anti-EGFR Antibodies In Patients Research Articles

TRIDENTE trial: A phase II study of rechallenge with encorafenib, binimetinib, and cetuximab in patients with RAS wild-type/BRAF V600E–mutant metastatic colorectal cancer.

TPS264 Background: The BEACON CRC trial demonstrated survival benefit of combination therapy with encorafenib (ENCO) + cetuximab (CET) +/- binimetinib (BINI) in patients with RAS wild-type (WT)/ BRAF V600E mutant metastatic colorectal cancer (mCRC). However, prognosis of those patients still be poor after the refractoriness to the BEACON combination therapy. One of resistant mechanisms to BRAF inhibitor has been reported as MAPK alterations including RAS mutation, similar to those for anti-EGFR antibody in patients with RAS/ BRAF V600E WT mCRC. Promising results of rechallenge therapy with anti-EGFR antibody in patients with RAS/ BRAF V600E WT mCRC and with combination of BRAF inhibitor + MEK inhibitor in patients with BRAF V600 mutant melanoma suggest the treatment strategy of rechallenge therapy with the BEACON triplet therapy in patients with RAS WT/ BRAF V600E mutant mCRC. Methods: TRIDENTE trial is a multicenter phase II trial to assess efficacy and safety of rechallenge therapy with ENCO + BINI + CET in patients with RAS WT/ BRAF V600E mutant mCRC after the refractoriness to either the BEACON doublet or triplet therapy. Key eligibility criteria includes RAS WT/ BRAF V600E mutant mCRC; ≥ 20 years old; ECOG PS 0-1; refractory or intolerant to at least one fluoropyrimidine-based regimen (including irinotecan or oxaliplatin); refractory or intolerant to anti-PD-1 antibody if patients with MSI-high; history of previous combination therapy containing ENCO + CET with at least partial response by RECIST v1.1; confirmed disease progression within 4 weeks after last administration of previous ENCO; ≥ 4 months of period between the last administration of previous ENCO and the start of study treatment. Enrolled patients receive the combination therapy with ENCO (300 mg, QD), BINI (45 mg, BID), and CET (initially 400 mg/m2, and subsequently 250 mg/m2, QW) as the study treatment. Primary endpoint is the objective response rate (ORR) by investigators’ assessment in patients receiving at least one dose of study treatment. A target sample size is calculated to be 21 on the hypothesis that the threshold ORR is 3% and expected ORR is 20%, with a significant level of 5% (one-sided) and power of 80%. Secondary endpoint includes progression-free and overall survivals, disease control rate, and safety. Exploratory molecular analysis is performed using targeted next-generation sequencing in circulating-tumor DNA at the timepoints of baseline and discontinuation of study treatment. As of September 19, 2022, 8 patients have been enrolled. Clinical trial information: jRCTs031210511 .

The effect of primary tumor location on second- or later-line treatment with anti-EGFR antibodies in patients with metastatic colorectal cancer: A single-center cohort study.

3541 Background: The guideline recommends anti-EGFR monoclonal antibodies (anti-EGFR Ab) as first-line treatment only for patients with left-sided RAS wild type (RASwt) metastatic colorectal cancer (mCRC). However, there are no recommendations on tumor sidedness in subsequent lines. This study aimed to evaluate the effect of primary tumor location on second- or later-line treatment outcomes in patients with KRASwt mCRC. Methods: Medical records of patients diagnosed with mCRC at Siriraj Hospital between 2008 and 2019 were retrospectively reviewed. Patients with KRASwt who received anti-EGFR Ab in second- or later-line treatment were included. The impact of tumor sidedness on progression-free survival (PFS) was determined using the Kaplan-Meier method and compared using the log-rank test. Results: Of 671 patients who had data on KRAS analysis, 396 patients (59%) had KRASwt. Of these, 210 patients received anti-EGFR Ab in second- or later-line treatment. Twenty-nine percent of patients (60 out of 210) had extended RAS analysis. Thirty patients (14%) had right-sided tumors, while 180 patients (86%) had left-sided tumors. Sixty-nine percent of patients (146 of 210) were treated in the third line, while 19% and 12% were treated in the second and the fourth line, respectively. Single-agent irinotecan was the most commonly used chemotherapy backbone (92%). Patients with right-sided tumors had non-significantly inferior PFS compared with patients with left-sided tumors (median PFS was 4.7 months, 95% CI 0.8–8.7 vs. 6 months, 95% CI 4.6–7.3; p = 0.55). Subgroup analysis on the impact of primary tumor location showed no difference in PFS when stratified by treatment lines. Conclusions: This study demonstrated that tumor sidedness has no impact on treatment outcomes in patients treated with anti-EGFR Ab in second- or later-line treatment. Therefore, there is not enough evidence to use tumor sidedness for treatment selection in these settings. A multi-center retrospective study is ongoing.

