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Accurately modeling tumor biology and testing novel therapies on patient-derived cells is critically important to developing therapeutic regimens personalized to a patient's specific disease. The vascularized micro-tumor (VMT), or "tumor-on-a-chip", is a physiologic preclinical cancer model that incorporates key features of the native human tumor microenvironment within a transparent microfluidic platform, allowing rapid drug screening in vitro. Herein we optimize methods for generating patient-derived VMT (pVMT) using fresh colorectal cancer (CRC) biopsies and surgical resections to test drug sensitivities at the individual patient level. In response to standard chemotherapy and TGF-βR1 inhibition, we observe heterogeneous responses between pVMT derived from 6 patient biopsies, with the pVMT recapitulating tumor growth, histological features, metabolic heterogeneity and drug responses of actual CRC tumors. Our results suggest that a translational infrastructure providing rapid information from patient-derived tumor cells in the pVMT, as established in this study, will support efforts to improve patient outcomes.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

PURPOSE Subset analyses from phase III evaluation of epidermal growth factor receptor inhibition (EGFRi) suggest improved outcomes in patients with EGFR-amplified gastroesophageal adenocarcinoma (GEA), but large-scale analyses are lacking. This multi-institutional analysis sought to determine the role of EGFRi in the largest cohort of patients with EGFR-amplified GEA to date. PATIENTS AND METHODS A total of 60 patients from 15 tertiary cancer centers in six countries met the inclusion criteria. These criteria required histologically confirmed GEA in the metastatic or unresectable setting with EGFR amplification identified by using a Clinical Laboratory Improvement Amendments–approved assay, and who received on- or off-protocol EGFRi. Testing could be by tissue next-generation sequencing, plasma circulating tumor DNA next-generation sequencing, and/or fluorescence in situ hybridization performed by a Clinical Laboratory Improvement Amendments approved laboratory. Treatment patterns and outcomes analysis was also performed using a deidentified clinicogenomic database (CGDB). RESULTS Sixty patients with EGFR-amplified GEA received EGFRi, including 31 of 60 patients (52%) with concurrent chemotherapy. Across treatment lines, patients achieved a 43% objective response rate with a median progression-free survival of 4.6 months (95% CI, 3.5 to 6.4). Patients receiving EGFRi in first-, second-, and third-line therapy achieved a median overall survival of 20.6 months (95% CI, 13.5 to not reached [NR]), 9 months (95% CI, 7.9 to NR), and 8.4 months (7.6 to NR), respectively. This survival far exceeded the 11.2-month (95% CI, 8.7 to 14.2) median overall survival from first-line initiation of non-EGFRi therapy in patients with EGFR-amplified GEA in the CGDB. Despite this benefit, analysis of the CGDB (January 2011-December 2020) suggests that only 5% of patients with EGFR-amplified GEA received EGFRi. CONCLUSION Patients with EGFR-amplified GEA derive significant benefit from EGFRi. Further prospective investigation of EGFRi in a well-selected patient population is ongoing in an upcoming trial of amivantamab in EGFR and/or MET amplified GEA.

Patients with advanced gastroesophageal cancer (mEG) and tumor mutational burden ≥10 mut/Mb (TMB ≥ 10) have more favorable outcomes on immune checkpoint inhibitor (ICPI) monotherapy compared with chemotherapy in subgroup analyses of randomized controlled trials. We sought to evaluate the robustness of these associations in real-world settings where patients and practices are more diverse. A total of 362 2 L and 692 1 L patients, respectively received ICPI (n = 99, 33) or chemotherapy (n = 263, 659) across approximately 280 U.S. academic or community-based cancer clinics March 2014–July 2021. Deidentified data were captured into a real-world clinico-genomic database. All patients underwent Foundation Medicine testing. Time to next treatment (TTNT) and overall survival (OS) comparing ICPI versus chemotherapy were adjusted for treatment assignment imbalances using propensity scores. 2L: TMB ≥ 10 had more favorable TTNT [median 24 vs. 4.1 months; HR: 0.19; 95% confidence interval (CI): 0.09–0.44; P = 0.0001] and OS (median 43.1 vs. 6.2 months; HR: 0.24; 95% CI: 0.011–0.54; P = 0.0005), TMB < 10 did not (P > 0.05). 1L: TMB ≥ 10 had more favorable TTNT (not reached vs. median 4.1 months; HR: 0.13; 95% CI: 0.03–0.48; P = 0.0024) and OS (not reached vs. median 17.1 months; HR: 0.30; 95% CI: 0.08–1.14; P = 0.078), TMB < 10 had less favorable TTNT (median 2.8 vs. 6.5 months; HR: 2.36; 95% CI: 1.25–4.45; P = 0.008) and OS (median 4.5 vs. 13.1 months; HR: 1.82, 95% CI: 0.87–3.81; P = 0.11). TMB ≥ 10 robustly identifies patients with mEG with more favorable outcomes on 2 L ICPI monotherapy versus chemotherapy. 1 L data are more limited, but effects are consistent with 2L. Significance: Using real-world data, we sought to evaluate robustness of these clinical associations using the same assay platform and biomarker cut-off point used in both clinical trials and pan-tumor CDx approvals for later treatment lines. TMB ≥ 10 robustly identified patients with mEG with more favorable outcomes on ICPI monotherapy versus chemotherapy and suggests this subset of patients could be targeted for further trial development.

