: Recurrent/metastatic CRC patients with unresectable liver metastases amenable to biopsy were eligible. Patients had prior treatment (Rx) with fluoropyrimidine, irinotecan, oxaliplatin, and an anti-EGFR targeted therapy (if RAS wt), or contra-indication to such. Patients received IFNα2b IV (20 million units/m2 IV daily) and rintatolimod (200 mg IV daily) plus oral celecoxib (200 mg twice daily) on days 1, 2, 3, 8, 9, 10, 15, 16, and 17. Response assessment was done via liver biopsies (pre-Rx and on day 24 ± 4 days) and CT imaging (RECIST v1.1) on D46. The primary endpoint was the change in CD8+ T-cells before Rx, with that seen post-Rx (measured by quantitative RT-PCR as a ratio of CD8α to a housekeeping gene, HPRT). With a sample size of N=12 evaluable pts, the study design had a 90% power to detect a 0.77 standard deviation increase (pre- to post Rx) at a significance level of 0.1 (ClinicalTrials.gov Identifier: NCT03403634). Results: Nineteen patients with microsatellite stable (MSS) CRC were enrolled between Apr 2018 and Oct 2020, 15 were treated and 12 were evaluable for the primary endpoint. Most were male (75%, N=9) and Caucasian (92%, N=11). The median age at diagnosis was 65 years. Most had previously received three or more prior lines of therapy (58%, N=7). The study's primary endpoint was met, evidenced by increased CD8a expression post-treatment (p=0.046). An increase in ratios of CD8a/CD4 (p=0.03), CD8a/FOXP3 (p Conclusion: Our study demonstrated that a combinatorial CKM regimen could re-program the immunosuppressive tumor microenvironment in MSS CRC patients with liver metastasis, raising the possibility that the CKM regimen can be used to enhance the effectiveness of ICIs in this group of patients. Citation Format: Sarbajit Mukherjee, Patrick M. Boland, Melissa Grimm, Ronald Slomba, Kristopher Attwood, Renuka Iyer, Pawel Kalinski. Initial results of a phase II study evaluating a chemokine-modulatory (CKM) regimen in patients with colorectal cancer metastatic to the liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT105.

e15587 Background: For resected stage II MSI-high (MSI-H) colorectal cancer (CRC) without high-risk features (HRF), standard of care recommendation per NCCN guidelines is observation. For tumors with HRF, the prognostic significance and impact of adjuvant therapy remains uncertain. The NCDB was queried to assess outcomes. Methods: Adult patients with stage II MSI-H CRC, surgically resected between 2010 and 2017, were identified in the NCDB. Univariate and multivariate Cox proportional hazards models and Kaplan Meier survival curves were generated to assess impact of HRF, clinical and demographic variables, and chemotherapy use on overall survival (OS). Results: 9634 patients with stage II MSI-H CRC who met criteria were identified. In multivariate analysis T4 tumor status, < 12 lymph nodes (LN) examined, perineural invasion (PNI) and positive margins were associated with worse OS, as were older age and increased comorbidity index (CDCC). Poor differentiation and lymphovascular invasion (LVI) were not associated with OS. No OS difference was observed between T4a vs T4b tumors or based upon tumor sidedness, tumor size, or sex. Excluding poor differentiation (33%) and LVI (17%), HRFs were present in 26% of cases: 21% (n = 2033) had 1 HRF and 5% (n = 505) had > 1 HRF. HRFs were associated with decreased 5-year OS. For 1 HRF OS was 66% (HR 1.48, p < 0.0001) and for > 1 HRF OS was 54% (HR 2.31, p < .0001), compared to 77% with 0 HRFs. HRF presence was associated with increased chemotherapy use: 46% for > 1 HRF, 26% for 1 HRF, and 8% with 0 HRFs. pT4 tumors (T4b > T4a) and younger age were also associated with increased chemotherapy use. By age group, chemotherapy was utilized in 31% of those < 50, 27% of those 51-60, 17% of those 61-70 and 6% of those > 70 years old. In the overall population, chemotherapy administration was associated with improved OS on multivariate analysis compared with no treatment (N = 1344; HR 0.76, p < 0.0001). 69% of patients received multi-agent chemotherapy. However, survival was similar between those receiving single-agent vs multi-agent chemotherapy (HR 1.2, p = 0.2016). In patients < 60 years old, chemotherapy use was associated with decreased OS (HR 1.4, p = 0.0156). Conclusions: In this retrospective, uncontrolled large database survey, HRFs are associated with decreased OS in stage II MSI-H CRC. This data indicates that poor differentiation and LVI do not independently worsen outcomes, but that the presence of multiple HRFs bears relevance. Though chemotherapy was associated with improved OS, the association is reversed in the < 60 year old population; this analysis cannot fully control for extent of disease and PS, among other factors. Chemotherapy should be judiciously administered in Stage II MSI-H CRC with HRFs. Immunotherapy trials are justified.[Table: see text]

