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While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAF V600E 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 BRAF V600E 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., (© 2023. The Author(s).)

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements., Significance: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397., (©2022 American Association for Cancer Research.)

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., Competing Interests: Nora K. HorickEmployment: Northwest BiotherapeuticsStock and Other Ownership Interests: Northwest BiotherapeuticsPatents, Royalties, Other Intellectual Property: An immediate family member holds patents, has patents pending, and receives royalties from a technology relating to health or medicine Lawrence S. BlaszkowskyStock and Other Ownership Interests: Pfizer Jeffrey W. ClarkResearch Funding: Pfizer David P. RyanStock and Other Ownership Interests: MPM Capital, Acworth Pharmaceuticals, Thrive Earlier Detection Corp, Exact SciencesHonoraria: UpToDate, Research to PracticeConsulting or Advisory Role: MPM Capital, Oncorus, Gritstone Oncology, Maverick Therapeutics, TCR2 Therapeutics, Twentyeight-Seven TherapeuticsResearch Funding: Stand Up To CancerPatents, Royalties, Other Intellectual Property: McGraw Hill Chapter Royalties, Johns Hopkins University PressExpert Testimony: Boehringer IngelheimOther Relationship: TCR2 Therapeutics Lipika GoyalConsulting or Advisory Role: Debiopharm Group, Alentis Therapeutics, QED Therapeutics, H3 Biomedicine, AstraZeneca, Klus Pharma, Agios, Taiho Pharmaceutical, Incyte, Sirtex Medical, Genentech, ExelixisUncompensated Relationships: Agios, Debiopharm Group, Taiho Pharmaceutical, Merck Joseph FransesStock and Other Ownership Interests: Merck, BiogenConsulting or Advisory Role: Foundation MedicineResearch Funding: Genentech/Roche Samuel J. KlempnerStock and Other Ownership Interests: TP TherapeuticsHonoraria: NateraConsulting or Advisory Role: Lilly, Boston Biomedical, Astellas Pharma, Foundation Medicine, Bristol Myers Squibb, Pieris Pharmaceuticals, Merck, Daiichi Sankyo/UCB JapanSpeakers' Bureau: Foundation MedicineResearch Funding: Leap Therapeutics, BeiGeneOther Relationship: NCCN Eric J. RoelandConsulting or Advisory Role: Napo Pharmaceuticals, AIM Specialty Health, Oragenics, BASF, Vector Oncology, Asahi Kasei, Heron, Pfizer/EMD Serono, Astellas Pharma, Helsinn TherapeuticsExpert Testimony: Regents of the University of California Colin D. WeekesHonoraria: Celgene, Merrimack, Lilly, BayerConsulting or Advisory Role: Celgene, Merrimack, IpsenResearch Funding: Millennium, Celgene, Bayer, Genentech/Roche, AbbVie, Lilly, AstraZeneca, Gilead Sciences, Ipsen, HalozymeTravel, Accommodations, Expenses: Lilly, Celgene, Bayer Theodore S. HongConsulting or Advisory Role: Merck, Synthetic Biologics, Novocure, SyndaxResearch Funding: Novartis, Taiho Pharmaceutical, AstraZeneca, IntraOp, Tesaro, Bristol Myers Squibb, Ipsen Emily E. Van SeventerEmployment: Blueprint MedicinesStock and Other Ownership Interests: Blueprint Medicines Ryan B. CorcoranStock and Other Ownership Interests: Avidity Biosciences, nRichDx, Fount Therapeutics, C4 Therapeutics, Revolution Medicines, Kinnate Biopharma, Remix Therapeutics, Erasca IncConsulting or Advisory Role: Avidity Nanomedicines, Taiho Pharmaceutical, Merrimack, Genentech, N-of-One, Astex Pharmaceuticals, Amgen, Bristol Myers Squibb, Roche, Shire, FOGPharma, Loxo, Roivant, Warp Drive Bio, Spectrum Pharmaceuticals, Symphogen, Array BioPharma, Chugai Pharma, nRichDx, Fount Therapeutics, Novartis, Shionogi, Elicio Therapeutics, C4 Therapeutics, Revolution Medicines, Zikani Therapeutics, Natera, Kinnate Biopharma, Ipsen, AbbVie, Asana Biosciences, Remix TherapeuticsResearch Funding: AstraZeneca, Sanofi, Asana Biosciences, Lilly Aparna R. ParikhConsulting or Advisory Role: PureTech, Foundation Medicine, Lilly, NateraResearch Funding: Plexxikon, Bristol Myers Squibb, Genentech, Guardant Health, Array BioPharma, Lilly, Novartis Pharmaceuticals UK Ltd, TesaroNo other potential conflicts of interest were reported.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC., (Copyright © 2021 the Author(s). Published by PNAS.)