Unadjusted for known treatment assignment imbalances, patients receiving 1st line ICPI vs. chemotherapy have more favorable outcomes when TMB ≥ 10 but not TMB < 10. Kaplan-Meier curves are unadjusted for imbalances (propensity weights applied). TTNT is shown by drug class for (A) TMB < 10, and (B) TMB ≥ 10. OS is shown by drug class for (C) TMB < 10, and (D) TMB ≥ 10. X-axis is truncated at 36 months. Overall survival estimates are left truncated (see Methods) with at-risk tables adjusted accordingly. Interaction models can be found in Supplemental Figure 9.

Summary of TMB ranges per cohort and MSI (TMB10+ only). The median and interquartile range of TMB is shown per group within the cohorts, grouped by MSI status.

Chemotherapy is ubiquitous in first-line treatment of advanced gastric cancer, yet responses are heterogeneous, and little is known about mediators of chemotherapy response. To move forward, an understanding of the effects of standard chemotherapy on the tumor–immune microenvironment (TME) is needed. Coupling whole-exome sequencing, bulk RNA and single-cell transcriptomics from paired pretreatment and on-treatment samples in treatment-naïve patients with HER2-positive and HER2-negative gastric cancer, we define features associated with response to platinum-based chemotherapy. Response was associated with on-treatment TME remodeling including natural killer (NK) cell recruitment, decreased tumor-associated macrophages, M1-macrophage repolarization, and increased effector T-cell infiltration. Among chemotherapy nonresponders, we observed low/absent PD-L1 expression or modulation, on-treatment increases in Wnt signaling, B-cell infiltration, and LAG3-expressing T cells coupled to an exodus of dendritic cells. We did not observe significant genomic changes in early on-treatment sampling. We provide a map of on-treatment TME modulation with standard chemotherapy and nominate candidate future approaches.Significance:Using paired pretreatment and on-treatment samples during standard first-line chemotherapy, we identify chemotherapy-induced NK-cell infiltration, macrophage repolarization, and increased antigen presentation among responders. Increased LAG3 expression and decreased dendritic cell abundance were seen in nonresponders, emphasizing remodeling of the TME during chemotherapy response and resistance.This article is highlighted in the In This Issue feature, p. 873

Supplementary Figure from The Genomics of Colorectal Cancer in Populations with African and European Ancestry

Supplementary Figure from Early Tumor–Immune Microenvironmental Remodeling and Response to First-Line Fluoropyrimidine and Platinum Chemotherapy in Advanced Gastric Cancer

Adding anti-PD1 antibodies to 5-FU/platinum chemotherapy improves survival in a subset of advanced gastroesophageal adenocarcinoma (GEA) patients. Beyond PD-L1 expression and mismatch repair status we have limited insight into molecular predictors of response or the relative contribution of PD-1 blockade. We conducted an investigator sponsored phase II trial (n = 47) sequentially adding pembrolizumab to standard 5-FU/platinum in previously untreated advanced GEA (ClinicalTrials.gov:NCT04249739). With an overall response rate of 67% the activity paralleled phase III chemoimmunotherapy trials. To understand on-treatment tumor and immune adaptations patients underwent serial biopsy of the primary tumor, including baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab. We leveraged transcriptional profiling from 358,067 cells to identify multicellular networks of malignant, stromal, and immune cells after chemotherapy and concurrent chemoimmunotherapy. The relative usage of pro-tumor and anti-tumor interaction hubs differed between fast and slow progressing patients. Chemotherapy induced early on-treatment formation of hubs centered on tumor-reactive T-cell and M1-oriented macrophage interactions with pro-inflammatory cytokines in slow progressors. Faster progression was characterized by increased MUC5A and MSLN containing programs in tumor cells and M2-oriented macrophages with immunosuppressive stromal interactions. After adding pembrolizumab we observed increased CD8 T-cell infiltration by scRNAseq and multiplex immunofluorescence and development of an immunity hub involving co-variation of the tumor-reactive CXCL13 program and epithelial interferon-stimulated gene programs enriched in slow progressors. Together this data provides prospective evidence of differential early on-treatment evolution of the gastric immune microenvironment and nominates candidate cellular interactions for clinical targeting.

