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In patients with locally advanced cancer without distant metastases, the neoadjuvant setting presents a platform to evaluate new drugs. For mismatch repair proficient/microsatellite stable (pMMR/MSS) colon and rectal cancer, immunotherapy has shown limited efficacy. Herein, we report exceptional responses observed with neoadjuvant botensilimab (BOT), an Fc-enhanced next-generation anti-CTLA-4 antibody, alongside balstilimab (BAL; an anti-PD-1 antibody) in two patients with pMMR/MSS colon and rectal cancer. The histological pattern of rapid immune response observed ("inside-out" (serosa-to-mucosa) tumor regression) has not been described previously in this setting. Spatial biology analyses (RareCyte Inc.) reveal mechanisms of actions of BOT, a novel innate-adaptive immune activator. These observations have downstream implications for clinical trial designs using neoadjuvant immunotherapy and potentially sparing patients chemotherapy., (© 2023. The Author(s).)

Background: Previous studies have shown that metastatic colorectal carcinoma (mCRC) patients treated with bevacizumab, experience variation in the plasma levels of angiogenesis growth factors and related cytokines, called angiogenic switch (AS). The aim of the present study was to analyze the relationship between AS and the clinical response during standard chemotherapy-bevacizumab treatment., Patients and Methods: Patients with Eastern Cooperative Oncology Group 0-1 mCRC were eligible. Patients received treatment with standard dose capecitabine plus either oxaliplatin or irinotecan and bevacizumab for 6 cycles. Initial treatment was followed by maintenance therapy with bevacizumab plus capecitabine until progression. Plasma levels of angiogenic-related cytokines (hepatocyte growth factor, placental growth factor, macrophage chemoattractant protein-3, MM-9, eotaxin, basic fibroblast growth factor, and interleukin 18) were prospectively analyzed at baseline and every 8 weeks. Progression-free survival (PFS) was calculated using the Kaplan-Meier method., Results: A total of 71 patients were enrolled. AS was observed in 45 patients (63.4%), 28 of whom experienced AS at the first evaluation after treatment start. Disease control, which includes partial/complete response and stable disease, was seen in 96% of AS patients (43/45), but only in 15/26 (58%) for the remaining patients without evidence of AS (P<0.001). The median PFS of AS patients was 11.4 months (95% confidence interval, 8.6-15.8) versus 8.3 months for patients without AS (95% confidence interval, 5.6-16.4; P=0.04)., Conclusions: Chemotherapy plus Bevacizumab combination in mCRC patients results in dynamic changes in plasma cytokines, which is associated with better disease control and longer PFS. These new findings support continuing studying AS as a potential marker of angiogenesis inhibitor effectiveness.