Overcoming intrinsic resistance to immune checkpoint blockade for microsatellite stable (MSS) colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) remains challenging. We conducted a single-arm, non-randomized, phase II trial (NCT03104439) combining radiation, ipilimumab and nivolumab to treat patients with metastatic MSS CRC (n = 40) and PDAC (n = 25) with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The primary endpoint was disease control rate (DCR) by intention to treat. DCRs were 25% for CRC (ten of 40; 95% confidence interval (CI), 13-41%) and 20% for PDAC (five of 25; 95% CI, 7-41%). In the per-protocol analysis, defined as receipt of radiation, DCR was 37% (ten of 27; 95% CI, 19-58%) in CRC and 29% (five of 17; 95% CI, 10-56%) in PDAC. Pretreatment biopsies revealed low tumor mutational burden for all samples but higher numbers of natural killer (NK) cells and expression of the HERVK repeat RNA in patients with disease control. This study provides proof of concept of combining radiation with immune checkpoint blockade in immunotherapy-resistant cancers., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 ., (©2021 American Association for Cancer Research.)

Background: Oligometastatic colorectal cancer (CRC) is potentially curable and demands individualised strategies., Methods: This single-centre retrospective study investigated if positron emission tomography (PET)/magnetic resonance imaging (MR) had a clinical impact on oligometastatic CRC relative to the standard of care imaging (SCI). Adult patients with oligometastatic CRC on SCI who also underwent PET/MR between 3/2016 and 3/2019 were included. The exclusion criterion was lack of confirmatory standard of reference, either surgical pathology, intraoperative gross confirmation or imaging follow-up. SCI consisted of contrast-enhanced (CE) computed tomography (CT) of the chest/abdomen/pelvis, abdominal/pelvic CE-MR, and/or CE whole-body PET/CT with diagnostic quality (i.e. standard radiation dose) CT. Follow-up was evaluated until 3/2020., Results: Thirty-one patients constituted the cohort, 16 (52%) male, median patient age was 53 years (interquartile range: 49-65 years). PET/MR and SCI results were divergent in 19% (95% CI 9-37%) of the cases, with PET/MR leading to management changes in all of them. The diagnostic accuracy of PET/MR was 90 ± 5%, versus 71 ± 8% for SCI. In a pairwise analysis, PET/MR outperformed SCI when compared to the reference standard (p = 0.0412)., Conclusions: These findings suggest the potential usefulness of PET/MR in the management of oligometastatic CRC., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer., Experimental Design: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results., Results: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy., Conclusions: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target., (©2021 American Association for Cancer Research.)

Background: The impact of primary tumor site on overall survival in patients with stage IV colorectal cancer (CRC) with single-organ metastases to the liver or lung has not been studied. Furthermore, the prognostic significance of commonly tested genetic variants such as KRAS mutation and microsatellite instability (MSI) are not well-described in this population., Materials and Methods: This National Cancer Database was used to identify 38,328 patients with CRC that presented with synchronous metastases to the liver or lung between 2010 and 2014. The primary outcome was overall survival, and groups were compared using Kaplan-Meier analyses and Cox proportional hazard models., Results: On unadjusted analysis, median survival was significantly longer for patients with lung metastases compared with those with liver metastases for left-sided (27 vs. 25 months; P = .02) and right-sided CRC (19 vs. 15 months; P < .001), whereas rectosigmoid and rectal cancers showed no difference. On multivariate analysis, patients with liver metastases demonstrated worse survival compared with those with lung metastasis (hazard ratio, 1.37; 95% confidence interval, 1.31-1.43; P < .001). These trends were confirmed in patients that received chemotherapy but did not have their primary tumor or metastases resected. In patients with genetic testing, both KRAS mutants and MSI tumors had worse survival in left-sided and rectal tumors with liver metastases, but had similar survival to KRAS wild type tumors and microsatellite stable tumors, respectively, across other primary site and metastatic patterns., Conclusions: For patients with single-organ metastases to the liver or lung, primary tumor site has an impact on overall survival. Further, KRAS mutation and MSI status are of prognostic importance in selected patients with single-organ metastases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The primary aim of the present study was to evaluate if PET/MR induced management changes versus standard of care imaging (SCI) in treated colorectal cancer patients. The secondary aim was to assess the staging performance of PET/MR and of SCI versus the final oncologic stage., Methods: Treated CRC patients who underwent PET/MR with 18 F-FDG and SCI between January 2016 and October 2018 were enrolled in this retrospective study. Their medical records were evaluated to ascertain if PET/MR had impacted on their clinical management versus SCI. The final oncologic stage, as reported in the electronic medical record, was considered the true stage of disease., Results: A total of 39 patients who underwent 42 PET/MR studies were included, mean age 56.7 years (range 39-75 years), 26 males, and 13 females. PET/MR changed clinical management 15/42 times (35.7%, standard error ± 7.4%); these 15 changes in management were due to upstaging in 9/42 (21.5%) and downstaging in 6/42 (14.2%). The differences in management prompted by SCI versus PET/MR were statistically significant, and PET/MR outperformed SCI (P value < 0.001; odds ratio = 2.8). In relation to the secondary outcome, PET/MR outperformed the SCI in accuracy of oncologic staging (P value = 0.016; odds ratio = 4.6)., Conclusions: PET/MR is a promising imaging tool in the evaluation of treated CRC and might change the management in these patients. However, multicenter prospective studies with larger patient samples are required in order to confirm these preliminary results.