Black people have a higher incidence of colorectal cancer and worse survival rates when compared with white people. Comprehensive genomic profiling was performed in 46,140 colorectal adenocarcinoma cases. Ancestry-informative markers identified 5,301 patients of African descent (AFR) and 33,770 patients of European descent (EUR). AFR were younger, had fewer microsatellite instability–high (MSI-H) tumors, and had significantly more frequent alterations in KRAS, APC, and PIK3CA. AFR had increased frequency of KRAS mutations, specifically KRASG12D and KRASG13. There were no differences in rates of actionable kinase driver alterations (HER2, MET, NTRK, ALK, ROS1, and RET). In patients with young-onset colorectal cancer (APC mutations by age, as well as differences in MAPK pathway alterations. These findings inform treatment decisions, impact prognosis, and underscore the need for model systems representative of the diverse U.S. population.Significance:KRAS (particularly KRASG12D/G13), APC, and PIK3CA were more frequently altered in AFR who had a lower frequency of MSI-H tumors. There were no differences in actionable kinase driver alterations. In young-onset colorectal cancer, both ancestries had a similar frequency of MSI-H/TMB-H tumors, but strikingly different trends in APC.See related commentary by Eng and Holowatyj, p. 1187.This article is highlighted in the In This Issue feature, p. 1171

Patients with advanced gastroesophageal cancer (mEG) and tumor mutational burden ≥10 mut/Mb (TMB ≥ 10) have more favorable outcomes on immune checkpoint inhibitor (ICPI) monotherapy compared with chemotherapy in subgroup analyses of randomized controlled trials. We sought to evaluate the robustness of these associations in real-world settings where patients and practices are more diverse. A total of 362 2 L and 692 1 L patients, respectively received ICPI (n = 99, 33) or chemotherapy (n = 263, 659) across approximately 280 U.S. academic or community-based cancer clinics March 2014–July 2021. Deidentified data were captured into a real-world clinico-genomic database. All patients underwent Foundation Medicine testing. Time to next treatment (TTNT) and overall survival (OS) comparing ICPI versus chemotherapy were adjusted for treatment assignment imbalances using propensity scores. 2L: TMB ≥ 10 had more favorable TTNT [median 24 vs. 4.1 months; HR: 0.19; 95% confidence interval (CI): 0.09–0.44; P = 0.0001] and OS (median 43.1 vs. 6.2 months; HR: 0.24; 95% CI: 0.011–0.54; P = 0.0005), TMB < 10 did not (P > 0.05). 1L: TMB ≥ 10 had more favorable TTNT (not reached vs. median 4.1 months; HR: 0.13; 95% CI: 0.03–0.48; P = 0.0024) and OS (not reached vs. median 17.1 months; HR: 0.30; 95% CI: 0.08–1.14; P = 0.078), TMB < 10 had less favorable TTNT (median 2.8 vs. 6.5 months; HR: 2.36; 95% CI: 1.25–4.45; P = 0.008) and OS (median 4.5 vs. 13.1 months; HR: 1.82, 95% CI: 0.87–3.81; P = 0.11). TMB ≥ 10 robustly identifies patients with mEG with more favorable outcomes on 2 L ICPI monotherapy versus chemotherapy. 1 L data are more limited, but effects are consistent with 2L.Significance:Using real-world data, we sought to evaluate robustness of these clinical associations using the same assay platform and biomarker cut-off point used in both clinical trials and pan-tumor CDx approvals for later treatment lines. TMB ≥ 10 robustly identified patients with mEG with more favorable outcomes on ICPI monotherapy versus chemotherapy and suggests this subset of patients could be targeted for further trial development.

Supplementary Table from Early Tumor–Immune Microenvironmental Remodeling and Response to First-Line Fluoropyrimidine and Platinum Chemotherapy in Advanced Gastric Cancer

Supplementary Tables S1-S7 Supplementary Figure S1

Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRASG12C-mutant cancers, including non–small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRASG12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS–MAPK signaling. Notably, a novel KRASY96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein–drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance.Significance:In one of the first reports of clinical acquired resistance to KRASG12C inhibitors, our data suggest polyclonal RAS–MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRASG12C inhibitor.See related commentary by Pinnelli and Trusolino, p. 1874.This article is highlighted in the In This Issue feature, p. 1861