RAS Mutations in Circulating Tumor DNA and Clinical Outcomes of Rechallenge Treatment With Anti-EGFR Antibodies in Patients With Metastatic Colorectal Cancer.

Several trials have evaluated the efficacy of rechallenge treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) in patients with metastatic colorectal cancer (mCRC). A recent trial indicated that RAS status in circulating tumor DNA (ctDNA) may potentially predict patients with RAS wild-type mCRC resistant to anti-EGFR mAb who would benefit from rechallenge treatment, and the findings should be further investigated. We enrolled patients whose plasma samples were collected in prospective phase II trials, the JACCRO CC-08 (n = 36) and CC-09 (n = 25), which evaluated rechallenge chemotherapy with anti-EGFR mAb for KRAS wild-type mCRC. RAS in ctDNA was analyzed at the time points of baseline, 8 weeks, and progression using OncoBEAM RAS CRC kit. Sixteen patients were enrolled in this study, with a response rate of 0% and a disease control rate (DCR) of 62.5%. RAS mutations were found at baseline in six patients. The DCR was 33% in patients with RAS mutations in ctDNA, whereas it was 80% in patients without RAS mutation at baseline. Patients with RAS mutation at baseline had significantly shorter progression-free survival (PFS) and overall survival (OS) than those without RAS mutation (median PFS, 2.3 v 4.7 months; hazard ratio [HR], 6.2; P = .013; median OS, 3.8 v 16.0 months; HR, 12.4; P = .0028). Six of 10 patients without RAS mutation at baseline acquired RAS mutations at progression. Postprogression survival after rechallenge treatment was numerically shorter in patients with RAS mutation at progression. RAS status in ctDNA was significantly associated with clinical outcomes in patients with mCRC receiving rechallenge treatment with anti-EGFR mAb. These findings could support the clinical utility of OncoBEAM RAS CRC kits for anti-EGFR mAb rechallenge in RAS wild-type mCRC.

Japanese Society of Medical Oncology Clinical Guidelines: Molecular Testing for Colorectal Cancer Treatment, 4th edition.

Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first‐line chemotherapy to assess the benefit of anti–epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)‐based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti–EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next‐generation sequencing‐based tests are weakly recommended because these tests have not been validated in clinical trials.

Perioperative use of anti-EGFR antibodies in resectable and potentially resectable liver metastases

The radical resection of all visible disease manifestations can significantly improve the prognosis of patients with metastatic colorectal cancer, however, complete metastasectomy is initially possible only in a small proportion of patients, with many of them subsequently developing relapse. Aiming to increase the number of patients subject to radical intervention and to reduce the recurrence risk, preoperative chemotherapy (with or without subsequent adjuvant treatment) is widely used in routine practice. In the hope of increasing immediate effectiveness (increasing the proportion of patients subject to R0 resection) and the long-term results of treatment, preoperative chemotherapy is often accompanied with biological agents (anti-EGFR and anti-VEGF monoclonal antibodies) continued after surgery in some cases. However, despite the evidence of increased resectability, the impact of this approach on long-term treatment outcomes has not yet been determined. The analysis of recent studies in this area doubts the safety of perioperative use of anti-EGFR antibodies in patients with potentially curable metastatic colorectal cancer.

RAS mutations in circulating tumor DNA (ctDNA) and clinical outcomes of rechallenge treatments with anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC)

RAS mutations in circulating tumor DNA (ctDNA) and clinical outcomes of rechallenge treatments with anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC)

Molecular dissection of effector mechanisms of RAS-mediated resistance to anti-EGFR antibody therapy.

Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), cetuximab and panitumumab, are a mainstay of metastatic colorectal cancer (mCRC) treatment. However, a significant number of patients suffer from primary or acquired resistance. RAS mutations are negative predictors of clinical efficacy of anti-EGFR antibodies in patients with mCRC. Oncogenic RAS activates the MAPK and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways in RAS-mutant CRC is less defined. A better mechanistic understanding of RAS-mediated resistance may guide development of rational intervention strategies. To this end we developed cancer models for functional dissection of resistance to anti-EGFR therapy in vitro and in vivo. To selectively activate MAPK- or AKT-signaling we expressed conditionally activatable RAF-1 and AKT in cancer cells. We found that either pathway independently protected sensitive cancer models against anti-EGFR antibody treatment in vitro and in vivo. RAF-1- and AKT-mediated resistance was associated with increased expression of anti-apoptotic BCL-2 proteins. Biomarkers of MAPK and PI3K/AKT pathway activation correlated with inferior outcome in a cohort of mCRC patients receiving cetuximab-based therapy. Dual pharmacologic inhibition of PI3K and MEK successfully sensitized primary resistant CRC models to anti-EGFR therapy. In conclusion, combined targeting of MAPK and PI3K/AKT signaling, but not single pathways, may be required to enhance the efficacy of anti-EGFR antibody therapy in patients with RAS-mutated CRC as well as in RAS wild type tumors with clinical resistance.

Abstract 5014: Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Abstract The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in patients with colorectal cancer, and EGFR is an important therapeutic target in such patients. To date, of the anti-EGFR monoclonal antibodies (mAbs), only cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). While treatment with a combination of antibodies and cytotoxic drugs improves response rate and median time to progression in some colorectal cancer patients, the duration of response is often limited. In addition, no clear association has been found between the expression level of the EGFR protein in the tumours, determined by the FDA-approved EGFR PharmDx kit, or other standard anti-EGFR antibodies, and the response to the EGFR inhibitors. We have previously shown that kits such as the EGFR PharmDx™ kit, used to determine the expression of EGFR, does not discriminate between the wild-type EGFR (wtEGFR) and the type III deletion mutant EGFR (EGFRvIII), and as such could have contributed to the lack of association between the expression of EGFR and response to anti-EGFR antibodies. Therefore, the aim of this study was to determine the predictive value of wtEGFR for response to therapy with cetuximab. The expression levels of wtEGFR, mutated EGFRvIII, and phosphorylated EGFR (pEGFR Tyr1068 & Tyr1173) were determined using immunohistochemistry and specific antibodies in 61 FFPE tumour specimens from patients with mCRC treated with cetuximab. Sections were scored by the percentage of positive tumour cells, location and intensity of staining. Their associations with response and progression free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test respectively. Overall 11% and 46% of the cases were found to express membranous and cytoplasmic EGFR, using mAb specific for wtEGFR (DAK-H1-WT). On the other hand using anti EGFR.113 clone antibody, which does not discriminate between the wtEGFR and EGFRvIII, 11.5% and 18% of the cases were found to express the cytoplasmic and membranous EGFR respectively. While all cases were negative for the pEGFR, the expression of EGFRvIII was mainly cytoplasmic, occurring in 41% of the cases examined. Of these, only the cytoplasmic expression of wtEGFR was found to be significantly associated with poor response to treatment (P = 0.002) and progression free survival (95% CI, 0-3 months vs 4.5-17 months, P = 0.001). Taken together, our results suggest that cytoplasmic expression of wtEGFR is associated with a significantly shorter progression free survival, and is a predictive marker for poor response to treatment with the anti-EGFR mAb cetuximab. These findings underlie the need for further investigations, in a larger population of patients, to validate the predictive value of wtEGFR for response to therapy with anti-EGFR antibodies in patients with mCRC. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Izhar Bagwan, Margaret Green, Alan Seddon, Helmout Modjtahedi. Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5014.

Final results of a first-in-human study evaluating the safety, pharmacology and initial efficacy of MM-151, an oligoclonal anti-EGFR antibody in patients with refractory solid tumors.