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR /VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: Duligotuzumab is a dual-action antibody directed against EGFR and HER3. Experimental Design: Metastatic colorectal cancer (mCRC) patients with KRAS ex2 wild-type received duligotuzumab or cetuximab and FOLFIRI until progression or intolerable toxicity. Mandatory tumor samples underwent mutation and biomarker analysis. Efficacy analysis was conducted in patients with RAS exon 2/3 wild-type tumors. Results: Of 134 randomly assigned patients, 98 had RAS ex2/3 wild-type. Duligotuzumab provided no progression-free survival (PFS) or overall survival (OS) benefit compared with cetuximab, although there was a trend for a lower objective response rate (ORR) in the duligotuzumab arm. No relationship was seen between PFS or ORR and ERBB3, NRG1, or AREG expression. There were fewer skin rash events for duligotuzumab but more diarrhea. Although the incidence of grade ≥3 AEs was similar, the frequency of serious AEs was higher for duligotuzumab. Conclusions: Duligotuzumab plus FOLFIRI did not appear to improve the outcomes in patients with RAS exon 2/3 wild-type mCRC compared with cetuximab + FOLFIRI. Clin Cancer Res; 24(10); 2276-84. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In colorectal carcinoma patients, distant metastatic disease is present at initial diagnosis in nearly 25% of them. The majority of patients with metastatic colorectal carcinoma have incurable disease; therefore, new therapies are needed. Agents derived from medicinal plants have already demonstrated therapeutic activities in human cancer cells. Antartina is an antitumor agent isolated from Deschampsia antarctica Desv. This study aimed to evaluate the antitumor properties of Antartina in colorectal carcinoma models. We used human and murine colorectal carcinoma cell lines for investigating proliferation, apoptosis, and cell-cycle effects of Antartina therapy in vitro Avatar and immunocompetent colorectal carcinoma animal models were applied for evaluating the effects of Antartina in vivo Immune response against colorectal carcinoma model was investigated using CTL assay, analyzing dendritic cell activation and intratumor T-cell subpopulation, and by tumor rechallenge experiments. Antartina inhibits in vitro human colorectal carcinoma cell proliferation; however, in vivo experiments in Avatar colorectal carcinoma model Antartina display a limited antitumor effect. In an immunocompetent colorectal carcinoma mice model, Antartina potently inhibited tumor growth and liver metastases, leading to complete tumor regressions in >30% of mice and increased animal survival. In addition, Antartina induced a potent specific cytotoxic T-cell response against colorectal carcinoma and a long-lasting antitumor immunity. Interestingly, Antartina increased tumor immunogenicity and stimulated dendritic cell activation. No toxic effects were observed at the doses employed. Our findings showed that Antartina has the ability to induce antitumor immunity against colorectal carcinoma and can be used to develop new tools for the treatment of colorectal carcinoma. Mol Cancer Ther; 17(5); 966-76. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Lessons Learned: Cobimetinib and duligotuzumab were well tolerated as single agents and in combination with other agents.The cobimetinib and duligotuzumab combination was associated with increased toxicity, most notably gastrointestinal, and limited efficacy in the patient population tested., Background: KRAS -mutant tumors possess abnormal mitogen-activated protein kinases (MAPK) pathway signaling, leading to dysregulated cell proliferation. Cobimetinib blocks MAPK signaling. The dual-action antibody duligotuzumab (MEHD7945A) inhibits ligand binding to both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3). Blockade of EGFR/HER3 and inhibition of mitogen-activated protein kinase (MEK) in KRAS -mutant tumors may provide additive benefit., Methods: Patients with KRAS -mutant solid tumors were eligible for this phase Ib dose-escalation study with a planned expansion phase. Duligotuzumab was given intravenously (IV) at 1,100 mg every 2 weeks (q2w), while cobimetinib was given orally in a standard 3 + 3 design to identify the recommended phase II dose (RP2D). The primary objective was to evaluate the safety and tolerability of this combination., Results: Twenty-three patients were enrolled. Dose-limiting toxicities (DLTs) included grade 4 hypokalemia and grade 3 mucosal inflammation, asthenia, and dermatitis acneiform. Seventy percent of patients experienced grade 3 or worse adverse events (AEs). Five (22%) and 12 (52%) patients missed at least 1 dose of duligotuzumab and cobimetinib, respectively, and 9 (39%) patients required a cobimetinib dose reduction. Three (13%) patients discontinued due to an AE. Best response was limited to 9 patients with stable disease and 13 patients with progressive disease., Conclusion: Given the limited tolerability and efficacy of this combination, the study did not proceed to expansion stage and closed for enrollment., (© AlphaMedPress; the data published online to support this summary is the property of the authors.)

Epigenetic control of gene expression is a major determinant of tumor phenotype and has been found to influence sensitivity to individual chemotherapeutic agents. Glutathione peroxidase 3 (GPX3, plasma glutathione peroxidase) is a key component of cellular antioxidant regulation and its gene has been reported to be methylated in specific tumor types. GPX3 role in oxidative damage has been associated with sensitivity to platinums in other tumors but its importance in colorectal cancer (CRC) has not been determined. We examined the role of GPX3 methylation in colorectal carcinoma in determining sensitivity to platinum drugs using primary tumor specimens, cell lines, knockdown cell lines, and tumor cell line xenografts. We find GPX3 promoter region methylation in approximately one third of CRC samples and GPX3 methylation leads to reduced GPX3 expression and increased oxaliplatin and cisplatin sensitivity. In contrast, in cell lines with high baseline levels of GPX3 expression or with the ability to increase GPX3 expression, platinum resistance is increased. The cisplatin IC 50 in GPX3-methylated cell lines is approximately 6-fold lower than that in GPX3-unmethylated lines. Additionally, knockdown cell lines with essentially no GPX3 expression require N-acetylcysteine to survive in culture underscoring the importance of GPX3 in redox biology. In vivo, GPX3 methylation predicts tumor xenograft sensitivity to platinum with regression of GPX3 knockdown xenografts with platinum treatment but continued growth of GPX3 wild type xenografts in the presence of platinum. These studies demonstrate the importance of GPX3 for CRC cells resistance to platinums and the potential utility of GPX3 methylation status as a predictive biomarker for platinum sensitivity in CRC.