Gastric carcinoma (GC) disproportionately affects Asian Americans. We examined whether history of upper gastrointestinal (GI) endoscopy was associated with lower stage at GC diagnosis among Asian Americans and whether origin of providers influenced referral for endoscopy. We employed Surveillance Epidemiology and End Results–Medicare data on Asian Americans diagnosed with GC in 2004–2013 (n = 1,554). Stage distribution, GI conditions at diagnosis, and history of endoscopy were compared between Asian ethnic groups. Multivariate logistic regression adjusting for age, sex, poverty level, tumor location, and histology was used to examine the association of ethnicity and endoscopic history with stage I disease at diagnosis of GC. Koreans were more likely to be diagnosed with stage I, T1a GC and have prior history of endoscopy, compared with other Asian ethnicities (24% vs. 8% for stage I, T1a; 40% vs. 15% for endoscopy). Patients with primary care providers of concordant ethnic origin were more likely to have history of endoscopy. Asian American patients with GC with history of endoscopy were more likely to be diagnosed with GC at stage I disease (adjusted OR, 3.07; 95% confidence interval, 2.34–4.02). Compared with other Asian Americans, Koreans were diagnosed with GC at earlier stages owing to common history of endoscopy, which was more often undergone by patients with primary care providers of concordant ethnic origin. Overall, upper GI endoscopy was associated with early detection of GC in Asian Americans. Novelty and Impact. It is well-established that Asian Americans in the United States are disproportionately affected by gastric cancer. In our study we found that Asian American patients treated by physicians of similar ethnic background are more likely to undergo upper GI endoscopy in the United States, leading to early detection of gastric cancer and longer survival. Given this, targeted endoscopic screening in Asian Americans should be considered for early detection of GC.

Prospective data examining the association of aspirin use, according to dose and duration, with long-term risk of gastric adenocarcinoma in non-Asian cohorts are lacking. We evaluated the association between aspirin use and risk of gastric adenocarcinoma in two large prospective U.S. cohort studies, the Nurses' Health Study and the Health Professionals Follow-up Study. Cox proportional hazards regression models were used to calculate multivariable adjusted HRs and 95% confidence intervals (CI). Among the 159,116 participants, we documented 316 gastric adenocarcinoma cases (176 women, 140 men) over 34 years encompassing 4.5 million person-years. Among women, regular aspirin use (at least two times or more per week) was significantly associated with lower risk of gastric adenocarcinoma (multivariable HR, 0.52; 95% CI, 0.37–0.73) compared with nonregular use. However, regular aspirin use was not associated with gastric adenocarcinoma risk among men (multivariable HR, 1.08; 95% CI, 0.77–1.52; Pheterogeneity for sex = 0.003). Among women, the lower risk of gastric adenocarcinoma was more apparent with increasing duration of aspirin use (Ptrend < 0.001) and more than five tablets per week (multivariable HR, 0.51; 95% CI, 0.31–0.84). Regular, long-term aspirin use was associated with lower risk of gastric adenocarcinoma among women, but not men. The benefit appeared after at least 10 years of use and was maximized at higher doses among women. The heterogeneity by sex in the association of aspirin use with risk of gastric adenocarcinoma requires further investigation.Prevention Relevance:Novel prevention is urgently needed to reduce incidence and mortality of gastric cancer. We found that regular aspirin use was associated with lower risk of gastric adenocarcinoma among women, but not men. The benefit appeared after at least 10 years of use and was maximized at higher doses among women.See related Spotlight, p. 213

Supplementary Data from Association Between Aspirin Use and Gastric Adenocarcinoma: A Prospective Cohort Study

Supplementary Materials

Black people have a higher incidence of colorectal cancer and worse survival rates when compared with white people. Comprehensive genomic profiling was performed in 46,140 colorectal adenocarcinoma cases. Ancestry-informative markers identified 5,301 patients of African descent (AFR) and 33,770 patients of European descent (EUR). AFR were younger, had fewer microsatellite instability–high (MSI-H) tumors, and had significantly more frequent alterations in KRAS, APC, and PIK3CA. AFR had increased frequency of KRAS mutations, specifically KRASG12D and KRASG13. There were no differences in rates of actionable kinase driver alterations (HER2, MET, NTRK, ALK, ROS1, and RET). In patients with young-onset colorectal cancer ( Significance: KRAS (particularly KRASG12D/G13), APC, and PIK3CA were more frequently altered in AFR who had a lower frequency of MSI-H tumors. There were no differences in actionable kinase driver alterations. In young-onset colorectal cancer, both ancestries had a similar frequency of MSI-H/TMB-H tumors, but strikingly different trends in APC. See related commentary by Eng and Holowatyj, p. 1187. This article is highlighted in the In This Issue feature, p. 1171