2518Background: MM-151 is a mixture of three IgG1 antibodies designed to bind simultaneously to non-overlapping EGFR epitopes and inhibit ligand-mediated signal amplification, downregulate EGFR expression, and enhance immune-effector activities (ADCC, CDC). A Phase 1 study was completed to assess safety, tolerability, pharmacology and preliminary clinical activity of MM-151 alone and in combination with irinotecan. Methods: This study evaluated MM-151 when administered as a monotherapy and in combination with irinotecan. An expansion cohort was also enrolled to evaluate clinical activity in EGFR-refractory metastatic CRC patients (pts). Subset analyses and additional biomarker evaluations were performed in EGFR-driven indications. Results: A total of 111 patients were enrolled (87 pts on monotherapy). The most common tumor types were CRC (43 [39%]), NSCLC (11 [10%]) and SCCHN (14 [13%]). Weekly dose selection was previously reported. Reported here are final safety and biomarker data. Most adverse events w...

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

BackgroundPatients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.MethodsWe developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.ResultsOverall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.ConclusionThe MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

P-284 Phase 3 RECOURSE trial of TAS-102 versus placebo with best supportive care in patients with metastatic colorectal cancer: European subgroup

Introduction: TAS-102 is comprised of an antineoplastic thymidine-based nucleoside analog, trifluridine, and the thymidine phosphorylase inhibitor, tipiracil hydrochloride, at a molar ratio of 1:0.5 (weight ratio, 1:0.471). The efficacy and safety of TAS-102 in patients with metastatic colorectal cancer refractory or intolerant to standard therapies were evaluated in the RECOURSE trial; enrollment criteria included ≥2 prior lines of standard chemotherapy (including prior fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and an anti-EGFR antibody in patients with KRAS wild-type tumors). The primary results of RECOURSE demonstrated a significant improvement in overall survival and progression-free survival with TAS-102 versus placebo (hazard ratio [HR] = 0.68 and 0.48 for overall survival and progression-free survival, respectively; both P < 0.0001). The aim of this analysis is to evaluate efficacy and safety in the European subgroup of RECOURSE. Methods: This prespecified analysis of RECOURSE compared the efficacy and safety of TAS-102 versus placebo in European patients with metastatic colorectal cancer in the RECOURSE trial. The primary endpoint (overall survival) and key secondary efficacy endpoint (progression-free survival) were evaluated using univariate and multivariate analyses for the European subgroup. Results: The European subgroup (n = 403) consisted of 271 patients in the TAS-102 arm and 132 patients in the placebo arm. Both groups had a mean age of 62 years and 62% were male. The median overall survival in the European subpopulation was 6.8 months versus 4.9 months in the TAS-102 and placebo groups, respectively (HR = 0.62; 95% confidence interval [CI]: 0.48-0.80; P = 0.0002). Median progression-free survival was 2.0 months and 1.7 months in the TAS-102 and placebo groups, respectively (HR = 0.41; 95% CI: 0.33-0.52; P < 0.0001), and best overall response (complete response, partial response, or stable disease) was 42.1% of TAS-102 patients versus 12.5% of placebo patients. Overall adverse events and adverse events ≥ Grade 3 (Gr 3+) are shown in the Table. The most common Gr 3+ treatment-related adverse event was neutropenia (29.3% TAS-102 versus 0% placebo). Conclusion: As in the overall RECOURSE study group, TAS-102 was associated with significantly improved overall survival and progression-free survival compared with placebo. No new safety signals were seen in this European subpopulation of patients with metastatic colorectal cancer refractory to standard therapies. Table: P-284

Hsa-miR-31-3p expression is linked to progression-free survival in patients with KRAS wild-type metastatic colorectal cancer treated with anti-EGFR therapy.

