Bart Biesmans

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis

BACKGROUND Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. METHODS 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. FINDINGS 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. INTERPRETATION While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population. FUNDING Belgian Federation against Cancer (Stichting tegen Kanker).

Prognostic Role of KRAS and BRAF in Stage II and III Resected Colon Cancer: Results of the Translational Study on the PETACC-3, EORTC 40993, SAKK 60-00 Trial

PURPOSE Mutations within the KRAS proto-oncogene have predictive value but are of uncertain prognostic value in the treatment of advanced colorectal cancer. We took advantage of PETACC-3, an adjuvant trial with 3,278 patients with stage II to III colon cancer, to evaluate the prognostic value of KRAS and BRAF tumor mutation status in this setting. PATIENTS AND METHODS Formalin-fixed paraffin-embedded tissue blocks (n = 1,564) were prospectively collected and DNA was extracted from tissue sections from 1,404 cases. Planned analysis of KRAS exon 2 and BRAF exon 15 mutations was performed by allele-specific real-time polymerase chain reaction. Survival analyses were based on univariate and multivariate proportional hazard regression models. RESULTS KRAS and BRAF tumor mutation rates were 37.0% and 7.9%, respectively, and were not significantly different according to tumor stage. In a multivariate analysis containing stage, tumor site, nodal status, sex, age, grade, and microsatellite instability (MSI) status, KRAS mutation was associated with grade (P = .0016), while BRAF mutation was significantly associated with female sex (P = .017), and highly significantly associated with right-sided tumors, older age, high grade, and MSI-high tumors (all P < 10(-4)). In univariate and multivariate analysis, KRAS mutations did not have a major prognostic value regarding relapse-free survival (RFS) or overall survival (OS). BRAF mutation was not prognostic for RFS, but was for OS, particularly in patients with MSI-low (MSI-L) and stable (MSI-S) tumors (hazard ratio, 2.2; 95% CI, 1.4 to 3.4; P = .0003). CONCLUSION In stage II-III colon cancer, the KRAS mutation status does not have major prognostic value. BRAF is prognostic for OS in MS-L/S tumors.

PIK3CA mutations are not a major determinant of resistance to the epidermal growth factor receptor inhibitor cetuximab in metastatic colorectal cancer.

Purpose: It has been reported that activating KRAS mutations negatively affect response to anti–epidermal growth factor receptor (EGFR) monoclonal antibodies in metastatic colorectal cancer. The mutation status of signaling molecules downstream of the EGFR target is thus crucial to predict clinical benefit to EGFR-targeted therapies. Other mechanisms of resistance to EGFR inhibitors could involve activating mutations of the other main EGFR effector pathway, i.e., the PI3K/PTEN/AKT pathway. Experimental Design: We analyzed the PIK3CA and KRAS mutation status in a large group (n = 200) of chemorefractory metastatic colorectal cancers treated with cetuximab (Erbitux) in monotherapy or in combination with irinotecan, and correlated the mutation status with outcome. Results: Twenty-three (12%) of the 200 samples carried 1 of the PIK3CA mutations included in our assay. We found no correlation between the presence of a PIK3CA mutation and impaired response to cetuximab. Conclusions: Our findings do not provide any evidence for a strong role of PIK3CA mutations as a single marker in determining response to cetuximab in chemorefractory metastatic colorectal cancer.

Clinical Usefulness of EGFR Gene Copy Number as a Predictive Marker in Colorectal Cancer Patients Treated with Cetuximab: A Fluorescent In situ Hybridization Study

Purpose: To evaluate the usefulness and the pitfalls inherent to the assessment of the epidermal growth factor receptor (EGFR) gene copy number (GCN) by fluorescence in situ hybridization (FISH) for outcome prediction to cetuximab in metastatic colorectal cancer. The value of testing KRAS mutation status, in addition to EGFR GCN, was also explored. Experimental Design: FISH analysis of 87 metastatic colorectal cancer patients treated with cetuximab was done, recording individual GCN per cell and using different samples per tumor. Performances of published cutoff points and different summaries of EGFR GCN distribution were assessed for response prediction. Results: In our data set, two published cutoff points performed less well than in their training set, yielding positive predictive values and negative predictive values between 40.0% and 48.3% and between 81.0% and 86.5%, respectively. Among summaries of GCN distribution explored, mean and right-tailed distribution of GCN yielded the highest performances. A mean EGFR GCN ≥2.83 provided an area under the curve of 0.71. Important heterogeneity of repeated measures of mean EGFR GCN was observed within tumors (intraclass correlation, 0.61; within-class SD, 0.40), leading to potential misclassifications of FISH status in 7 of 18 (38.8%) patients if a cutoff point were used. In multivariable analysis, EGFR GCN testing provided significant information independent of the KRAS status to predict response (P = 0.016) and overall survival (P = 0.005). Conclusions: We confirm the association between increased EGFR GCN and outcome after cetuximab. However, because of reproducibility concerns, any decision making based on published cutoff points is not warranted.

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

Background Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. Methodology/Principal Findings Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). Conclusions/Significance Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

Clinical biomarkers in oncology: focus on colorectal cancer.