Gastric cancer is the third leading cause of cancer-related deaths worldwide. Over 95% of gastric cancers are adenocarcinomas, which are typically classified based on anatomic location and histologic type. Gastric cancer generally carries a poor prognosis because it is often diagnosed at an advanced stage. Systemic therapy can provide palliation, improved survival, and enhanced quality of life in patients with locally advanced or metastatic disease. The implementation of biomarker testing, especially analysis of HER2 status, microsatellite instability (MSI) status, and the expression of programmed death-ligand 1 (PD-L1), has had a significant impact on clinical practice and patient care. Targeted therapies including trastuzumab, nivolumab, and pembrolizumab have produced encouraging results in clinical trials for the treatment of patients with locally advanced or metastatic disease. Palliative management, which may include systemic therapy, chemoradiation, and/or best supportive care, is recommended for all patients with unresectable or metastatic cancer. Multidisciplinary team management is essential for all patients with localized gastric cancer. This selection from the NCCN Guidelines for Gastric Cancer focuses on the management of unresectable locally advanced, recurrent, or metastatic disease.

Supplemental Appendix 7: Paired pre- and post- treatment SNaPshot molecular profiling available for 11 patients.

Supplementary Figures

Purpose:We performed a NCI-sponsored, prospective study of neoadjuvant FOLFIRINOX followed by chemoradiation with carboplatin/paclitaxel followed by surgery in patients with locally advanced gastric or gastroesophageal cancer.Patients and Methods:The primary objective was to determine completion rate of neoadjuvant FOLFIRINOX × 8 followed by chemoradiation. Secondary endpoints were toxicity and pathologic complete response (pCR) rate. Exploratory analysis was performed of circulating tumor DNA (ctDNA) to treatment response.Results:From October 2017 to June 2018, 25 patients were enrolled. All patients started FOLFIRINOX, 92% completed all eight planned cycles, and 88% completed chemoradiation. Twenty (80%) patients underwent surgical resection, and 7 had a pCR (35% in resected cohort, 28% intention to treat). Tumor-specific mutations were identified in 21 (84%) patients, of whom 4 and 17 patients had undetectable and detectable ctDNA at baseline, respectively. Presence of detectable post-chemoradiation ctDNA (P = 0.004) and/or postoperative ctDNA (P = 0.045) were associated with disease recurrence.Conclusions:Here we show neoadjuvant FOLFIRINOX followed by chemoradiation for locally advanced gastroesophageal cancer is feasible and yields a high rate of pCR. ctDNA appears to be a promising predictor of postoperative recurrence.See related commentary by Catenacci, p. 6281

Purpose:Stimulation of effector T cells is an appealing immunotherapeutic approach in oncology. OX40 (CD134) is a costimulatory receptor expressed on activated CD4+ and CD8+ T cells. Induction of OX40 following antigen recognition results in enhanced T-cell activation, proliferation, and survival, and OX40 targeting shows therapeutic efficacy in preclinical studies. We report the monotherapy dose-escalation portion of a multicenter, phase I trial (NCT02315066) of ivuxolimab (PF-04518600), a fully human immunoglobulin G2 agonistic monoclonal antibody specific for human OX40.Patients and Methods:Adult patients (N = 52) with selected locally advanced or metastatic cancers received ivuxolimab 0.01 to 10 mg/kg. Primary endpoints were safety and tolerability. Secondary/exploratory endpoints included preliminary assessment of antitumor activity and biomarker analyses.Results:The most common all-causality adverse events were fatigue (46.2%), nausea (28.8%), and decreased appetite (25.0%). Of 31 treatment-related adverse events, 30 (96.8%) were grade ≤2. No dose-limiting toxicities occurred. Ivuxolimab exposure increased in a dose-proportionate manner from 0.3 to 10 mg/kg. Full peripheral blood target engagement occurred at ≥0.3 mg/kg. Three (5.8%) patients achieved a partial response, and disease control was achieved in 56% of patients. Increased CD4+ central memory T-cell proliferation and activation, and clonal expansion of CD4+ and CD8+ T cells in peripheral blood were observed at 0.1 to 3.0 mg/kg. Increased immune cell infiltrate and OX40 expression were evident in on-treatment tumor biopsies.Conclusions:Ivuxolimab was generally well tolerated with on-target immune activation at clinically relevant doses, showed preliminary antitumor activity, and may serve as a partner for combination studies.