To identify microRNAs (miRNA) that predict response to anti-EGFR antibodies in patients with wild-type KRAS metastatic colorectal cancer (mCRC). miRNA profiling was performed in a training set of 87 patients with mCRC refractory to chemotherapy treated with anti-EGFR antibodies. This included 33 fresh-frozen (FF) and 35 formalin-fixed paraffin-embedded (FFPE) samples retrospectively collected and 19 prospectively collected FF samples. An independent validation cohort consisting of 19 FF and 26 FFPE prospectively collected samples from patients with mCRC treated with anti-EGFR antibodies was used to confirm our findings. After screening the expression of 1,145 miRNAs in FF samples from the training set, we identified that hsa-miR-31-3p expression level was significantly associated with progression-free survival (PFS). Statistical models based on miRNA expression discriminated between high and low risk of progression for both FF and FFPE samples. These models were confirmed in the validation cohort for both FF [HR, 4.1; 95% confidence interval (CI), 1.1-15.3; P < 0.04] and FFPE samples (HR, 2.44; 95% CI, 1.1-5.4; P = 0.028). The percentage of variation of RECIST criteria in the validation series was significantly associated with the expression level of hsa-miR-31-3p (r(2) = 0.49; P = 0.0035) and risk status determined by hsa-miR-31-3p expression level (P = 0.02, Kruskal-Wallis rank test). Nomograms were built and validated to predict PFS-depending on hsa-miR-31-3p expression level. Following in vitro studies, we identified 47 genes regulated by hsa-miR-31-3p. Hsa-miR-31-3p seems to be a new mCRC biomarker whose expression level allows for the identification of patients with wild-type KRAS mCRC who are more likely to respond to anti-EGFR therapy.

Clinical relevance of KRAS mutations in codon 13: Where are we?

Recent advances in molecular diagnosis and the trend towards personalized medicine have made colorectal cancer one of the tumors where therapies have significantly improved patient survival after metastasis development. KRAS mutations in codon 12 and 13 are recognized biomarkers that are analyzed in clinic previously for anti-EGFR therapies administration. Since originally mutations in both codons were considered as a predictor of lack of response to cetuximab or panitumumab, the European Medicines Agency and the US Food and Drug Administration suggested that patients harboring any of those mutations should be excluded from the treatment. However, subsequent retrospective analysis has shown that mutations in codon 12 and codon 13 of KRAS gene could be different in their biological characteristics and as a result could confer variable effects in patients. In addition and increasing and sometimes contradictory number of solutions have been published demonstrating that patients with mutations in codon 13 could have worse outcome but could obtain a significant clinical benefit from anti-EGFR therapies. Here, we review and update the latest data on the biological role leading to a predictive outcome and benefit from anti-EGFR antibodies in patients with specific KRAS mutations in codon 13.

O2-038 - Mutations in NRAS Codon 61 AND KRAS Codon 146 are Poor Prognostic Factors in Patients who Received Anti-EGFR Monoclonal Antibody for Metastatic Colorectal Cancer

ABSTRACT Background Previous studies showed that gene mutations (NRAS, BRAF, PIK3CA) are associated with a poor prognosis or resistance of anti-EGFR antibody in metastatic colorectal cancer (mCRC) patients with wild-type (WT) of KRAS codon 12/13 (KRAS-WT). However, the significance of these biomarkers has not been clarified. In addition, EGFR immunohistochemistry (IHC) and EGFR gene amplification to evaluate the efficacy of anti-EGFR antibody treatment have not been reported for mCRC. Method We evaluated tumor response and survival in patients who received anti-EGFR antibody by mutation analysis of KRAS, NRAS, BRAF, and PIK3CA in KRAS-WT patients with mCRC. Tumor DNA samples are obtained from patients treated in our hospital with anti-EGFR antibody between August 2008 and August 2011. Results A total of 117 patients were enrolled in this analysis, including 100 KRAS-WT patients. Seventy-one patients (60.7%) were all WT for KRAS, NRAS, BRAF, and PIK3CA, and 46 patients (39.3%) had at least one mutation or had insufficient DNA samples to analyze. Mutations of KRAS codon 61 (2 patients), KRAS codon 146 (5), BRAF V600E (2), PIK3CA exon9 (8), NRAS codon 12/13 (2), and NRAS codon 61 (5) were detected. No patients had a mutation of PIK3CA exon 20. Patients with at least one mutation had no response. Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E were associated with a shorter progression-free survival (PFS) compared with all WT patients (P = 0.049, P = 0.004, P = 0.036, respectively). Twelve patients (12% of KRAS-WT patients) with a mutation of KRAS codon 146, BRAF V600E, NRAS codon 61 had poor prognosis compared with the other KRAS-WT patients (PFS, 6.4 versus 2.0 months, P Conclusion Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E could be a strong prognostic factor of anti-EGFR antibody in patients with mCRC. Combination of IHC and DISH of EGFR could identify patients with a tumor response to anti-EGFR antibody in patients that are all wild type for KRAS, NRAS, BRAF, and PIK3CA.