PURPOSE/OBJECTIVE(S) Neoadjuvant chemoradiotherapy (CRT) serves to downstage and increase the rate of surgical resection for borderline resectable and resectable pancreatic adenocarcinoma ((B)RPC). Although conventional staging provides prognostic information, it remains a challenge to predict which patients will proceed with resection. We hypothesize that the severity of vessel involvement and the trajectory of CA19-9 levels after neoadjuvant treatment would associate with proceeding to resection. MATERIALS/METHODS Medical records were retrospectively reviewed at a single center for patients diagnosed with (B)RPC and treated with neoadjuvant CRT between 1/2005 and 12/2020. Progress notes were evaluated to determine if a patient proceeded to surgery and the trajectory of CA 19-9 levels in response to treatment. Radiologist notes were evaluated to determine the extent of mesenteric vessel (superior mesenteric artery, celiac axis, portal vein, hepatic artery, and superior mesenteric vein) involvement. A vessel involvement score was calculated as the sum of all vessels in contact with tumor: 1 point for each vessel, with partial (< 180 degrees) encasement of artery counting as 2 points. Odds ratios (OR) of proceeding to resection were computed with 95% confidence intervals (CI) for CA19-9 response to treatment. Ordinal logistic regression models were fit to estimate the impact of greater vessel involvement on the odds of NOT proceeding with surgery. RESULTS Forty-two (n = 42) patients were included in the analysis. The median age was 65 years (IQR: 61 to 71). 73% of patients were treated with 3600 cGy in 15 fractions (fx), 24% of patients were treated with 4500-5040 cGy in 25-28 fx, and 3% of patients were treated with 3300 cGy in 5 fx. 55% of patients were treated with neoadjuvant FOLFIRINOX and 45% of patients were treated with other regimens. 88% of patients were considered borderline resectable and 12% were resectable at the time of diagnosis. Patients with any decrease in CA19-9 levels after CRT had higher odds of proceeding with resection (OR = 21.0, 95% CI: 3.2 to 139.2). However, decrease in CA19-9 levels after upfront chemotherapy was not significantly associated with proceeding with resection (P = 0.91). The odds of NOT proceeding with resection after neoadjuvant CRT were 1.81 times higher (95% CI: 0.97 to 3.39) for each increase in vessel involvement score, however these findings were not statistically significant (P = 0.062). CONCLUSION Patients diagnosed with (B)RPC proceeding with CRT have different odds of proceeding to surgery based on their response to CRT and extent of vessel involvement. Post-CRT CA19-9 can help predict which patients may proceed to resection. Future investigations will develop nomograms for estimating odds of proceeding to surgery in larger datasets to help aid in patient counseling and multidisciplinary discussion.

Purpose/Objective(s) Radiation therapy (RT) is a critical modality for the treatment of unresectable hepatocellular carcinoma (HCC). Little is known about how the etiology of HCC impacts overall and progression free survival (OS and PFS) in patients treated with definitive RT. We hypothesize that patients treated with definitive RT for HCC secondary to hepatitis B and hepatitis C viruses (viral group) would have favorable OS and PFS when compared to patients with HCC secondary to other etiologies (non-viral group). Materials/Methods Medical records were retrospectively reviewed at a single institution for patients diagnosed with unresectable HCC and treated with definitive RT between 1/2011 and 12/2020. Patients treated with prior surgery or radiofrequency ablation were excluded. Progress notes were reviewed to determine date of last follow-up, progression, and death. Notes were also assessed to determine the etiology of HCC, age at diagnosis, Child-Pugh classification (CPC), AJCC 8th edition T stage, RT strategy, and planning target volume (PTV). Descriptive statistics were quantified for baseline characteristics. Survival curves were estimated via Kaplan-Meier method; statistical difference was determined using the log-rank test. Multivariate (MVA) Cox proportional hazards analysis was conducted to estimate hazard ratios (HR) when controlling for confounding factors. Results Forty-six patients were included in the analysis. 30 (65%) patients were in the viral group, while 16 (35%) patients were in the non-viral group. Median ages at diagnosis for the viral and non-viral groups were 64 (interquartile range (IQR): 60 to 69) and 71 years (IQR: 66.5 to 76.5), respectively (P = 0.014). Treatment with hypofractionated RT (P = 0.559), proton therapy (P = 0.686), performance status (P = 0.222), CPC (P = 0.270), T stage (P = 0.639), and PTV (P = 0.472) were not different between groups. There was a trend toward improved Median OS for the viral group compared to the non-viral group: 17.9 months (95% confidence interval (CI): 9.7 to not-reached (NR)) vs. 8.8 months (95% CI: 6.1 to NR) (P = 0.072). Similarly, PFS was not different between viral and non-viral groups: 9.3 (95% CI: 7.4 to 19.0) vs. 8.0 (95% CI: 4.0 to 10.3), P = 0.314. However, after controlling for confounding factors with MVA Cox proportional hazards models, the viral group had superior OS (HR = 4.20, 95% CI: 1.47 to 12.02, P = 0.008); the viral group also had superior PFS (HR = 2.48, 95% CI: 1.06 to 5.81, P = 0.037). Conclusion Our retrospective review of patients with unresectable HCC treated with definitive RT provides evidence that viral etiologies of HCC may fare better in OS and PFS when compared to non-viral etiologies. The etiology of HCC development for virally vs non-virally mediated tumors may have implications for treatment and response in the setting of radiation therapy.

The immune tumor microenvironment (TME) of colorectal cancer (CRC) is a crucial contributor to disease biology, making it an important target for therapeutic intervention. The diversity of immune cell populations within various subsets of CRC has led to the discovery that immune characterization of the TME has both prognostic and predictive value for patients. The convergence of improved molecular and cellular characterization of CRC along with the widespread use of immunotherapy in solid tumors has led to a revolution in the approach to clinical care. Monoclonal antibodies (mAbs) which target key immune checkpoints, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), have demonstrated remarkable clinical activity in microsatellite instability-high (MSI-H) CRCs and are now used in routine practice. The observation that MSI-H cancers are highly infiltrated with immune cells and carry a high neoantigen load led to the successful targeting of these cancers with immunotherapy. More recently, the Food and Drug Administration (FDA) approved a PD-1 inhibitor for microsatellite stable (MSS) cancers with high tumor mutation burden. However, the anti-tumor activity of immunotherapy is rare in the majority of CRC. While immune cell characterization does provide prognostic value in these patients, these observations have not yet led to therapeutic interventions. By delineating factors that predict efficacy, resistance, and therapeutic targets, ongoing research will inform the development of effective combination strategies for the vast majority of MSS CRC and immunotherapy-resistant MSI-H cancers.