Background: Patients with microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) metastatic colorectal cancer (mCRC) show a significant response to checkpoint inhibitor therapies, but the economic impact of these therapies is unknown. A decision analytic model was used to explore the effectiveness and cost burden of MSI-H/dMMR mCRC treatment., Methods: The treatment of hypothetical patients with MSI-H/dMMR mCRC was simulated in 2 treatment scenarios: a third-line treatment and an exploratory first-line treatment. The treatments compared were nivolumab, ipilimumab and nivolumab, trifluridine and tipiracil (third-line treatment), and mFOLFOX6 and cetuximab (first-line treatment). Disease progression, drug toxicity, and survival rates were based on the CheckMate 142, study of TAS-102 in patients with metastatic colorectal cancer refractory to standard chemotherapies (RECOURSE), and Cancer and Leukemia Group B/Southwest Oncology Group 80405 trials. The analyzed outcomes included survival (life-years), quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICERs)., Results: Ipilimumab with nivolumab was the most effective strategy (10.69 life-years and 9.25 QALYs for the third line; 10.69 life-years and 9.44 QALYs for the first line) in comparison with nivolumab (8.21 life-years and 6.76 QALYs for the third line; 8.21 life-years and 7.00 QALYs for the first line), trifluridine and tipiracil (0.74 life-years and 0.07 QALYs), and mFOLFOX6 and cetuximab (2.72 life-years and 1.63 QALYs). However, neither checkpoint inhibitor therapy was cost-effective in comparison with trifluridine and tipiracil (nivolumab ICER, $153,000; ipilimumab and nivolumab ICER, $162,700) or mFOLFOX6 and cetuximab (nivolumab ICER, $150,700; ipilimumab and nivolumab ICER, $158,700)., Conclusions: This modeling analysis found that both single and dual checkpoint blockade could be significantly more effective for MSI-H/dMMR mCRC than chemotherapy, but they were not cost-effective, largely because of drug costs. Decreases in drug pricing and/or the duration of maintenance nivolumab could make ipilimumab and nivolumab cost-effective. Prospective clinical trials should be performed to explore the optimal duration of maintenance nivolumab., (© 2018 American Cancer Society.)

Clonal heterogeneity associated with acquired resistance presents a critical therapeutic challenge. Whole-exome sequencing of paired tumor biopsies and targeted sequencing of cell-free DNA (cfDNA) from patients with BRAF V600E colorectal cancer receiving BRAF inhibitor combinations identified 14 distinct alterations in MAPK pathway components driving acquired resistance, with as many as eight alterations in a single patient. We developed a pooled clone system to study clonal outgrowth during acquired resistance, in vitro and in vivo In vitro , the dynamics of individual resistant clones could be monitored in real time in cfDNA isolated from culture media during therapy. Outgrowth of multiple resistant clones was observed during therapy with BRAF, EGFR, and MEK inhibitor combinations. However, ERK inhibition, particularly in combination with BRAF and EGFR inhibition, markedly abrogated clonal outgrowth in vitro and in vivo Thus, convergent, up-front therapy may suppress outgrowth of heterogeneous clones harboring clinically observed resistance alterations, which may improve clinical outcome. Significance: We observed heterogeneous, recurrent alterations in the MAPK pathway as key drivers of acquired resistance in BRAF V600E colorectal cancer, with multiple concurrent resistance alterations detectable in individual patients. Using a novel pooled clone system, we identify convergent up-front therapeutic strategies capable of intercepting multiple resistance mechanisms as potential approaches to suppress emergence of acquired resistance. Cancer Discov; 8(4); 417-27. ©2018 AACR. See related commentary by Janku, p. 389 See related article by Corcoran et al., p. 428 This article is highlighted in the In This Issue feature, p. 371 ., (©2018 American Association for Cancer Research.)