Purpose:Detection of persistent circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who will ultimately recur. Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. We evaluated a plasma-only ctDNA assay integrating genomic and epigenomic cancer signatures to enable tumor-uninformed MRD detection.Experimental Design:A total of 252 prospective serial plasma specimens from 103 patients with colorectal cancer undergoing curative-intent surgery were analyzed and correlated with recurrence.Results:Of 103 patients, 84 [stage I (9.5%), II (23.8%), III (47.6%), IV (19%)] had evaluable plasma drawn after completion of definitive therapy, defined as surgery only (n = 39) or completion of adjuvant therapy (n = 45). In “landmark” plasma drawn 1-month (median, 31.5 days) after definitive therapy and >1 year follow-up, 15 patients had detectable ctDNA, and all 15 recurred [positive predictive value (PPV), 100%; HR, 11.28 (P < 0.0001)]. Of 49 patients without detectable ctDNA at the landmark timepoint, 12 (24.5%) recurred. Landmark recurrence sensitivity and specificity were 55.6% and 100%. Incorporating serial longitudinal and surveillance (drawn within 4 months of recurrence) samples, sensitivity improved to 69% and 91%. Integrating epigenomic signatures increased sensitivity by 25%–36% versus genomic alterations alone. Notably, standard serum carcinoembryonic antigen levels did not predict recurrence [HR, 1.84 (P = 0.18); PPV = 53.9%].Conclusions:Plasma-only MRD detection demonstrated favorable sensitivity and specificity for recurrence, comparable with tumor-informed approaches. Integrating analysis of epigenomic and genomic alterations enhanced sensitivity. These findings support the potential clinical utility of plasma-only ctDNA MRD detection.See related commentary by Bent and Kopetz, p. 5449

Supplemental Figure 1. Distribution of EGFR extracellular domain (ECD) mutations in ctDNA samples from patients with CRC. Supplemental Figure 2. Response to crizotinib in a patient with small bowel adenocarcinoma and GOPC-ROS1 fusion. Supplemental Figure 3. Detection of copy number amplification is associated with increased ctDNA fraction. Supplemental Table 1. List of 62 genes sequenced in this study using the FoundationACT ctDNA assay. Supplemental Table 2. Genomic alterations detected in temporally matched ctDNA and tissue samples.

Supplementary Data from A Phase I, Open-Label, Dose-Escalation Study of the OX40 Agonist Ivuxolimab in Patients with Locally Advanced or Metastatic Cancers

Purpose: Genomic profiling of tumor biopsies from advanced gastrointestinal and anal cancers is increasingly used to inform treatment. In some cases, tissue biopsy can be prohibitive, and we sought to investigate whether analysis of blood-derived circulating tumor DNA (ctDNA) may provide a minimally invasive alternative.Experimental Design: Hybrid capture–based genomic profiling of 62 genes was performed on blood-based ctDNA from 417 patients with gastrointestinal carcinomas to assess the presence of genomic alterations (GA) and compare with matched tissue samples.Results: Evidence of ctDNA was detected in 344 of 417 samples (82%), and of these, ≥1 reportable GA was detected in 89% (306/344) of samples. Frequently altered genes were TP53 (72%), KRAS (35%), PIK3CA (14%), BRAF (8%), and EGFR (7%). In temporally matched ctDNA and tissue samples available from 25 patients, 86% of alterations detected in tissue were also detected in ctDNA, including 95% of short variants, but only 50% of amplifications. Conversely, 63% of alterations detected in ctDNA were also detected in matched tissue. Examples demonstrating clinical utility are presented.Conclusions: Genomic profiling of ctDNA detected potentially clinically relevant GAs in a significant subset of patients with gastrointestinal carcinomas. In these tumor types, most alterations detected in matched tissue were also detected in ctDNA, and with the exception of amplifications, ctDNA sequencing routinely detected additional alterations not found in matched tissue, consistent with tumor heterogeneity. These results suggest feasibility and utility of ctDNA testing in advanced gastrointestinal cancers as a complementary approach to tissue testing, and further investigation is warranted. Clin Cancer Res; 24(8); 1881–90. ©2018 AACR.

Supplementary Tables

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.Statement of SignificanceLINE-1 ORF1p transposon protein is pervasively expressed in many cancers and a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 μL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring.

Chemotherapy is ubiquitous in first-line treatment of advanced gastric cancer, yet responses are heterogeneous, and little is known about mediators of chemotherapy response. To move forward, an understanding of the effects of standard chemotherapy on the tumor–immune microenvironment (TME) is needed. Coupling whole-exome sequencing, bulk RNA and single-cell transcriptomics from paired pretreatment and on-treatment samples in treatment-naïve patients with HER2-positive and HER2-negative gastric cancer, we define features associated with response to platinum-based chemotherapy. Response was associated with on-treatment TME remodeling including natural killer (NK) cell recruitment, decreased tumor-associated macrophages, M1-macrophage repolarization, and increased effector T-cell infiltration. Among chemotherapy nonresponders, we observed low/absent PD-L1 expression or modulation, on-treatment increases in Wnt signaling, B-cell infiltration, and LAG3-expressing T cells coupled to an exodus of dendritic cells. We did not observe significant genomic changes in early on-treatment sampling. We provide a map of on-treatment TME modulation with standard chemotherapy and nominate candidate future approaches. Significance: Using paired pretreatment and on-treatment samples during standard first-line chemotherapy, we identify chemotherapy-induced NK-cell infiltration, macrophage repolarization, and increased antigen presentation among responders. Increased LAG3 expression and decreased dendritic cell abundance were seen in nonresponders, emphasizing remodeling of the TME during chemotherapy response and resistance. This article is highlighted in the In This Issue feature, p. 873