Background CRC remains the 2nd most common cause of cancer-related death in the US. Hepatic metastases develop in 20–50% of CRC patients.1 Median overall survival (OS) of patients with metastatic CRC is poor, even with the advent of biologics. A high density of CRC-infiltrating effector cytotoxic T lymphocytes (Teff; CTL) is known to predict long-term outcomes and the responsiveness of tumors to immune checkpoint inhibitors (ICI). In our ex vivo tumor explant models and CRC-bearing experimental animals, the combination of toll-like receptor-3 (TLR3) ligands with interferon (IFN)-α with cyclooxygenase (COX)-2 inhibitors resulted in increased production of Teff attracting chemokines CXCL10 and CCL5, along with suppression of regulatory T cells (Treg) attracting chemokine, CCL22 in the tumor microenvironment.2,3 A combination of all three factors was needed to uniformly elevate the desirable chemokines and counteract CCL22 induction. Based on these studies and on prior clinical safety data, we developed this phase IIa study combining IFNα2b, celecoxib (COX-2 inhibitor) and rintatolimod (selective TLR3 agonist) as a chemokine-modulating (CKM) regimen for CRC patients with unresectable liver-metastatic disease. We aim to study the immunological impact, potential clinical efficacy and safety of this CKM regimen in a non-randomized, single-arm prospective phase II trial. Methods Eligible patients have recurrent/metastatic unresectable CRC with hepatic metastases that are amenable to biopsy. Enrolled patients have prior treatment with or contra-indication to a fluoropyrimidine, irinotecan, oxaliplatin, anti-VEGF treatment, and an anti-EGFR targeted therapy (if RAS wt), as well as a PD-1 or PD-L1 targeted drug if MSI-H/dMMR. Patients receive celecoxib (200 mg orally PO BID), IFNα2b IV (20 million units/m2 IV QD), and rintatolimod (200 mg IV QD) on days 1, 2, 3, 8, 9, 10, 15, 16 and 17 in the absence of disease progression or unacceptable toxicity. Response assessment via liver biopsies (pre-treatment and on D20) and CT imaging (RECIST v1.1) on D46. If stable disease/response is demonstrated during repeat CT imaging, patients will continue to follow-up with CT imaging q8 weeks until progression, clinical deterioration, or withdrawal from the study. Primary endpoint assessment compares the change in CD8+ T-cells before treatment, with that seen post-treatment (measured by quantitative RT-PCR and expressed as a ratio of CD8α to a housekeeping gene). Secondary endpoints include objective response rate and safety profile. Subjects are monitored continuously for safety, based on Bayesian analysis. Exploratory endpoints include progression-free survival and overall survival. With a sample size of n=12 evaluable pts, the study design has a 90% power to detect a 0.77 standard deviation increase (pre- to post treatment) at a significance level of 0.1. Results N/A Conclusions N/A Trial Registration ClinicalTrials. gov Identifier: NCT03403634. Ethics Approval The study was approved by Roswell Park Comprehensive Cancer Center‘s Institutional Review Board, approval number: MOD00006722/I-52917. Consent N/A References Lahr C.J, et al., A multifactorial analysis of prognostic factors in patients with liver metastases from colorectal carcinoma. J Clin Oncol, 1983. 1(11): p. 720–6. Muthuswamy R, et al. NF-κB hyperactivation in tumor tissues allows tumor-selective reprogramming of the chemokine microenvironment to enhance the recruitment of cytolytic T effector cells. Cancer Res. 2012;72(15):3735–3743. Obermajer N, et al. Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents. Nat Protoc13, 335–357 ( 2018).