Purpose: Optimal margin size when resecting colorectal liver metastases (CRLM) remains unclear, particularly in the setting of perioperative chemotherapy. We evaluated the prognostic significance of margin size in patients who received neoadjuvant FOLFOX and/or FOLFIRI prior to resection of CRLM., Methods: Clinicopathologic factors were collected for patients who underwent curative liver resections for CRLM between 4/2004-1/2016 and received neoadjuvant oxaliplatin and/or irinotecan and 5-FU (FOLFOX, FOLFIRI). Margins were categorized as < 1, ≥ 1-< 5, ≥ 5-< 10, or ≥ 10 mm and evaluated for association with overall survival (OS) and disease-free survival (DFS) by Cox multiple regression analysis. Margin status was classified as positive (< 1 mm) or negative (≥ 1 mm) and similarly evaluated., Results: Of 227 patients, the median age was 58 years and most had synchronous (80%) lesions. The majority had colon cancers (75%). Margin sizes were 13% < 1 mm, 27% ≥ 1-< 5 mm, 23% ≥ 5-< 10 mm, 36% ≥ 10 mm. Most (63%) received chemotherapy post-liver resection. Five-year OS and DFS were 54% (95% CI 46-62%) and 22% (95% CI 16-28%), respectively. Positive margins significantly increased the risk of death without post-liver resection chemotherapy (HR = 3.32, p = 0.0077), but not with post-liver resection chemotherapy (HR = 1.00, p = 0.99). Negative margin sizes of ≥ 1-< 5, ≥ 5-< 10, and ≥ 10 mm were not significant predictors of OS (p > 0.05)., Conclusion: Patients undergoing liver resection for CRLM should receive post-resection chemotherapy if negative margins (≥ 1 mm) cannot be achieved. For patients receiving FOLFOX and/or FOLFIRI chemotherapy, wider margins did not improve OS.

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6C low monocytes and not Ly6C high monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6C low monocytes ( Cx3cr1 -/- mice), Ly6C high monocytes ( CCR2 -/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6C low monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G + neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6C low monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies., Competing Interests: Conflict of interest statement: R.K.J. received consultant fees from Merck, Ophthotech, Pfizer, SPARC, SynDevRx, and XTuit; owns equity in Enlight, Ophthotech, SynDevRx, and XTuit; and serves on the Board of Directors of XTuit and the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund. Neither any reagent nor any funding from these organizations was used in this study.

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy., (Copyright © 2016, American Association for the Advancement of Science.)

Colorectal cancer represents a major cause of cancer morbidity and mortality, with approximately 1.2 million cases and 600,000 deaths worldwide each year. Because of the anatomic continuity of the colon into the rectum, cancers affecting these organs have historically been considered equivalent. In this Prospective, we discuss the clinical and experimental data suggesting that colon cancer and rectal cancer are highly related, but distinct, diseases. Reconsidering the relationship between these cancers has implications for the development of new therapeutic paradigms., (©2012 AACR.)