1649P - Mutations in NRAS Codon 61 and KRAS Codon 146 are Poor Prognostic Factors in Patients who Received Anti-EGFR Monoclonal Antibody for Metastatic Colorectal Cancer

ABSTRACT Background Previous studies showed that gene mutations (NRAS, BRAF, PIK3CA) are associated with a poor prognosis or resistance of anti-EGFR antibody in metastatic colorectal cancer (mCRC) patients with wild-type (WT) of KRAS codon 12/13 (KRAS-WT). However, the significance of these biomarkers has not been clarified. In addition, EGFR immunohistochemistry (IHC) and EGFR gene amplification to evaluate the efficacy of anti-EGFR antibody treatment have not been reported for mCRC. Method We evaluated tumor response and survival in patients who received anti-EGFR antibody by mutation analysis of KRAS, NRAS, BRAF, and PIK3CA in KRAS-WT patients with mCRC. Tumor DNA samples are obtained from patients treated in our hospital with anti-EGFR antibody between August 2008 and August 2011. Results A total of 117 patients were enrolled in this analysis, including 100 KRAS-WT patients. Seventy-one patients (60.7%) were all WT for KRAS, NRAS, BRAF, and PIK3CA, and 46 patients (39.3%) had at least one mutation or had insufficient DNA samples to analyze. Mutations of KRAS codon 61 (2 patients), KRAS codon 146 (5), BRAF V600E (2), PIK3CA exon9 (8), NRAS codon 12/13 (2), and NRAS codon 61 (5) were detected. No patients had a mutation of PIK3CA exon 20. Patients with at least one mutation had no response. Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E were associated with a shorter progression-free survival (PFS) compared with all WT patients (P = 0.049, P = 0.004, P = 0.036, respectively). Twelve patients (12% of KRAS-WT patients) with a mutation of KRAS codon 146, BRAF V600E, NRAS codon 61 had poor prognosis compared with the other KRAS-WT patients (PFS, 6.4 versus 2.0 months, P Conclusion Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E could be a strong prognostic factor of anti-EGFR antibody in patients with mCRC. Combination of IHC and DISH of EGFR could identify patients with a tumor response to anti-EGFR antibody in patients that are all wild type for KRAS, NRAS, BRAF, and PIK3CA.

Mutations in NRAS codon 61, KRAS codon 146, and BRAF V600E as prognostic factors in patients who received anti-EGFR antibody for metastatic colorectal cancer.

e14126 Background: Previous studies showed that gene mutations (NRAS, BRAF, PIK3CA) are associated with a poor prognosis or resistance of anti-EGFR antibody in metastatic colorectal cancer (mCRC) patients with wild type (WT) of KRAS codon 12/13 (KRAS-WT). However the significance of these biomarkers has not been clarified. In addition, EGFR immunohistochemistry (IHC) and EGFR gene amplification to evaluate the efficacy of anti-EGFR antibody treatment has not been reported for mCRC. Methods: We evaluated tumor response and survival in patients who received anti-EGFR antibody by mutation analysis of KRAS, NRAS, BRAF, and PIK3CA in KRAS-WT patients with mCRC. Tumor DNA samples are obtained from patients treated in our hospital with anti-EGFR antibody between August 2008 and August 2011. Results: A total of 117 patients were enrolled in this analysis, including 100 KRAS-WT patients. Seventy-one patients (60.7%) were all WT for KRAS, NRAS, BRAF, and PIK3CA, and 46 patients (39.3%) had at least 1 mutation or had insufficient DNA samples to analyze. Mutations of KRAS codon 61 (2 patients), KRAS codon 146 (5), BRAF V600E (2), PIK3CA exon9 (8), NRAS codon 12/13 (2), and NRAS codon 61 (5) were detected. No patients had a mutation of PIK3CA exon 20. Patients with at least 1 mutation had no response. Mutations of NRAS codon 61, KRAS codon 146, and BRAF V600E were associated with a shorter progression free survival (PFS) compared with all WT patients (p=0.049, p=0.004, p=0.036, respectively). Twelve patients (12% of KRAS-WT patients) with a mutation of NRAS codon 61, KRAS codon146, and BRAF V600E had poor prognosis compared with the other KRAS-WT patients (PFS, 6.4 vs 2.0 months, p&lt;0.001; overall survival (OS), 13.7 vs 7.9 months, p=0.012). In all WT patients, moderate to strong EGFR IHC was associated with a better response rate than negative and weak IHC (p=0.046). Conclusions: Mutations of NRAS codon 61, KRAS codon 146, and BRAF V600E could be a strong prognostic factor of anti-EGFR antibody in patients with mCRC. Combination of IHC and DISH of EGFR could identify patients with a tumor response to anti-EGFR antibody in patients that are all WT for KRAS, NRAS, BRAF, and PIK3CA.