BACKGROUND: Adjuvant chemotherapy is a standard treatment option for patients with stage III and high-risk stage II colon cancer. Sex is one of several factors responsible for the wide inter-patient variability in drug responses. Amalgamated data on the effect of sex on the toxicity of current standard adjuvant treatment for colorectal cancer are missing. METHODS: The objective of our study was to compare incidence and severity of major toxicities of fluoropyrimidine- (5FU or capecitabine) based adjuvant chemotherapy, with or without oxaliplatin, between male and female patients after curative surgery for colon cancer. Adult patients enrolled in 27 relevant randomized trials included in the ACCENT (Adjuvant Colon Cancer End Points) database, a large, multi-group, international data repository containing individual patient data, were included. Comparisons were conducted using logistic regression models (stratified by study and treatment arm) within each type of adjuvant chemotherapy (5FU, FOLFOX, capecitabine, CAPOX, and FOLFIRI). The following major toxicities were compared (grade III or IV and grade I-IV, according to National Cancer Institute Common Terminology Criteria [NCI-CTC] criteria, regardless of attribution): nausea, vomiting, nausea or vomiting, stomatitis, diarrhea, leukopenia, neutropenia, thrombocytopenia, anemia, and neuropathy (in patients treated with oxaliplatin). RESULTS: Data from 34 640 patients were analyzed. Statistically significant and clinically relevant differences in the occurrence of grade III or IV nonhematological {especially nausea (5FU: odds ratio [OR] = 2.33, 95% confidence interval [CI] = 1.90 to 2.87, P < .001; FOLFOX: OR = 2.34, 95% CI = 1.76 to 3.11, P < .001), vomiting (5FU: OR = 2.38, 95% CI = 1.86 to 3.04, P < .001; FOLFOX: OR = 2.00, 95% CI = 1.50 to 2.66, P < .001; CAPOX: OR = 2.32, 95% CI = 1.55 to 3.46, P < .001), and diarrhea (5FU: OR = 1.35, 95% CI = 1.21 to 1.51, P < .001; FOLFOX: OR = 1.60, 95% CI = 1.35 to 1.90, P < .001; FOLFIRI: OR = 1.57, 95% CI = 1.25 to 1.97, P < .001)} as well as hematological toxicities (neutropenia [5FU: OR = 1.55, 95% CI = 1.37 to 1.76, P < .001; FOLFOX: OR = 1.96, 95% CI = 1.71 to 2.25, P < .001; FOLFIRI: OR = 2.01, 95% CI = 1.66 to 2.43, P < .001; capecitabine: OR = 4.07, 95% CI = 1.84 to 8.99, P < .001] and leukopenia [5FU: OR = 1.74, 95% CI = 1.40 to 2.17, P < .001; FOLFIRI: OR = 1.75, 95% CI = 1.28 to 2.40, P < .001]) were observed, with women being consistently at increased risk. CONCLUSIONS: Our analysis confirms that women with colon cancer receiving adjuvant fluoropyrimidine-based chemotherapy are at increased risk of toxicity. Given the known sex differences in fluoropyrimidine pharmacokinetics, sex-specific dosing of fluoropyrimidines warrants further investigation.

Cotargeting vascular endothelial growth factor and programmed cell death 1 or programmed cell death ligand 1 may produce anticancer activity in refractory metastatic colorectal cancer (mCRC). The clinical benefit of atezolizumab combined with chemotherapy and bevacizumab remains unclear for the treatment of mCRC.To assess whether the addition of atezolizumab to capecitabine and bevacizumab therapy improves progression-free survival (PFS) among patients with refractory mCRC and to perform exploratory analyses among patients with microsatellite-stable (MSS) disease and liver metastasis.This double-blind phase 2 randomized clinical trial enrolled 133 patients between September 25, 2017, and June 28, 2018 (median duration of follow-up for PFS, 20.9 months), with data cutoff on May 4, 2020. The study was conducted at multiple centers through the Academic and Community Cancer Research United network. Adult patients with mCRC who experienced disease progression while receiving fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and anti-epidermal growth factor receptor antibody therapy (if the patient had a RAS wild-type tumor) were included.Patients were randomized (2:1) to receive capecitabine (850 or 1000 mg/m2) twice daily on days 1 to 14 and bevacizumab (7.5 mg/kg) on day 1 plus either atezolizumab (1200 mg; investigational group) or placebo (placebo group) on day 1 of each 21-day cycle.The primary end point was PFS; 110 events were required to detect a hazard ratio (HR) of 0.65 with 80% power (1-sided α = .10). Secondary end points were objective response rate, overall survival (OS), and toxic effects.Of 133 randomized patients, 128 individuals (median age, 58.0 years [IQR, 51.0-65.0 years]; 77 men [60.2%]) were assessed for efficacy (82 in the investigational group and 46 in the placebo group). Overall, 15 patients (11.7%) self-identified as African American or Black, 8 (6.3%) as Asian, 1 (0.8%) as Pacific Islander, 101 (78.9%) as White, 1 (0.8%) as multiple races (Asian, Native Hawaiian/Pacific Islander, and White), and 2 (1.6%) as unknown race or unsure of race. Microsatellite-stable disease was present in 110 patients (69 in the investigational group and 41 in the placebo group). Median PFS was 4.4 months (95% CI, 4.1-6.4 months) in the investigational group and 3.6 months (95% CI, 2.2-6.2 months) in the placebo group (1-sided log-rank P = .07, a statistically significant result; HR, 0.75; 95% CI, 0.52-1.09). Among patients with MSS and proficient mismatch repair, the HR for PFS was 0.66 (95% CI, 0.44-0.99). The most common grade 3 or higher treatment-related adverse events in the investigational vs placebo groups were hypertension (6 patients [7.0%] vs 2 patients [4.3%]), diarrhea (6 patients [7.0%] vs 2 patients [4.3%]), and hand-foot syndrome (6 patients [7.0%] vs 2 patients [4.3%]). One treatment-related death occurred in the investigational group. In the investigational group, the response rate was higher among patients without liver metastasis (3 of 13 individuals [23.1%]) vs with liver metastasis (4 of 69 individuals [5.8%]). The benefit of atezolizumab for PFS and OS was greater among patients without vs with liver metastasis (primary analysis of PFS: HR, 0.63 [95% CI, 0.27-1.47] vs 0.77 [95% CI, 0.51-1.17]; OS: HR, 0.33 [95% CI, 0.11-1.02] vs 1.14 [95% CI, 0.72-1.81]).In this randomized clinical trial, the addition of atezolizumab to capecitabine and bevacizumab therapy provided limited (ie, not clinically meaningful) clinical benefit. Patients with MSS and proficient mismatch repair tumors and those without liver metastasis benefited more from dual inhibition of the vascular endothelial growth factor and programmed cell death 1 or programmed cell death ligand 1 pathways.ClinicalTrials.gov Identifier: NCT02873195.