Purpose: Cetuximab and panitumumab are monoclonal antibodies that target the epidermal growth factor receptor (EGFR) and are approved for the treatment of patients with KRAS wild-type meta-static colorectal cancer. There are no data that describe the activity of panitumumab in patients with progressive disease on cetuximab. We performed a single-arm phase II trial of panitumumab in patients with KRAS wild-type metastatic colorectal cancer that had progressed on prior cetuximab., Patients and Methods: We used a two-stage study design to treat patients with panitumumab at 6 mg/kg every 14 days (cycle length = 28 days). Treatment was continued until disease progression, death, inability to tolerate panitumumab, or study withdrawal. The primary endpoint was response rate; secondary endpoints included progression-free survival and overall survival. Twenty patients were treated in the first stage, with plans to treat an additional twelve patients if there was at least one objective response. We collected blood samples at baseline and prior to cycles 2 and 3 to evaluate for the presence of anti-cetuximab and anti-panitumumab antibodies., Results: We treated twenty patients for a median of two cycles (range 1-4). No patients responded, and 45% had a best response of stable disease (no progression for at least two cycles). Median progression-free survival was 1.7 months and median overall survival was 5.2 months. Panitumumab was well tolerated. Thirteen patients (65%) had grade 1-2 dry skin or rash, and three patients had treatment-related grade 3 toxicities (one each with hyperglycemia, hyperbilirubinemia, and hypokalemia). No patients had detectable anti-cetuximab antibodies at any time point; one patient developed anti-panitumumab antibodies., Conclusions: Panitumumab has minimal benefit in patients with KRAS wild-type metastatic colorectal cancer that has progressed on prior cetuximab. Discussion Both cetuximab and panitumumab competitively inhibit ligand binding to EGFR, thereby promoting receptor internalization and blocking receptor-mediated signaling. Although the two agents have never been compared directly in a randomized clinical trial, they produce similar response rates when used alone as well as in combination with cytotoxic agents. Cetuximab is a chimeric antibody with approximately 30% murine protein, while panitumumab is a fully human monoclonal antibody. Correspondingly, rates of severe hypersensitivity reactions are somewhat increased with cetuximab (3%) compared to panitumumab (1%). However, the potential efficacy of panitumumab in patients who have developed disease progression on cetuximab has been an open question. Metges et al. (PANERB trial) prospectively treated 32 KRAS wild-type metastatic colorectal cancer patients with cetuximab and irinotecan followed by panitumumab monotherapy after progression. Remarkably, the authors reported an objective response rate of 22% to panitumumab, including a disease control rate (objective response plus stable disease) of 73% in 11 patients who had previously responded to cetuximab and irinotecan. In contrast, we found no responders and a stable disease rate of 45% with a median duration of only 1.7 months in our trial of 20 patients. Moreover, no patients had detectable anti-cetuximab antibodies at baseline. It is not clear to what extent the PANERB trial included patients without objective disease progression on cetuximab or for whom cetuximab-containing regimens may have been ceased due to toxicity in the absence of disease progression. In both circumstances, retreatment with panitumumab may be expected to demonstrate some degree of clinical activity. In our study, disease progression after at least 4 weeks of cetuximab documented radiographically or by increased carcinoembryonic antigen (CEA) levels was required for inclusion in order to ensure that the study population demonstrated unequivocal evidence of progression on cetuximab. While it remains possible that a small subset of patients may benefit from panitumumab after progression on cetuximab, our results suggest that this approach should not be adopted until predictive biomarkers for panitumumab response in this setting have been discovered and validated. Until then, patients who develop progression on cetuximab should be enrolled in trials of novel agents.

A proliferation of new cytotoxic and biologic agents has led to improved survival in patients with metastatic colorectal cancer (mCRC). The ability of surgery to increase long-term survival in patients with liver and/or lung metastases also has been firmly established. It has become increasingly difficult as the numbers and types of treatment options have expanded to identify optimal drug combinations, sequences, and duration and the best way to integrate systemic chemotherapy with potentially curative surgery for metastatic lesions. For this review, the authors examined how recent clinical trials have addressed some pertinent questions regarding the use of systemic chemotherapy and biologic agents in patients with mCRC.

Purpose: To determine the maximum tolerated doses (MTD), toxicities, efficacy, and pharmacokinetics (PK) of gefitinib combined with irinotecan, 5-fluorouracil (5-FU) and leucovorin (IFL) in patients with previously untreated advanced colorectal cancer., Experimental Design: Starting doses were gefitinib 250 mg/day orally without interruption, irinotecan 100 mg/m(2) as a 90 min intravenous (i.v.) infusion, 5-FU 400 mg/m(2) bolus i.v. and leucovorin 20 mg/m(2) i.v. on days 1 and 8 of a 21-day cycle. Dose escalations involved increasing gefitinib to 500 mg then increasing irinotecan to 125 mg/m(2) and 5-FU to 500 mg/m(2)., Results: Twenty-four patients received therapy. The starting doses proved to be the MTD, as attempts to increase the dose of either gefitinib or the chemotherapeutic agents resulted in dose-limiting toxicities. Gastrointestinal effects and bone marrow suppression were the principal toxicities; however, only 1/17 (6%) patients treated with the MTD had severe (grades 3-4) diarrhea and severe neutropenia occurred in only two (12%) patients. Partial responses occurred in 10/17 patients receiving the MTD and another five had stable disease. Median progression-free and overall survivals were 12.2 and 26.6 months, respectively. In ten patients treated with the MTD, the steady-state PK of gefitinib was not affected by IFL nor did gefitinib appear to influence the PK of either irinotecan or 5-FU., Conclusions: Gefitinib can be safely combined with an intermittent weekly schedule of IFL. Evidence of promising activity should encourage further clinical evaluation of epidermal growth factor receptor tyrosine kinase inhibitors, such as gefitinib, combined with multiagent chemotherapy for metastatic colorectal cancer.