Molecular dissection of effector mechanisms of RAS-induced resistance to monoclonal anti-EGFR antibodies.

501 Background: RAS mutations are negative predictors of efficacy of anti-EGFR antibodies in patients with colorectal cancer (CRC). Oncogenic RAS activates the MAPK- and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways and their downstream effectors in RAS-mutant CRC is less defined. To this end we conducted functional studies using transgenic cancer models in vitro and in vivo. In addition, we analyzed pathway activation in primary tumor samples from metastatic CRC patients. Methods: Anti-EGFR-antibody sensitive cancer cell lines were retrovirally transduced to stably express (i) oncogenic RAS, (ii) conditionally active mutant RAF-1 or (iii) AKT. The impact of oncogene expression on cell proliferation, induction of apoptosis, clonal survival and tumor growth in NOD/SCID mice was analyzed in relation to treatment with cetuximab or panitumumab. Cell lines with endogenous KRAS or PI3KCA mutations were used as additional controls. Primary tumor samples from 47 patients with metastatic CRC were immunohistochemically analyzed for activation of the MAPK and PI3K/AKT pathways in relation to KRAS status. Results: Transgenic RAS, RAF-1 and AKT protected anti-EGFR-sensitive CRC cells against anti-proliferative and pro-apoptotic effects of the antibodies in vitro and in vivo. This was in line with immunohistochemical analyses of primary tumour samples, which revealed a strong correlation of MAPK and PI3K/AKT pathway activation and mutant KRAS (regression 0.921, p&lt;0.0001). Sustained activation of both pathways caused upregulation of anti-apoptotic BCL-2 proteins. Transgenic expression of these BCL-2 proteins could substitute oncogenic RAS in mediating antibody resistance. Cotreatment with BH3 mimetics neutralizing anti-apoptotic BCL-XL and BCL-2 sensitized RAS-mutant cancer cells to anti-EGFR therapy. Conclusions: Activation of the MAPK and PI3K/AKT pathways mediates resistance to anti-EGFR therapies in cancer models in vitro and in vivo. Simultaneous or cross activation of both pathways is observed in KRAS mutant primary CRC. These results may guide clinical strategies to overcome RAS-mediated antibody resistance.

Predictive value of KRAS mutations in chemoresistant CRC

On the basis of the results of KRAS analysis from the CO.17 randomized, controlled, phase III study by Karapetis et al., KRAS mutations have emerged as a valid predictive marker associated with resistance to cetuximab and lack of survival benefit from this anti-EGFR antibody in patients with chemoresistant colorectal cancer.

The Marriage of Growth Factor Inhibitors and Chemotherapy: Bliss or Bust?