TPS259 Background: Detection of circulating tumor DNA (ctDNA) shed into the bloodstream represents a highly specific and sensitive approach for identifying microscopic or residual tumor cells after surgical resection. For patients (pts) with colon cancer (CC), the detection of ctDNA is associated with persistent disease after resection and outperforms traditional clinical and pathological features in prognosticating risk for recurrence. However, for pts with stage II CC, there are currently no validated biomarkers predicting benefit in identifying pts whose residual disease cancer be cleared by adjuvant chemotherapy. We hypothesize that for pts whose stage II colon cancer has been resected and who have no traditional high-risk features, a positive ctDNA status may identify those who will benefit from adjuvant chemotherapy. Methods: In this prospective phase II/III clinical trial, pts (N=1,408) with resected stage II CC without traditional high-risk features and whom the evaluating oncologist deems suitable for active surveillance (i.e., not needing adjuvant chemotherapy) will be randomized 1:1 into 2 arms: standard-of-care/observation (Arm A), or prospective testing for ctDNA (Arm B). Postoperative blood will be analyzed for ctDNA with the Guardant Reveal assay, covering CC-relevant mutations and CC-specific methylation profiling. Pts in Arm B with ctDNA detected will be treated with 6 months of adjuvant (FOLFOX) chemotherapy. For all pts in Arm A, ctDNA status will be analyzed retrospectively at the time of endpoint analysis. The primary endpoints are clearance of ctDNA with adjuvant chemotherapy (phase II) and recurrence-free survival (RFS) for “ctDNA-detected” pts treated with or without adjuvant chemotherapy (phase III). Secondary endpoints will include time-to-event outcomes (OS, RFS, TTR) by ctDNA marker status and treatment, prevalence of detectable ctDNA in stage II CC, and rates of compliance with assigned intervention. Archived normal and matched tumor and blood samples will be collected for exploratory correlative research. Enrollment continues across North America to the 540-patient phase II endpoint. Support: U10CA180868, -180822; UG1CA189867; GuardantHealth. Clinical trial information: NCT04068103 .

PURPOSE/OBJECTIVE(S) Neoadjuvant chemoradiotherapy is a critical treatment for borderline resectable and resectable pancreatic adenocarcinoma ((B)RPC). The PREOPANC trials employ a short course chemoradiation (SCCRT) regimen of 3600 cGy in 15 fractions with concurrent gemcitabine (GEM)-based chemotherapy, but little is known about the relative efficacy of capecitabine (CAPE)-based concurrent chemotherapy in this population. We hypothesize that patients treated with SCCRT with CAPE-based concurrent chemotherapy will have superior overall survival (OS) compared to GEM-based strategies. MATERIALS/METHODS Medical records were retrospectively reviewed at a single center for patients diagnosed with (B)RPC between 1/2005 and 12/2020. Patients treated with neoadjuvant SCCRT were included in the analysis. Descriptive statistics were quantified for baseline characteristics. OS was estimated using Kaplan-Meier estimates, statistical difference was determined using the log-rank test. Multivariate Cox proportional hazards analysis was conducted to estimate hazard ratios (HR) when controlling for confounding factors. RESULTS Thirty-one (n = 31) patients were included in the analysis. 71% of patients (n = 22) were treated with CAPE-based concurrent chemotherapy, while 29% of patients (n = 9) were treated with GEM-based concurrent chemotherapy. Median age at diagnosis was 65 (interquartile range (IQR): 60 to 71) for the CAPE group, compared to 66 (IQR: 63 to 71) for the GEM group, P = 0.414. 100% of patients in the CAPE group were borderline resectable, compared to 82% of patients in the GEM group, P = 0.171. 44% of patients in the CAPE group were treated with neoadjuvant FOLFIRINOX, compared to 14% in the GEM group, P = 0.016. Patients treated with concurrent 5-CAPE-based chemotherapy had a longer median OS than other concurrent chemotherapy approaches: Not reached (95% confidence interval (CI): 24 to not reached) vs. 17 months (95% CI: 6 to 22 months), P < 0.0001. Multivariate Cox proportional hazards models, controlling for borderline vs. resectable status, age at diagnosis, and use of neoadjuvant FOLFIRINOX, demonstrated that CAPE-based concurrent chemotherapy had a HR of death of 0.081 (95% CI: 0.018 to 0.369, P = 0.0012) compared to GEM-based concurrent chemotherapy. CONCLUSION Patients diagnosed with (B)RPC have many therapeutic options; this study suggests a benefit to CAPE-based concurrent chemotherapy when treating patients with SCCRT, even after controlling for significant confounding by greater use of neoadjuvant FOLFIRINOX with CAPE-based concurrent chemotherapy. SCCRT may be more effective with concurrent capecitabine instead of concurrent gemcitabine for (B)RPC.