Cancer genomics and translational medicine rely on the molecular profiling of patient's tumor obtained during surgery or biopsy. Alternatively, blood is a less invasive source of tumor DNA shed, amongst other ways, as cell-free DNA (cfDNA). Highly-sensitive assays capable to detect cancer genetic events from patient's blood plasma became popularly known as liquid biopsy (LqB). Importantly, retrospective studies including small number of selected patients with metastatic colorectal cancer (mCRC) patients treated with anti-EGFR therapy have shown LqB capable to detect the acquired clonal mutations in RAS genes leading to therapy resistance. However, the usefulness of LqB in the real-life clinical monitoring of these patients still lack additional validation on controlled studies. In this context, we designed a prospective LqB clinical trial to monitor newly diagnosed KRAS wild-type (wt) mCRC patients who received a standard FOLFIRI-cetuximab regimen. We used BEAMing technique for evaluate cfDNA mutations in KRAS, NRAS, BRAF, and PIK3CA in twenty-five patients during a 2-y period. A total of 2,178 cfDNA mutation analyses were performed and we observed that: a) continued wt circulating status was correlated with a prolonged response; b) smoldering increases in mutant cfDNA were correlated with acquired resistance; while c) mutation upsurge/explosion anticipated a remarkable clinical deterioration. The current study provides evidences, obtained for the first time in an unbiased and prospective manner, that reinforces the utility of LqB for monitoring mCRC patients.

ABT-751 is an orally bioavailable sulfonamide with antimitotic properties. A nonrandomized phase 1 dose-escalation study of ABT-751 in combination with CAPIRI (capecitabine and irinotecan) and bevacizumab was conducted to define the maximum tolerated dose, dose-limiting toxicity (DLT), and pharmacokinetics in patients with advanced colorectal cancer. Patients were treated with ABT-751 daily for 7 days (alone) and then began 21-day cycles of treatment with ABT-751 daily and capecitabine twice daily for 14 days plus irinotecan on day 1 intravenously. Bevacizumab was added as standard of care at 7.5 mg/kg on day 1 after the first 2 dose levels. Because of intolerance to the regimen, a reduced dose of ABT-751 was also explored with reduced-dose and full-dose CAPIRI with bevacizumab. ABT-751 and irinotecan pharmacokinetics, ABT-751 glucuronidation, and protein binding were explored. Twenty-four patients were treated over 5 dose levels. The maximum tolerated dose was ABT-751 125 mg combined with full-dose CAPIRI and bevacizumab 7.5 mg/kg on day 1. DLTs were hypokalemia, elevated liver tests, and febrile neutropenia. ABT-751 is metabolized by UGT1A8 and to a lesser extent UGT1A4 and UGT1A1. Irinotecan and APC exposure were increased, SN-38 exposure was similar, and SN-38 glucuronide exposure was decreased. Clinically relevant alterations in ABT-751 and irinotecan pharmacokinetics were not observed. Despite modest efficacy, the combination of ABT-751, CAPIRI, and bevacizumab will not be studied further in colorectal cancer., (© 2016, The Authors. The Journal of Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.)