The CpG island methylator phenotype (CIMP) is a distinct epigenetic phenotype in colorectal carcinoma with concordant methylation in multiple promoter CpG islands. The relationship between CpG island methylation and clinical outcomes among colorectal cancer patients treated with chemotherapy has been a controversial subject. Utilizing real-time polymerase chain reaction (PCR; MethyLight technology), we quantified DNA methylation in 13 CpG island loci (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, MINT1, MINT31, IGFBP3, MGMT, and WRN) in 30 metastatic microsatellite stable colorectal carcinomas in phase I/II clinical trials of combination chemotherapy (5-fluorouracil, irinotecan, leucovorin, and gefitinib). Tumor response was assessed by CT scans performed at baseline and every 6 weeks thereafter. Overall CIMP-high status (either >or=9/13 or >or=7/13 methylated markers; identifying 3 or 5 CIMP-high tumors, respectively) and methylation in CACNA1G, IGF2, MLH1, NEUROG1, RUNX3, MINT31, and WRN were associated with worse survival (all p < 0.01). Although not statistically significant, there was a trend toward resistance to chemotherapy among tumors with CpG island methylation. In conclusion, CpG island methylation may predict poor survival in metastatic microsatellite stable colorectal carcinoma treated with chemotherapy. Additional studies are necessary to examine the role of DNA methylation in treatment efficacy.

Background: In this study, the maximum tolerated dose and toxicity profile of FOLFIRI (infusional fluorouracil [5-FU]/leucovorin/irinotecan) plus gefitinib (an oral inhibitor of the epidermal growth factor receptor) were evaluated as first-line therapy in patients with metastatic colorectal cancer., Patients and Methods: Sixteen patients participated in this study. Oral gefitinib was administered at 250 mg or 500 mg daily in 2 dose-escalation cohorts. FOLFIRI was administered without dose escalation on a 14-day cycle with treatment on day 1 with irinotecan 180 mg/m2, leucovorin 200 mg/m2, and 5-FU 400 mg/m2 bolus, followed by 5-FU 2400 mg/m2 continuous infusion over 46 hours., Results: The maximum tolerated dose of gefitinib was 250 mg, with diarrhea and neutropenia noted as the principal dose-limiting toxicities. Dose reductions in 5-FU and irinotecan were required in 4 patients because of diarrhea and 1 patient because of neutropenia. A partial response was observed in 25% of patients, and 56% had stable disease for > 12 weeks, corresponding to a disease control rate of 81%., Conclusion: These findings suggest that gefitinib can be safely combined with FOLFIRI as first-line treatment of metastatic CRC and support the safety of further investigations of EGFR tyrosine kinase inhibitors with multiagent chemotherapy in this patient population.

Purpose: We studied the efficacy of cetuximab therapy in patients with metastatic colorectal cancer (mCRC) previously treated with an oral inhibitor of the tyrosine kinase domain of the epidermal growth factor receptor., Patients and Methods: We reviewed the posttrial records of 73 patients with mCRC who participated in 1 of 3 clinical trials that examined a combination of gefitinib or erlotinib with standard cytotoxic chemotherapy. Medical and pharmacy records were used to identify patients who were subsequently treated with cetuximab-based therapy. Computed tomography scans during cetuximab-based therapy were reviewed, and the clinical activity of cetuximab was assessed by response rate using Response Evaluation Criteria in Solid Tumors and progression-free survival., Results: Twenty-four patients with mCRC previously treated with gefitinib or erlotinib and combination cytotoxic chemotherapy who subsequently received cetuximab-based therapy were identified. While receiving cetuximab-based therapy, no patient experienced a partial or complete response; however, 3 patients (16% of patients with available scans for formal measurements) had a minor response, defined as a 15%-29.9% decrease in the sum of longest dimensions of target lesions, and 72% had stable disease. The progression-free survival was 5.1 months for all patients and 6 months for patients who had documented progression of disease while previously receiving gefitinib- or erlotinib-based therapy., Conclusion: Cetuximab appears to have clinical benefit in patients with mCRC previously treated with a chemotherapy regimen that included an oral tyrosine kinase inhibitor of epidermal growth factor receptor. Whether these results apply to other cancer types is unknown but worthy of further study.