Purpose/Objective(s) Radiation therapy (RT) remains a critical treatment modality for unresectable hepatocellular carcinoma (HCC), with proton beam therapy (PBT) being widely utilized due to its decreased exit dose compared to photon-based RT. We hypothesize that patients with HCC receiving definitive PBT would experience decreased acute toxicity when compared to those receiving photon-based RT. Materials/Methods Electronic medical records were retrospectively reviewed at a single institution for patients diagnosed with unresectable HCC and treated with definitive RT between 1/2011 and 12/2020. Patients treated with prior surgery or radiofrequency ablation were excluded. Progress notes and treatment summaries were reviewed to determine baseline characteristics including age at diagnosis, Child-Pugh classification (CPC), AJCC 8th edition T stage, performance status, and RT dose. Acute treatment toxicities were evaluated using Common Terminology Criteria for Adverse Events v5.0. Descriptive statistics were quantified for baseline characteristics. Odds ratios (OR) with 95% confidence intervals (CI) were generated to determine the association between PBT vs. photon therapy and acute radiation toxicities in bivariate and multivariate logistic regression models. Results Forty-six patients were included in the analysis. 24 (52%) patients received PBT while 22 (48%) received photon-based RT. Median age for the PBT and photon-based RT groups were 67 (interquartile range (IQR): 58.6 to 72.5) and 65.5 years (IQR: 61.0 to 73.0), respectively (P = 0.991). 50% of patients who received PBT had a T stage greater than T2, compared to 71% of patient in the photon group (P = 0.148). 42% of the PBT group had CPC B or worse, compared to 38% in the photon group (P = 0.813). Performance status and total dose did not significantly differ between groups (P = 0.351 and P = 0.773, respectively). PBT was associated with higher odds of any grade radiation dermatitis (RD) (OR = 6.00, 95% CI: 1.13 to 31.9) and lower odds of any grade anorexia (OR = 0.21, 95% CI 0.05 to 0.91). Odds of any grade diarrhea and fatigue, did not significantly differ between groups (P = 0.086 and P = 0.958, respectively). After controlling for performance status, total dose, T stage, CPC, and age at diagnosis, PBT was still associated with higher odds of RD (OR = 11.07, 95% CI: 1.14 to 107.71) and lower odds of anorexia (OR = 0.14, 95% CI: 0.02 to 0.93). Conclusion This study provides evidence that although PBT appears to have a superior safety profile in regard to acute anorexia, patients treated with PBT have higher odds of acute RD; these effects persist even after controlling for relevant confounding factors. PBT may result in fewer GI toxicities at the cost of greater skin toxicity.

Objectives: Preoperative chemotherapy and radiation for localized esophageal cancer produces cure rates near 30% when combined with surgical resection. Vandetanib, a small molecule receptor tyrosine kinase inhibitor of VEGFR-2, VEGFR-3, RET, and EGFR, demonstrated synergy with radiation and chemotherapy in preclinical models. We conducted a phase I study to assess the safety and tolerability of vandetanib when combined with preoperative chemoradiation in patients with localized esophageal carcinoma who were surgical candidates. Methods: Patients with stage II-III esophageal and gastroesophageal junction carcinoma without prior therapy were enrolled in a 3+3 phase I design. Patients received once-daily vandetanib (planned dosing levels of 100, 200, and 300 mg) with concomitant daily radiotherapy (1.8 Gy/d, 45 Gy total) and chemotherapy, consisting of infusional 5-FU (225 mg/m2/d over 96 h, weekly), paclitaxel (50 mg/m2, days 1, 8, 15, 22, 29) and carboplatin (AUC of 5, days 1, 29). Results: A total 9 patients were enrolled with 8 having either distal esophageal or gastroesophageal junction carcinomas. All patients completed the planned preoperative chemoradiation and underwent esophagectomy. Nausea (44%) and anorexia (44%) were the most common acute toxicities of any grade. One grade 4 nonhematologic toxicity was observed (gastrobronchial fistula). One additional patient suffered a late complication, a fatal aortoenteric hemorrhage, not definitively related to the investigational regimen. Five (56%) patients achieved a pathologic complete response. Three (33%) additional patients had only microscopic residual disease. Five (56%) patients remain alive and disease free with a median follow-up of 3.7 years and median overall survival of 3.2 years. The maximum tolerated dose was vandetanib 100 mg/d. Conclusions: Vandetanib at 100 mg daily is tolerable in combination with preoperative chemotherapy (5-FU, paclitaxel, carboplatin) and radiation therapy with encouraging efficacy worthy of future study.

TPS4121 Background: There are currently no validated predictive biomarkers for stage II resected colon cancer (CC) after adjuvant chemotherapy. However, circulating tumor DNA (ctDNA) that is shed into the bloodstream represents a highly specific and sensitive approach for identifying microscopic or residual tumor cells. For patients (pts) with CC, the detection of ctDNA is associated with persistent disease after resection and may outperform traditional clinical and pathological features as a prognostic factor to assess risk for recurrence. We hypothesize that for pts whose stage II colon cancer has been resected and who have no traditional high-risk features, a positive ctDNA status may identify those who will benefit from adjuvant chemotherapy. Methods: In this prospective phase II/III clinical trial, pts (N=1,408) with resected stage II CC without traditional high-risk features and whom the evaluating oncologist deems suitable for no adjuvant chemotherapy will be randomized 1:1 into 2 arms: standard-of-care/observation (Arm A), or prospective testing for ctDNA (Arm B). Postoperative blood will be analyzed for ctDNA with the GuardantHealth LUNAR panel, covering CC-relevant mutations and CC-specific methylation profiling. Pts in Arm B with ctDNA detected will be treated with 6 months of adjuvant (FOLFOX) chemotherapy. For all pts in Arm A, ctDNA status will be analyzed retrospectively at the time of endpoint analysis. The primary endpoints are clearance of ctDNA with adjuvant chemotherapy (phase II) and recurrence-free survival (RFS) for “ctDNA-detected” pts treated with or without adjuvant chemotherapy (phase III). Secondary endpoints will include time-to-event outcomes (OS, RFS, TTR) by ctDNA marker status and treatment, prevalence of detectable ctDNA in stage II CC, and rates of compliance with assigned intervention. Archived normal and matched tumor and blood samples will be collected for exploratory correlative research. The trial is actively accruing towards the phase II endpoint across all US and Canadian cooperative groups. Support: U10-CA-180868, -180822; UG1CA-189867; GuardantHealth. Clinical trial information: NCT04068103 .