Purpose: The aim of this study was to investigate the feasibility and efficacy of personalizing treatment of patients with advanced untreated colorectal cancer (CRC)., Patients and Methods: Patients with untreated metastatic CRC, performance status 0-1, and candidates for systemic chemotherapy were eligible. Tumor tissues were analyzed for KRAS, BRAF, and PI3K mutations and expression of topoisomerase-1 (Topo-1), excision repair cross-complementing gene 1 (ERCC1), thymidylate synthase (TS), and thymidine phosphorylase (TP). Patients with Topo-1 expression received irinotecan, whereas patients with negative Topo-1 and ERCC1 expression received oxaliplatin. Otherwise, patients received physician's choice of treatment. If TS was positive, no fluoropyrimidine was administered and if negative, 5-flurorouracil if TP was negative, or capecitabine if TP was positive. KRAS-mutated patients were treated with bevacizumab, whereas KRAS-native received cetuximab. The primary endpoint of the study was progression-free survival (PFS)., Results: A total of 74 patients were enrolled and 67 received personalized treatment including irinotecan (n=27), oxaliplatin (n=16), FOLFIRI (n=12), and FOLFOX (n=12). Thirty-eight patients received cetuximab and 29 bevacizumab. With a median follow-up time of 18.3 months (95% confidence interval [CI], 4-36), the overall median PFS was 8.3 months (95% CI, 6.9-9.7), representing a 12-month PFS rate of 36.5% (95% CI, 25-48). Overall clinical benefit, including response rate and disease stabilization, was 86% (95% CI, 73%-97%). The overall median survival was 21 months (95% CI, 11-40)., Conclusions: Real-time target-guided personalized first-line treatment of patients with advanced CRC is feasible but, with the approached used, did not result in a clear improvement in PFS to warrant phase III testing.

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations., (©2015 American Association for Cancer Research.)

Background This phase Ib study was designed to determine the maximum tolerated doses (MTD) and dose limiting toxicities (DLTs) of irinotecan and cetuximab with sorafenib. Secondary objectives included characterizing the pharmacokinetics and pharmacodynamics and evaluating preliminary antitumor activity in patients with advanced colorectal cancer (CRC). Methods Patients with metastatic, pretreated CRC were treated at five dose levels. Results Eighteen patients were recruited with median age 56.5 years. In the first five patients treated, 2 irinotecan related DLTs were observed. With reduced dose intensity irinotecan, there were no further DLTs. The most common toxicities were diarrhea, nausea/vomiting, fatigue, anorexia and rash. DLTs included neutropenia and thrombocytopenia. Two patients had partial responses (one with a KRAS mutation) and 8 had stable disease (8-36 weeks). The median progression free survival (PFS) and overall survival (OS) were 2.5 and 4.7 months respectively. Pharmacokinetic analyses suggest sorafenib and metabolite exposure correlate with OS and DLTs. Conclusions The recommended phase II dose (RP2D) is irinotecan 100 mg/m(2) i.v. days 1, 8; cetuximab 400 mg/m(2) i.v. days 1 and 250 mg/m(2) i.v. weekly; and sorafenib 400 mg orally twice daily in advanced, pretreated CRC. The combination resulted in a modest response rate.

The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors.

New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444-2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor., Experimental Design: Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts., Results: In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups., Conclusion: These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects., (©2011 AACR.)

Unlabelled: Single-agent cetuximab is safe and active in elderly patients with advanced colorectal cancer (CRC). A cetuximab-capecitabine combination has not previously been tested in elderly patients with advanced CRC., Material and Methods: Sixty-six patients with advanced CRC were treated with cetuximab as a 400 mg/m2 i.v. infusion followed by 250 mg/m2 i.v. weekly plus capecitabine at a dose of 1,250 mg/m2 every 12 hours. After the inclusion of 27 patients, the protocol was amended for safety reasons, reducing the dose of capecitabine to 1,000 mg/m2 every 12 hours. Thirty-nine additional patients were treated with the reduced dose of capecitabine., Results: The overall response rate was 31.8%. KRAS status was determined in 58 patients (88%). Fourteen of 29 patients with wild-type KRAS tumors responded (48.3%; 95% confidence interval [CI], 29.4%-67.5%), compared with six of 29 patients with mutant KRAS tumors (20.7%; 95% CI, 8.0%-39.7%). The median progression-free survival (PFS) interval was 7.1 months. The median PFS interval for patients whose tumors were wild-type KRAS was significantly longer than for those with mutant KRAS tumors (8.4 months versus 6.0 months; p = .024). The high incidence of severe paronychia (29.6%) declined (7.7%) after capecitabine dose adjustment., Conclusions: Cetuximab plus capecitabine at a dose of 1,000 mg/m2 every 12 hours may be an alternative to more aggressive regimens in elderly patients with advanced wild-type KRAS CRC.