Purpose: To investigate the combination of erlotinib, capecitabine, and oxaliplatin in patients who were previously treated for metastatic colorectal cancer., Patients and Methods: Patients were eligible if they had metastatic colorectal cancer that progressed, were intolerant to first-line chemotherapy, or had disease recurrence within 1 year of adjuvant therapy for early-stage disease. Each 21-day cycle consisted of daily oral erlotinib at 150 mg, oral capecitabine at 1,000 mg/m2 (reduced to 750 mg/m2 after the first 13 patients) twice a day on days 1 to 14, and intravenous oxaliplatin at 130 mg/m2 on day 1., Results: Thirty-two patients were enrolled onto this phase II study. By intention-to-treat analyses, eight patients (25%) experienced a partial response and 14 patients (44%) had stable disease for at least 12 weeks. The median progression-free survival was 5.4 months and the median overall survival was 14.7 months. These results were essentially unchanged when limited to the cohort of patients (78%) who received prior irinotecan for metastatic colorectal cancer. Most common grade 3 to 4 toxicities included diarrhea (38%), nausea/emesis (19%), fatigue (16%), dehydration (16%), and dermatitis (13%); grade 3 or 4 toxicities were reduced with a lower starting dose of capecitabine., Conclusion: The combination of capecitabine, oxaliplatin, and erlotinib seems to have promising activity against metastatic colorectal cancer in patients who received prior chemotherapy, with a relatively higher response rate and progression-free survival compared with previous reports of either infusional FU, leucovorin, and oxaliplatin or capecitabine and oxaliplatin in similar patient populations.

Background: With the increasing survival of patients with metastatic colorectal cancer (CRC) there is a growing need for effective second- and third-line agents. We conducted a multicenter, phase II study to examine the combination of capecitabine and thalidomide (Cape/Thal) in patients with refractory metastatic CRC., Methods: Patients with previously treated stage IV CRC were eligible. Treatment consisted of capecitabine at 1,000 mg/m2 po BID for 14 days every 3 weeks and thalidomide, starting at 200 mg po QD continuously. Thalidomide was escalated individually to 600 mg po QD as tolerated. We analyzed overall survival (OS), progression-free survival, response, and toxicity rates., Results: Thirty-four eligible patients were enrolled. The median age was 57 years, and most patients had a normal performance status (65%). All patients had received prior chemotherapy and 19 (56%) had received 2 or 3 prior regimens. The median number of Cape/Thal cycles administered was 3 (range, 1-15). Grade 3/4 toxicities included fatigue (15%), venous thromboembolic events (12%), somnolence (12%), and constipation (9%). Grade 2 hand-foot syndrome occurred in 5 (15%) patients. There were no radiographic responses; 13 patients (38%) achieved stable disease. The median PFS was 2.6 months (95% confidence interval [CI] = 2.2-3.9) and the median OS was 7.1 month (95% CI = 5.2-12.0)., Conclusions: Though well-tolerated, the combination of capecitabine and thalidomide was not associated with objective tumor responses in a population of patients with previously treated metastatic CRC.

Purpose: Recently, activating mutations of the epidermal growth factor receptor (EGFR) gene were discovered in non-small cell lung cancers sensitive to gefitinib (ZD1839, an EGFR tyrosine kinase inhibitor) but not in gefitinib-resistant cancers. Abnormalities of EGFR and related pathways may have an effect on responsiveness of advanced colorectal cancer to combination chemotherapy with gefitinib., Experimental Design: We examined patients with previously untreated metastatic colorectal cancer, who were enrolled into two phase I/II trials of combination chemotherapy (irinotecan, leucovorin, and 5-fluorouracil) and daily oral gefitinib. We obtained paraffin tissue blocks of primary tumors from 31 patients, sequenced the EGFR, KRAS, and BRAF genes, and did immunohistochemistry for EGFR, phosphorylated AKT1, p53, p21, and p27., Results: Twelve (39%) of the 31 patients experienced a partial objective response to the therapy. A novel EGFR mutation in exon 18 (c.2170G>A, p.Gly724Ser) was identified in only one patient who did not experience an objective tumor response. EGFR immunohistochemistry was not predictive of responsiveness. In contrast, loss of p21 was associated with a higher response rate to therapy (P = 0.05). Moreover, the response rate among patients whose tumors maintained p21 expression and possessed a mutation in p53 was only 9% (1 of 11, P = 0.005). Overexpression of phosphorylated AKT1 also seemed to predict a trend towards resistance to the therapy., Conclusions: p21 expression in colorectal cancer, especially in combination with p53 mutation, is a predictor of resistance to the combination chemotherapy with gefitinib. Activating EGFR mutations are rare in colorectal cancer and do not seem to confer sensitivity to gefitinib and chemotherapy.