3040 Background: Pembrolizumab was recently FDA approved across solid tumors for TMB scores ≥ 10mut/Mb as assessed by next-generation sequencing (NGS) of tissue (tTMB). A prior study of advanced cancer patients treated with immunotherapy found that higher somatic TMB, as defined by the 80th percentile in each histology, was associated with better overall survival. Previously, bTMB assessed by ctDNA from patients with newly diagnosed advanced NSCLC at a score of 16 mut/MB correlated with a tTMB score of 10 mut/MB. TMB levels vary by cancer type, line of treatment, and therapy received; the distribution of bTMB scores across solid tumor types has not been well characterized. Here we report the distribution of bTMB scores in patients with advanced malignancies. Methods: We queried 5,610 samples from patients with different cancer types undergoing clinical cell-free DNA testing (Guardant360; Redwood City, CA) and assessed bTMB scores from October 2020 - January 2021. bTMB score was derived via a previously described computational algorithm examining the total number of synonymous and non-synonymous SNVs and indels across a 1.0MB genomic footprint. We assessed the success rate of bTMB evaluation, overlap with microsatellite instability (MSI) status, and defined the distribution of bTMB levels across indications in this dataset. Results: bTMB score was successfully assessed in 4,275/5,610 (76.3%) samples (Table). The majority of samples (58%) were tested at disease progression as compared to initial diagnosis (42%). The median turnaround time from sample receipt to clinical reporting was 11 days and decreased to 9 days over the course of the study. For the majority of cancer types the 80th percentile TMB was ≥ 16 mut/MB tissue equivalency. Conclusions: Our analysis demonstrates the feasibility of measuring bTMB using a commercially available liquid biopsy assay. bTMB scores trended higher than tTMB previously reported in these cancer types, reflecting the ability of ctDNA to better capture tumor heterogeneity. cfDNA may allow for exploration of bTMB evolution throughout treatment. TMB should be interpreted in the context of disease, treatment, and method; these data establish a pan-cancer benchmark for bTMB which will serve as a resource for further studies.[Table: see text]

124 Background: Microsatellite stable (MSS) colorectal cancer and CRCLM are highly resistant to current immunotherapeutic approaches. Understanding the TME and the impact of standard chemotherapeutics is of utmost importance in developing optimal therapeutic strategies. Data is limited on the impact of NACT in the TME. In this study, we sought to explore the effects of chemotherapy on the proportions of different immune cell populations [Total leukohematopoetic cells, CD45+, T cell markers, CD3+, CD8+, and macrophage (M) markers, CD163+, CD68+)], as well as to assess their prognostic capacity. Methods: CRCLM pts who underwent liver resection were divided in 2 groups: pts who had NACT within 4 months of liver resection [NACT (+)] and no NACT [NACT (-)]. Whole slide staining of metastatectomy specimen for CD3, CD8, CD68, CD163, and CD45 was done. Cellular density within representative tumor core sections was analyzed. Retrospective chart review was done to obtain clinical data. Disease free (DFS), relapse free (RFS), and overall survival (OS) were analyzed using Kaplan Meir methods. Association between markers and outcomes was evaluated using Cox Regression Models. Results: A total of 43 pts were studied [NACT (+), n= 14; NACT (-), n=29). There were no significant differences in baseline characteristics between the two groups, including age, primary site, T stage, N stage, and grade. Median OS was 48 months. Outcomes were not significantly different with use of NACT. Within the NACT (+) CRCLM significant increases were seen in densities of CD3, CD8, CD45 and CD163 cells. Densities of CD8 cells were strongly correlated to CD163 cell densities ([r] 0.77, p < .0001). The NACT (+) cohort saw significantly increased ratios of T-cells/macrophages as compared to NACT (-) (CD3/CD68, CD3/CD163, CD8/CD163). High CD45 density was associated with improved OS (HR = 0.28, p = 0.006). CD3, CD8, CD68, and CD163 were not independently associated with OS. However, both the ratio of CD3/CD68 and CD3/163 were associated with improved OS (HR 0.44, p=0.03 and HR 0.27, p=0.03 respectively). Conversely, CD68/45 and CD163/CD45 ratios were associated with a worse OS (HR 1.42, p=0.05 and HR 1.43, p=0.03 respectively). No difference was seen in cellular populations based on the type of NACT received: oxaliplatin vs irinotecan. Conclusions: Our study highlights that the administration of NACT is associated with favorable changes in LM TME. While there is a general increase in leukocytes (CD45), the density of CD3+ and CD8+ T cells is particularly increased. Further, this increase is proportionately greater than the concurrent increases on monocytoid populations, reflected by increased CD3/CD163 and CD8/CD163 ratios. Ratios of T-cells/ macrophages are predictive of survival. This study was partly supported by the American Cancer Society Grant, 126771-IRG-14-194-11-IRG.