Background and Objectives: Early age of onset is a marker of a possible hereditary component in colorectal cancer (CRC). We evaluated whether early age of onset is a good marker to identify Lynch syndrome, especially retrospectively, and if there is any other feature that could improve this identification., Methods: We selected patients with CRC aged 45 years or younger from the pathological reports of three different institutions and different periods of time. Clinical information, family history, and tumor samples were obtained. Cases were classified according to mismatch repair (MMR) proficiency., Results: Of 133 tumors, 22 showed microsatellite instability (MSI). In 15 MSI cases, a germline mutation in 1 of the MMR genes was identified, 7 of which were not identified before. The positive predictive value (PPV) of right colon CRC for a positive genetic MMR test is 30.6%, whereas "signet ring" cells and fulfillment Amsterdam II criteria have PPVs of 42.9% and 47.8%, respectively. Combining right-sided CRC with mucin production, with fulfilling Amsterdam II criteria, or with "signet ring" cells, PPVs are 54.5, 64.3, and 100%. The probability of the absence of a mutation when CRC is located in the left colon is 94.7%, whereas absence of aggregation for Lynch-related neoplasm has a 100% probability., Conclusions: Early age of onset is an effective method to identify retrospectively Lynch syndrome. Taking into account the location and histology features of the tumor, and the familial history of the cases, we notably increase the a priori probability of detecting a germline MMR mutation.

Rationale: This phase I study was conducted to determine the maximum tolerated dose (MTD) of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, with 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX4) in patients with advanced colorectal cancer (CRC). Bevacizumab was later included as standard of care at the MTD., Patients and Methods: Patients received FOLFOX4 with escalating doses of erlotinib: dose level (DL) 1, 50 mg; DL 2, 100 mg; and DL 3, 150 mg once daily continuously. Bevacizumab 5 mg/kg days 1 and 15 was added at the MTD upon Food and Drug Administration approval. Correlative studies included pharmacokinetics, pharmacodynamics was assessed in paired skin biopsies, and fluorodeoxyglucose positron emission tomography scans., Results: Fifteen patients received 60 cycles (120 FOLFOX treatments). Two dose-limiting toxicities (DLTs) were seen at DL 3: intolerable grade 2 rash (Common Terminology Criteria for Adverse Events version 2) lasting > 1 week, and grade 4 neutropenia. Dose level 2 was expanded to 6 more patients, this time adding bevacizumab, and 1 DLT of grade 3 mucositis occurred. As expected, the primary toxicities were cytopenias, diarrhea, rash, and fatigue. There were 2 occurrences of pneumatosis. One patient experienced an unrelated grade 4 myocardial infarction before starting chemotherapy. No pharmacokinetic drug interactions were observed. The Response Evaluation Criteria in Solid Tumors response rate was 11 of 14 (78%), median progression-free survival was 9.5 months, and median overall survival was 30 months. Three patients are currently alive > 3 years, with 1 having no evidence of disease., Conclusion: The MTD of erlotinib with FOLFOX4 with or without bevacizumab is 100 mg daily. The regimen appeared to increase toxicity but showed activity in patients with CRC.

Aim: To characterize clinicopathological and familial features of early-onset colorectal cancer (CRC) and compare features of tumors with and without microsatellite instability (MSI)., Methods: Forty-five patients with CRC aged 45 or younger were included in the study. Clinical information, a three-generation family history, and tumor samples were obtained. MSI status was analyzed and mismatch repair genes were examined in the MSI families. Tumors were included in a tissue microarray and an immunohistochemical study was carried out with a panel of selected antibodies., Results: Early onset CRC is characterized by advanced stage at diagnosis, right colon location, low-grade of differentiation, mucin production, and presence of polyps. Hereditary forms represent at least 21% of cases. Eighty-one percent of patients who died during follow-up showed a lack of expression of cyclin E, which could be a marker of poor prognosis. beta-catenin expression was normal in a high percentage of tumors., Conclusion: Early-onset CRC has an important familial component, with a high proportion of tumors showing microsatellite stable. Cyclin E might be a poor prognosis factor.

KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post- PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%).