The causes of interpatient variation in severe toxicity resulting from treatment with weekly 5-fluorouracil (5-FU)/ leucovorin (LV) are poorly understood. This study was undertaken to examine the contribution of commonly occurring polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene to interpatient variability in 5-FU pharmacokinetics and toxicity. Patients with stage III/IV colorectal cancer were treated by bolus intravenous (I.V.) injection with 500 mg/m2 doses of 5-FU and LV once every week. The pharmacokinetics of 5-FU was determined on weeks 1 and 4. Genotyping assays were developed for 8 polymorphisms in the DPYD gene. A well-characterized functional polymorphism in the 5' untranslated region of the thymidylate synthase (TS) gene was also analyzed. A cohort of 22 patients (15 male, 7 female) with a median age of 61 years was evaluated. Although there was no relationship between the area under the plasma concentration time curve (AUC) for the first dose of 5-FU and worst-grade toxicity during the first cycle of therapy, 3 of the 4 patients in whom the AUC on week 4 was more than equal to 5 microgram/h/mL greater than the value for the first dose experienced grade 3/4 toxicity during subsequent treatment. Among the 8 polymorphisms in the DPYD gene, 7 were found to vary in the study population but none were significantly associated with the AUC of 5-FU. There was no relationship between the DPYD and TS genotypes examined and 5-FU toxicity. Extensive polymorphism in the DPYD gene was observed; however, no conclusive correlations existed between the DPYD and TS genotype and 5-FU pharmacokinetics or toxicity. Decreases in 5-FU clearance in certain patients may provide insight into the increased toxicity following repetitive cycles of treatment with weekly I.V. bolus 5-FU. The present study offers useful themes for undertaking larger prospective pharmacogenetic studies in the future.

Purpose: To evaluate the toxicity and efficacy of a modified deGramont regimen of 5-fluorouracil (5-FU), leucovorin, and oxaliplatin in patients with advanced colorectal cancer who have progressed on at least one but not more than two prior chemotherapy regimens., Patients and Methods: Patients with stage 4 colorectal cancer were treated with oxaliplatin 85 mg/m2 by a 2-hour intravenous infusion, followed by leucovorin 500 mg/m2 by a 2-hour intravenous infusion, followed by 5-FU 400 mg/m2 by bolus injection, followed by 5-FU 2.4 g/m2 administered by a 46-hour continuous infusion. Cycles were administered every 2 weeks., Results: Seventy patients were treated and 68 patients had previously received irinotecan. Eleven percent of patients had a partial response, 33% of CEA-evaluable patients had a > or = 50% drop in their CEA level. The median time to progression was 6.2 months, and the median overall survival was 8.7 months. Toxicity was mild to moderate, as 14% of patients experienced grade 3 or 4 neutropenia and 3% of patients experienced grade 3 neuropathy., Conclusion: The modified deGramont regimen of 5-FU, leucovorin, and oxaliplatin is tolerable and is associated with a modest degree of antitumor activity in patients who have progressed on both 5-FU and irinotecan.

Purpose: Troglitazone, a potent activator of the peroxisome proliferator-activated receptor-gamma, induces tumor differentiation in human liposarcomas and causes regression of tumors that are derived from human colon cancer cells in nude mice. We therefore assessed the efficacy of troglitazone in the treatment of metastatic colon cancer in humans., Methods: Twenty-five patients with metastatic colorectal cancer were treated with oral troglitazone. Patients were followed up for evidence of toxicity, tumor response, and survival., Results: The treatment was well tolerated: no grade 3/4 treatment-related toxicities were observed. However, no objective tumor responses were noted, and all 25 patients had progressive disease as their best response to therapy. The median progression-free survival time was only 1.6 months, and the median survival time was 3.9 months., Discussion: Troglitazone is not an active agent for the treatment of metastatic colorectal cancer.