Immunotherapy targeting immune checkpoints has become a common approach for many types of cancer but currently there are no reliable biomarkers to predict and monitor treatment response. Immunohistochemical staining of PD-L1, which was initially developed as a companion diagnostic, is problematic due to tumor heterogeneity, variations in specimen preparation and multiple scoring schemes. There is, therefore, a critical need for a better biomarker to guide the use of immunotherapy to improve clinical outcomes. We previously reported that higher levels of Bim and CX3CR1/granzyme B in CD8+ T cells were associated with increased response to PD-1 blockade therapy in patients with melanoma; high levels of Bim were also prognostic of poor survival in melanoma patients. In this study, we investigated the roles of Bim and CX3CR1/granzyme B in patients receiving PD-1 blockade therapy (i.e. pembrolizumab) for small bowel adenocarcinoma as part of planned correlative analysis of the ACCRU clinical trial, NCT02949219. Patients with unresectable or metastatic biopsy-proven small bowel adenocarcinoma (excluding ampulla of Vater and appendix) who had at least one prior line of systemic chemotherapy were eligible for the trial. Pembrolizumab (200 mg) was administered over 30 minutes intravenously every 3 weeks until unacceptable toxicity, disease progression, or patient refusal. All patients underwent peripheral blood collection at baseline prior to initiation of treatment and then after 3 cycles of treatment. Levels of Bim and CX3CR1/granzyme B in circulating T cells were quantified by flow cytometry using patients' peripheral blood mononuclear cells. Data was analyzed via Cox proportional hazards model, Wilcoxon Rank-Sum test, and Kaplan Meier curves using SAS 9.4. A total of 35 eligible patients were included in the analysis. Three patients had confirmed response (all partial responses) and 10 had stable disease as their best overall response. Seven patients had disease progression around the second blood draw. The median time from baseline to the second blood draw was 9 weeks. Lower levels of Bim in CD8+CD11ahi T cells at baseline on the percentage scale were associated with treatment response (median 54.5% vs. 79.5%; p=0.0087) and disease control (partial response plus stable disease; median 71.2% vs. 81.3%; p=0.0104) but higher levels led to worse progression-free survival (hazard ratio=1.05, 95% confidence interval 1.01-1.08; p=0.0051). In addition, positive changes in CX3CR1/granzyme B in CD8+CD11ahi T cells from baseline were associated with better overall survival (median 20.2 months vs. 3.7 months, p=0.0086). The levels of PD-1 and Ki-67 in CD8+CD11ahi T cells were not associated with treatment response or survival. In conclusion, Bim and CX3CR1/granzyme B are biomarkers that associate with response and survival benefits respectively of PD-1 blockade therapy. This preliminary data calls for a larger-scale, prospective study to validate their predictive and prognostic utilities. Citation Format: Mojun Zhu, Henan Zhang, Nathan R. Foster, Haidong Dong, Tanios S. Bekaii-Saab, Brandy L. Jaszewski, Patrick M. Boland, Michael J. Overman, Katrina Pedersen, Robert R. McWilliams, Academic and Community Cancer Research United (AACRU). Bim and CX3CR1/granzyme B in circulating CD8+ T cells are predictive biomarkers for PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4467.

4032 Background: Pembrolizumab provides effective antitumor immunity and durable responses in patients (pts) with advanced, colorectal cancer (CRC) with microsatellite instability-high (MSI-H) tumors. We present data on antitumor immunity with pembrolizumab in pts from the phase 2, KEYNOTE-164 study who had approximately 3 years of follow-up, and in pts re-treated after disease progression. Methods: KEYNOTE-164 enrolled pts with metastatic MSI-H CRC, MSI-H status confirmed locally by IHC or PCR, and ≥2 (cohort A) or ≥1 (cohort B) prior lines of therapy (fluoropyrimidine, oxaliplatin, irinotecan, or anti VEGF/EGFR). Eligible pts received pembrolizumab 200 mg Q3W for 2y (35 administrations) or until progression, unacceptable toxicity, or withdrawal. Pts who stopped pembro due to a confirmed CR or after completing 2y of treatment and who progressed after stopping were eligible for re-treatment with up to 17 administrations in the second-course phase, at investigator discretion. Tumor response was assessed Q9W per RECIST v1.1 by independent review. The primary endpoint was ORR. Secondary endpoints included DOR, PFS, OS, and safety. The data cutoff date was Sep 9, 2019. Results: At data cutoff, the median follow-up was 31.4 mo (range, 0.2-47.8) for 61 pts in cohort A and 36.1 mo (0.1-39.3) for 63 pts in cohort B. ORR was 32.8% (3CR, 17PR; 95% CI% 21.3-46.0) for cohort A and 34.9% (8CR, 14PR; 95% CI 23.3-48.0) in cohort B. Median DOR was not reached (NR [range, 6.2-41.3+]) and not reached (range, 3.9+ to 37.1+), respectively. Fifteen pts in cohort A and 17 in cohort B had ongoing responses at data cutoff. Median PFS was 2.3 mo (95% CI 2.1-8.1) with 3-yr PFS rate of 31% in cohort A and was 4.1 mo (2.1-18.9) with 3-yr PFS rate of 34% in cohort B. Median OS was 31.4 mo (21.4-NR) with 3-yr OS rate of 49% in cohort A and was not reached (19.2-NR) with 3-yr OS rate of 52% in cohort B. Nine pts (6 in cohort A, 3 in cohort B) had a second course of treatment. The best response in second course was PR in 1 patient each in cohort A and B. Grade 3-4 drug-related adverse events occurred in 10 (16%) pts in cohort A and 8 (13%) pts in cohort B. No grade 5 drug-related events occurred. Conclusions: After approximately 3 y of follow-up, pembrolizumab continues to provide effective long-term antitumor immunity with durable responses, with small numbers of drug-related adverse events and no drug-related deaths in pts with advanced, MSI-H CRC. Clinical trial information: NCT02460198 .