PURPOSE: Colorectal cancer (CRC) incidence in patients younger than 50 years of age, commonly defined as early-onset (EO-CRC), is rising. EO-CRC often presents with distinct clinicopathologic features. However, data on prognosis are conflicting and outcomes with modern treatment approaches for metastatic disease are still limited. MATERIALS AND METHODS: We prospectively enrolled patients with metastatic CRC (mCRC) to a biobanking and clinical data collection protocol from 2014 to 2018. We grouped the cohort based on age at initial diagnosis: < 40 years, 40-49 years, and ≥ 50 years. We used regression models to examine associations among age at initial diagnosis, treatments, clinicopathologic features, and survival. RESULTS: We identified 466 patients with mCRC (45 [10%] age < 40 years, 109 [23%] age 40-49 years, and 312 [67%] age ≥ 50 years). Patients < 40 years of age were more likely to have received multiple metastatic resections (odds ratio [OR], 3.533; P = .0066) than their older counterparts. Patients with EO-CRC were more likely to receive triplet therapy than patients > 50 years of age (age < 40 years: OR, 6.738; P = .0002; age 40-49 years: OR, 2.949; P = .0166). Patients 40-49 years of age were more likely to have received anti-EGFR therapy (OR, 2.633; P = .0016). Despite differences in care patterns, age did not predict overall survival. CONCLUSION: Despite patients with EO-CRC receiving more intensive treatments, survival was similar to the older counterpart. However, EO-CRC had clinical and molecular features associated with worse prognoses. Improved biologic understanding is needed to optimize clinical management of EO-CRC. The cost-benefit ratio of exposing patients with EO-CRC to more intensive treatments has to be carefully evaluated.

Purpose: We performed a NCI-sponsored, prospective study of neoadjuvant FOLFIRINOX followed by chemoradiation with carboplatin/paclitaxel followed by surgery in patients with locally advanced gastric or gastroesophageal cancer. Patients and Methods: The primary objective was to determine completion rate of neoadjuvant FOLFIRINOX × 8 followed by chemoradiation. Secondary endpoints were toxicity and pathologic complete response (pCR) rate. Exploratory analysis was performed of circulating tumor DNA (ctDNA) to treatment response. Results: From October 2017 to June 2018, 25 patients were enrolled. All patients started FOLFIRINOX, 92% completed all eight planned cycles, and 88% completed chemoradiation. Twenty (80%) patients underwent surgical resection, and 7 had a pCR (35% in resected cohort, 28% intention to treat). Tumor-specific mutations were identified in 21 (84%) patients, of whom 4 and 17 patients had undetectable and detectable ctDNA at baseline, respectively. Presence of detectable post-chemoradiation ctDNA (P = 0.004) and/or postoperative ctDNA (P = 0.045) were associated with disease recurrence. Conclusions: Here we show neoadjuvant FOLFIRINOX followed by chemoradiation for locally advanced gastroesophageal cancer is feasible and yields a high rate of pCR. ctDNA appears to be a promising predictor of postoperative recurrence. See related commentary by Catenacci, p. 6281

BACKGROUND: Adagrasib, an oral small-molecule inhibitor of mutant KRAS G12C protein, has shown clinical activity in pretreated patients with several tumor types, including colorectal cancer. Preclinical studies suggest that combining a KRAS G12C inhibitor with an epidermal growth factor receptor antibody could be an effective clinical strategy. METHODS: In this phase 1–2, open-label, nonrandomized clinical trial, we assigned heavily pretreated patients with metastatic colorectal cancer with mutant KRAS G12C to receive adagrasib monotherapy (600 mg orally twice daily) or adagrasib (at the same dose) in combination with intravenous cetuximab once a week (with an initial loading dose of 400 mg per square meter of body-surface area, followed by a dose of 250 mg per square meter) or every 2 weeks (with a dose of 500 mg per square meter). The primary end points were objective response (complete or partial response) and safety. RESULTS: As of June 16, 2022, a total of 44 patients had received adagrasib, and 32 had received combination therapy with adagrasib and cetuximab, with a median follow-up of 20.1 months and 17.5 months, respectively. In the monotherapy group (43 evaluable patients), a response was reported in 19% of the patients (95% confidence interval [CI], 8 to 33). The median response duration was 4.3 months (95% CI, 2.3 to 8.3), and the median progression-free survival was 5.6 months (95% CI, 4.1 to 8.3). In the combination-therapy group (28 evaluable patients), the response was 46% (95% CI, 28 to 66). The median response duration was 7.6 months (95% CI, 5.7 to not estimable), and the median progression-free survival was 6.9 months (95% CI, 5.4 to 8.1). The percentage of grade 3 or 4 treatment-related adverse events was 34% in the monotherapy group and 16% in the combination-therapy group. No grade 5 adverse events were observed. CONCLUSIONS: Adagrasib had antitumor activity in heavily pretreated patients with metastatic colorectal cancer with mutant KRAS G12C, both as oral monotherapy and in combination with cetuximab. The median response duration was more than 6 months in the combination-therapy group. Reversible adverse events were common in the two groups. (Funded by Mirati Therapeutics; KRYSTAL-1 ClinicalTrials.gov number, NCT03785249.)