To examine the reaction of tumour arteries to endothelin-1, we obtained arteries supplying blood flow to colorectal tumours from patients, as well as mesenteric arteries supplying the normal colon tissue from the same patients and mesenteric arteries from patients without a colorectal tumour pathology. The contraction in response to endothelin-1 and the relaxation produced by bradykinin was recorded in each of these arteries. Accordingly, the sensitivity to endothelin-1 but not the maximal response, was higher in the arteries supplying colorectal tumours than in mesenteric arteries supplying normal colon or in mesenteric arteries from patients with no tumour pathology. The contraction produced by endothelin-1 was not modified by exposure to L-NAME or meclofenamate in arteries supplying both the tumour and the normal colon. The endothelin ET(A) andET(B) receptors were expressed similarly in arteries supplying the tumour or normal colon. However, the antagonist of the endothelin ET(B) receptors BQ788 (10(-6) M) decreased the contractions in the arteries supplying the tumour but not in those supplying the normal colon. By contrast, the antagonist of endothelin ET(A) receptors BQ123 (10(-6) M) reduced the contraction equally in both these types of arteries. Likewise, in arteries precontracted with U46619, the relaxation in response to bradykinin was similar in all three types of arteries. Together, these results suggest that the arteries supplying human colorectal tumours are more sensitive to endothelin-1, which could be due to the enhanced activity of endothelin ET(B) receptors in the absence of any change in the modulatory effect of nitric oxide or prostanoids in the arterial response to this peptide.

Purpose: The antiepidermal growth factor receptor (EGFR) antibody cetuximab shows activity in multiple epithelial tumor types; however, responses are seen in only a subset of patients. This study was conducted to identify markers that are associated with disease control in patients treated with cetuximab., Patients and Methods: One hundred ten patients with metastatic colorectal cancer were enrolled onto a cetuximab monotherapy trial. Transcriptional profiling was conducted on RNA from mandatory pretreatment metastatic biopsies to identify genes whose expression correlates with best clinical responses. EGFR and K-ras mutation analyses and EGFR gene copy number analyses were performed on DNA from pretreatment biopsies., Results: Gene expression profiles showed that patients with tumors that express high levels of the EGFR ligands epiregulin and amphiregulin are more likely to have disease control with cetuximab (EREG, P = .000015; AREG, P = .000025). Additionally, patients whose tumors do not have K-ras mutations have a significantly higher disease control rate than patients with K-ras mutations (P = .0003). Furthermore, patients with tumors that have high expression of EREG or AREG also have significantly longer progression-free survival (PFS) than patients with low expression (EREG: P = .0002, hazard ratio [HR] = 0.47, and median PFS, 103.5 v 57 days, respectively; AREG: P < .0001, HR = 0.44, and median PFS, 115.5 v 57 days, respectively)., Conclusion: Patients with tumors that have high gene expression levels of epiregulin and amphiregulin and patients with wild-type K-ras are more likely to have disease control on cetuximab treatment. The identified markers could be developed further to select patients for cetuximab therapy.

The Epidermal Growth Factor Receptor (EGFR) plays a role in multiple tumor cell processes and is targeted by several anticancer therapies. Although EGFR mutations may determine tumor susceptibility in a small proportion of patients, knowledge of the EGFR signaling pathway status in tumors may help guide further drug development and hypothesis-driven combination studies. We aimed to validate and apply a novel computer-aided immunohistochemical (IHC) technique to characterize the status of EGFR signaling in matched colorectal tumor and normal colon tissue samples. Tissue Microarrays (TMA)were made from both cancerous and normal colorectal tissue in 18 patients and stained with antibodies against EGFR, phospho-EGFR (pEGFR), Akt, pAkt, MAPK, and pMAPK. TMA's were quantitatively scored using the Automated Cellular Imaging System (ACIS II, Chromavision, Inc). ACIS was compared against cell line Western blotting, ELISA, and visual scoring (0-3+) by a pathologist. We found that ACIS analysis was highly reproducible and results were well correlated with other techniques. A post-scan "image microdissection" technique of analyzing heterogeneous human samples showed good correlation between paired human samples [Pearson correlation for tumors, 0.922 (p < .001)]. Cancer samples had markedly higher staining of pEGFR, Akt, pAkt, MAPK, and pMAPK. We conclude that ACIS IHC of human tissue samples is quantitative, reproducible, and correlates with Western blots and ELISA in cell line pellets as well as pathologist's scores of human samples. Colorectal tumors show higher staining of pEGFR and downstream effectors compared to matched normal colorectal tissues.