Many clinical trials have demonstrated a benefit associated with the combination of growth factor, growth factor receptor, or signaling pathway inhibitors with cytotoxic chemotherapy; however, there are instances where such combinations have proven futile or antagonistic. Among the most successful combinations have been those utilizing antibodies targeting the extracelluar domain of the epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER-2) with cetuximab and trastuzumab, respectively. As our ability to screen individual tumors for specific receptor and signaling pathway alterations becomes more facile, patterns that can serve to effectively guide our therapy are emerging. Recent investigations in patients with advanced colorectal cancer have identified KRAS mutation as a critical predictor of lack of clinical benefit from the anti-EGFR antibodies panitumumab and cetuximab. On further analysis, these same studies suggest a possible negative interaction between these antibodies and the fluoropyrimidines fluorouracil (FU) or capecitabine (CAP), which is most apparent in KRASmutated patients. Activating mutations in KRAS, a key regulator of cellular growth and proliferation, have been identified in 30% to 50% of patients with advanced colorectal cancer. The benefit of antiEGFR antibodies is almost entirely associated with patients whose colon cancers develop in the absence of KRAS mutation. Amado et al enrolled 423 patients with advanced and refractory colorectal cancer treated with either single-agent panitumumab or best supportive care with the option to cross over to panitumumab at the time of disease progression. Patients with wild-type KRAS enjoyed a median progression-free survival (PFS) of 12.3 months and an overall response rate of 17% in those treated with panitumumab versus a PFS of 7.3 months for the best supportive care group (HR, 0.45). Those with mutant KRAS enjoyed no benefit in PFS from the use of panitumumab (HR, 0.99) and had an overall response rate of 0%. Additional phase II investigations using cetuximab alone or with various chemotherapeutic agents in patients with advanced colorectal cancer also reported a negligible response rate in patients whose cancers contained mutant KRAS. Clinical results have recently been reported from four large, mature, multi-institutional, randomized clinical trials in which anti-EGFR antibodies were added to chemotherapy in approximately 1,500 untreated patients with advanced colorectal cancer and with defined KRAS status. The relevant outcomes data from these trials are listed in Table 1. The Oxaliplatin and Cetuximab in First-Line Treatment of mCRC (OPUS) and Capecitabine, Irinotecan, Oxaliplatin (CAIRO II) studies used oxaliplatin combined with fluoropyrimidine-based chemotherapy with or without cetuximab. Both studies demonstrate a reduction in response rate and PFS, as well as survival for CAIRO II, with the addition of the anti-EGFR antibody in patients whose cancers possess mutant KRAS and little if any additional benefit in PFS for patients with wild-type KRAS. Both the Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer (CRYSTAL) and Panitumumab Advanced Colorectal Cancer Evaluation (PACCE) investigations, using irinotecan-based chemotherapy, similarly demonstrate a decrement in benefit with the addition of the anti-EGFR antibody in patients with mutant KRAS tumors, although the effect is more modest, particularly with regard to PFS. Why might there be a decrement in benefit with the addition of anti-EGFR antibodies to chemotherapy in patients with mutant KRAS–containing cancers, particularly with oxaliplatin-based chemotherapy? Given that the negative trends occur in four independent trials with only two invariable constituents, namely a fluoropyrimidine and the anti-EGFR antibody, one hypothesis that could explain the clinical observation is a negative interaction at the cellular level between EGFR inhibition and the fluoropyrimidine. The greater negative effect with oxaliplatin versus irinotecan may be the result of oxaliplatin’s greater dependence on FU for its clinical activity. Preclinical investigations, as well as the study in the second-line setting reported by Rothenberg et al, clearly support the concept that the addition of FU to oxaliplatin results in greater than an additive interaction, whereas the single-agent activity of oxaliplatin is modest, particularly in contrast to irinotecan, which has respectable single-agent activity in patients with advanced colorectal cancer. The additional benefit of adding cetuximab to single-agent irinotecan in the Bowel Oncology Cetuximab Antibody (BOND) trial also suggests that any negative interaction between the anti-EGFR antibody and chemotherapy is related to the presence of a fluoropyrimidine in the regimen. The proposed antagonistic interaction between fluoropyrimidines and anti-EGFR antibodies may be explained by the fact that FU requires metabolically active cells in which to undergo the requisite multistep anabolism to the active forms of the drug. In wild-type KRAS cells, the anti-EGFR antibody may effectively control malignant cell growth, overshadowing a possible negative interaction between the antibody and chemotherapy. In KRAS-mutated cells, with no JOURNAL OF CLINICAL ONCOLOGY COMMENTS AND CONTROVERSIES VOLUME 27 NUMBER 10 APRIL 1 2009