PURPOSE Circulating tumor DNA (ctDNA) analyses allow for postoperative risk stratification in patients with curatively treated colon and breast cancers. Use of ctDNA in esophagogastric cancers (EGC) is less characterized and could identify high-risk patients who have been treated with curative intent. METHODS In this retrospective analysis of real-world data, ctDNA levels were analyzed in the preoperative, postoperative, and surveillance settings in patients with EGC using a personalized multiplex polymerase chain reaction–based next-generation sequencing assay. Plasma samples (n = 943) from 295 patients at > 70 institutions were collected before surgery, postoperatively, and/or serially during routine clinical follow-up from September 19, 2019, to February 21, 2022. ctDNA detection was annotated to clinicopathologic features and recurrence-free survival. RESULTS A total of 295 patients with EGC were analyzed, and 212 patients with stages I-III disease were further explored. Pretreatment ctDNA was detected in 96% (23/24) of patients with preoperative time points. Postoperative ctDNA was detected in 23.5% (16/68) of patients with stage I-III EGC within 16 weeks (molecular residual disease window) after surgery without receiving systemic therapy. ctDNA detection at any time point after surgery (hazard ratio [HR], 23.6; 95% CI, 10.2 to 66.0; P < .0001), within the molecular residual disease window (HR, 10.7; 95% CI, 4.3 to 29.3; P < .0001), and during the surveillance period (HR, 17.7; 95% CI, 7.3 to 50.7; P < .0001) was associated with shorter recurrence-free survival. In multivariable analysis, ctDNA status and clinical stage of disease were independently associated with outcomes. CONCLUSION Using real-world data, we demonstrate that postoperative tumor-informed ctDNA detection in EGC is feasible and allows for enhanced patient risk stratification and prognostication during curative-intent therapy.

Therapeutic combinations targeting innate and adaptive immunity and predictive biomarkers of response in esophagogastric cancer (EGC) are needed. We assessed safety and clinical utility of DKN-01 (a novel DKK1-neutralizing IgG4 antibody) combined with pembrolizumab and retrospectively determined DKK1 tumoral expression as a biomarker. Patients with advanced EGC received intravenous DKN-01 (150 or 300 mg) on days 1 and 15 with pembrolizumab 200 mg on day 1 in 21-day cycles. Clinical response was assessed by RECIST v1.1. Association of tumoral DKK1 mRNA expression (H-score: high ≥ upper-tertile, low < upper-tertile) with response was assessed with PD-L1 levels as a covariate. Sixty-three patients received DKN-01 150 mg (n = 2) or 300 mg (n = 61) plus pembrolizumab. Common adverse events were fatigue, anemia, blood alkaline phosphatase elevation, aspartate aminotransferase elevation, and hyponatremia. Among evaluable anti-PD-1/PD-L1-naïve patients receiving DKN-01 300 mg and pembrolizumab, objective response rate (ORR) was 11.4% (5/44) and 18.5% (5/27) in patients with gastroesophageal junction or gastric cancer (GEJ/GC). Among response-evaluable anti-PD-1/PD-L1-naïve patients with GEJ/GC and known tumoral DKK1 expression, ORR was 50% in DKK1-high and 0% in DKK1-low patients, median PFS was 22.1 vs. 5.9 weeks (HR, 0.24; 95% CI, 0.08–0.67), respectively, and median OS was 31.6 weeks vs. 17.4 weeks (HR, 0.41; 95% CI, 0.16–1.07), respectively. Association of DKK1 expression with PFS was independent of PD-L1 expression (adjusted HR, 0.21; 95% CI, 0.06–0.69). DKN-01 combined with pembrolizumab was well tolerated with no new safety signals. Antitumor activity was enriched in anti-PD-1/PD-L1-naïve patients with GEJ/GC whose tumors expressed high DKK1.