Rapidly growing insight into the molecular biology of colorectal cancer has led to high hopes for the identification of molecular markers to be used in optimized and tailored treatment regimens. However, many of the published data on gene-specific biomarkers are contradictory in their findings, and no tests are currently used in clinical practice, with the exception of microsatellite instability (MSI) and guanylyl cyclase C (GCC) testing in the adjuvant setting, and in Europe KRAS mutation testing is used in the setting of epidermal growth factor receptor (EGFR)-targeted therapy for metastatic disease. There are many reasons for the failure of the initial marker hypothesis-driven approach. Although supported by a good biologic rationale, single markers such as tumor protein p53 (TP53) gene mutations, when applied to a complex tumor type containing many synchronous alterations, do not perform well in predicting outcome. Many markers also suffer from technical shortcomings, resulting from the lack of quantitative techniques to capture the impact of the molecular alteration. The impact of markers obtained from microarray expression profiling needs to be further investigated in studies based on much larger cohorts, and cross-validation studies will be essential.Recently, mutations in the KRAS gene were shown to be strong negative predictors of response to EGFR inhibitors in metastatic disease. It has also been suggested that BRAF gene mutations may be predictive of EGFR inhibitor resistance, and there are some conflicting data regarding the role of the PIK3CA gene. Further studies are needed to help integrate the latest findings into clinically useful tools for personalized medicine.

Implementation of Formalin-Fixed, Paraffin-Embedded Cell Line Pellets as High-Quality Process Controls in Quality Assessment Programs for KRAS Mutation Analysis

In recent years, the mutational status of the KRAS oncogene has become incorporated into standard medical care as a predictive marker for therapeutic decisions related to patients with metastasized colorectal cancer. This is necessary, because these patients benefit from epidermal growth factor receptor (EGFR)-targeted therapy with increased progression-free survival only if the tumor does not carry a mutation in KRAS. Many different analytical platforms, both those commercially available and those developed in house, have been used within pathology laboratories to assess KRAS mutational status. For a testing laboratory to become accredited to perform such tests, it is essential that they perform reliability testing, but it has not previously been possible to perform this kind of testing on the complete workflow on a large scale without compromising reproducibility or the mimicry of the control sample. We assessed a novel synthetic control for formalin-fixed, paraffin-embedded (FFPE) tumor samples in a blind study conducted within nine laboratories across Europe. We show that FFPE material can, at least in part, mimic clinical samples and we demonstrate this control to be a valuable tool in the assessment of platforms used in testing for KRAS mutational status.

DUSP 4 expression identifies a subset of colorectal cancer tumors that differ in MAPK activation, regardless of the genotype

Abstract As dual-specificity phosphatase (DUSP) expression has been correlated to sensitivity to MEK inhibitors, DUSP expression levels may indicate activation of the mitogen-activated protein kinase (MAPK) pathway in many tumor types. In this study, we investigate if DUSP levels can indicate different levels of MAPK activation within colorectal cancer (CRC) patients. In three different CRC patient microarray datasets, we analyzed the expression of DUSP1. DUSP4 and DUSP6 according to mutational status, their correlation with survival and their association with different clinical characteristics. DUSP4 was significantly differentially expressed between all mutational subgroups with the highest expression in BRAF mutated tumors. Moreover, high DUSP4 expression was associated with a worse overall survival and with clinical characteristics typical for BRAF mutant patients. The use of DUSP expression as a predictive biomarker towards MAPK targeted therapy in CRC patients needs further investigation.

DUSP4 expression level in colorectal primaries predicts overall survival benefit in Kras wild-type and Kras mutant colorectal cancer after treatment with cetuximab for metastatic disease

De Roock, Wendy ; Janssens, Marc ; Biesmans, Bart ; Jacobs, Bart ; De Schutter, Jef ; et. al. DUSP4 expression level in colorectal primaries predicts overall survival benefit in Kras wild-type and Kras mutant colorectal cancer after treatment with cetuximab for metastatic disease.20th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics ((Switzerland) Geneva, du 24/10/2008 au 28/10/2008). In: EJC Supplements, Vol. 6, no. 12, p. 33 (2008) http://hdl.handle.net/2078.1/59065 -DOI : 10.1016/ S1359-6349(08)72030-5

Single Nucleotide Polymorphism (rs4932178) in the P1 Promoter of FURIN Is Not Prognostic to Colon Cancer

High expression of the proprotein processing enzyme FURIN has been associated with tumor progression and metastasis. A SNP (rs4932178) in the promoter of FURIN has been reported to affect expression in liver, with the T allele resulting in higher expression than the C allele. In this study we have investigated the association of this SNP with prognostic and biological subgroups of colorectal cancer (CRC). In a panel of 1382 patients with CRC, this SNP had no impact on overall survival or on postoperative risk of relapse. This SNP also could not be linked with FURIN expression levels in CRC samples from the patients. Furthermore, we demonstrate in luciferase reporter experiments in the colon cancer cell lines Caco-2 and SW480 and in the hepatocellular carcinoma cell line Huh 7 that expression is not affected by the SNP. Since, FURIN inhibition in human colon cancer cell lines has previously been shown to repress tumor metastases, association between FURIN gene expression levels and postoperative relapse-free survival was also investigated. However, no association could be found. Altogether, we could not confirm an effect of the SNP on FURIN expression in vitro and no correlations could be found in vivo with FURIN expression or outcome.