Purpose: This phase I study was conducted to establish the dose-limiting toxicities and maximum-tolerated dose of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, in combination with FOLFIRI, a standard regimen of irinotecan, leucovorin, and infusional 5-fluorouracil (5-FU) in patients with advanced colorectal cancer., Experimental Design: The trial used a dose-escalation design beginning with 100 mg/day erlotinib continuously and dose-reduced FOLFIRI (150 mg/m2 i.v. day 1 irinotecan, 200 mg/m2 i.v. leucovorin, 320 mg/m2 i.v. bolus days 1 to 2 5-FU, and 480 mg/m2 i.v. 5-FU infusion over 22 hours, days 1 to 2) administered in 6-week cycles (three FOLFIRI treatments). Plasma sampling was performed for irinotecan, erlotinib, and 5-FU for pharmacokinetic analysis during cycle 1., Results: The study was halted after six patients at the lowest dose level due to unexpectedly severe toxicities, including disfiguring grade 2 rash (three patients), grade 3 diarrhea (three patients), and grade > or = 3 neutropenia (three patients). All patients required some dose interruption or reduction of either erlotinib or FOLFIRI, and only one patient completed two 6-week cycles of therapy. Five patients had stable disease after one cycle, and one patient had a partial response. No plasma pharmacokinetic interaction was observed that could explain the observed increased toxicity., Conclusions: FOLFIRI combined with erlotinib causes excessive toxicity at reduced doses. These findings contrast with available data regarding the optimal safety profile of trials combining small molecule epidermal growth factor receptor inhibitors with other conventional chemotherapy and highlight the need to perform safety-oriented studies of such combinations.

Colorectal carcinoma is the third most common malignancy worldwide. The use of currently available therapies results in only a modest impact on overall survival of patients with advanced-stage disease. New approaches for the treatment of colorectal cancer are urgently needed. The epidermal growth factor receptor (EGFR) is frequently dysregulated in colorectal carcinoma, and overexpression of the receptor confers a poor prognosis. Targeting the EGFR has become a rational approach for the treatment of colorectal carcinoma. Several strategies to inhibit the EGFR and its downstream signaling pathways are currently being investigated in preclinical and clinical studies, including monoclonal antibodies directed against the extracellular domain of the receptor and small-molecule inhibitors of its tyrosine kinase activity. Some of these drugs have already been tested in colorectal cancer and have shown preliminary evidence of antitumor efficacy. Important issues to elucidate in the future include the definition of the biologic context in which these drugs are more likely to be effective and the integration of the different agents in current therapeutic strategies for colorectal cancer. This article will provide a comprehensive review of the current available preclinical and clinical data on EGFR-targeted therapies in colorectal cancer.

Recent advances in the treatment of colorectal cancer have lead to significant gains in response rates and survival. The combination of newer agents such as irinotecan and oxaliplatin with 5-fluorouracil/leucovorin using various dosing schedules in the metastatic setting has resulted in a steady improvement in the outcome of patients with colorectal cancer. Experimental therapies such as epidermal growth factor receptor inhibitors, vascular endothelial growth factor inhibitors and cyclooxygenase-2 inhibitors, have shown promise in early clinical trials and have acceptable toxicity profiles. Efforts towards improving risk-stratification of stage II colorectal cancer patients and optimising therapy in patients with advanced disease, have focused on molecular and genetic markers. It is hoped that the addition of new therapies to existing drug combinations, as well as further advances in the understanding of colorectal cancer biology, will lead to further improvement in survival and quality of life for patients.

In patients with locally advanced cancer without distant metastases, the neoadjuvant setting presents a platform to evaluate new drugs. For mismatch repair proficient/microsatellite stable (pMMR/MSS) colon and rectal cancer, immunotherapy has shown limited efficacy. Herein, we report exceptional responses observed with neoadjuvant botensilimab (BOT), an Fc-enhanced next-generation anti-CTLA-4 antibody, alongside balstilimab (BAL; an anti-PD-1 antibody) in two patients with pMMR/MSS colon and rectal cancer. The histological pattern of rapid immune response observed (inside-out (serosa-to-mucosa) tumor regression) has not been described previously in this setting. Spatial biology analyses (RareCyte Inc.) reveal mechanisms of actions of BOT, a novel innate-adaptive immune activator. These observations have downstream implications for clinical trial designs using neoadjuvant immunotherapy and potentially sparing patients chemotherapy.