Jeroen R. Dijkstra

Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab

BACKGROUND Anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) treatment are only effective in patients with KRAS wild type tumours. Here we assess the predictive value of other potential relevant markers involved in the epidermal growth factor receptor (EGFR) signalling pathways for response to cetuximab-based treatment. MATERIALS AND METHODS Formalin-fixed paraffin-embedded colorectal cancer tissue of the primary tumour was obtained from 559 mCRC patients treated with chemotherapy and bevacizumab with or without cetuximab (phase III CAIRO2 study). DNA was isolated for mutation analysis of BRAF (V600E), KRAS (codon 12 and 13) and PIK3CA (exon 9 and 20). Tissue microarrays (TMAs) were constructed for the assessment of EGFR and HER2 gene copy number (GCN), and EGFR and PTEN protein expression. The results of these markers, individually or in combination, were correlated with progression-free survival (PFS) and overall survival (OS) in the subgroup of patients with a KRAS wild type tumour treated in the cetuximab-arm. KRAS wild type patients treated without cetuximab were used as a control group. RESULTS A total of 208 tumours (39.4%) contained a KRAS mutation, 8.7% a BRAF mutation and 9.9% a PIK3CA mutation. Loss of PTEN expression and the presence EGFR protein expression were observed in 42.0% and 61.7% of the samples, respectively. An increased EGFR GCN was observed in 15.3% of the samples, and 11.5% of the evaluable samples contained an increased HER2 GCN. In KRAS wild type patients treated with cetuximab a BRAF mutation was significantly and independently associated with PFS and OS. In patients treated without cetuximab the PFS and OS were also associated with the BRAF genotype. No prognostic or predictive value was observed for any of the other markers when tested individually or in combination. CONCLUSIONS BRAF genotype is correlated with PFS and OS in KRAS wild type mCRC patients, which is independent of cetuximab treatment. PIK3CA mutation, loss of PTEN expression, EGFR GCN and HER2 GCN have no predictive value for response to treatment with cetuximab, neither individually nor in combination with other markers.

High sensitivity of both sequencing and real-time PCR analysis of KRAS mutations in colorectal cancer tissue.

The KRAS mutation status predicts the outcome of treatment with epidermal growth factor receptor targeted agents, and therefore the testing for KRAS mutations has become an important diagnostic procedure. To optimize the quality of this test, we compared the results of the two most commonly used KRAS mutation tests, cycle sequencing and a real‐time PCR‐based assay, in DNA extracted from formalin‐fixed paraffin‐embedded (FFPE) colorectal cancer samples of 511 patients. The results were interpreted in the context of the tumour cell percentage and the assay parameters. In 510 samples KRAS mutation status assessment was successful. A KRAS mutation was detected in 201 tumours (39.4%). Sequencing and the real‐time PCR‐based assay generated the same result in 486 samples (95.3%). The sequencing result was considered false positive in one (0.2%) and false negative in nine samples (1.8%). The assay result was considered false positive in six (1.2%) and false negative in seven samples (1.4%). Explanations for discrepant test results were a higher sensitivity of the assay in samples with a low tumour cell percentage, occurrence of mutations that are not covered by the assay and δ Ct values approximating the cut‐off value of the assay. In conclusion, both sequencing and the real‐time PCR‐based assay are reliable tests for KRAS mutation analysis in FFPE colorectal cancer samples, with a sensitivity of 95.5% (95% confidence interval [CI] 91.7–97.9%) and 96.5% (95% CI 93.0–98.6%), respectively. The real‐time PCR based assay is the method of choice in samples with a tumour cell percentage below 30%.

Beyond KRAS mutation status: influence of KRAS copy number status and microRNAs on clinical outcome to cetuximab in metastatic colorectal cancer patients

BackgroundKRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab.MethodsFormalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n = 17) or poor (n = 17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR.ResultsCopy number loss of the KRAS locus was observed in the tumour of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild-type KRAS tumours were associated with a poor PFS. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good PFS. In wild-type KRAS patients, miRNA expression did not correlate with PFS in a multivariate model.ConclusionsOur results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of mCRC eligible for anti-EGFR therapy.

COL3A1 2209G>A is a predictor of pelvic organ prolapse

Introduction and hypothesisA familial tendency has been demonstrated in the etiology of pelvic organ prolapse (POP), but the specific genetic defects have not been identified. Type III collagen is an important factor in the repair of connective tissue, and gene polymorphisms may impair the tensile strength. We hypothesized that polymorphisms in the alpha I chain of the type III collagen protein-encoding gene (COL3A1) pose women at risk for POP.MethodsIn this case–control study, the prevalence of type III collagen polymorphisms was compared in women with and without signs and symptoms of POP.ResultsTwo hundred and two POP patients and 102 normal parous controls were included. A homozygous single-nucleotide substitution in the coding region of type III collagen (COL3A1 2209G>A, rs1800255) was identified in 27 (13%) POP patients and three (3%) controls (odds ratio, 5.0; 95% confidence interval, 1.4–17.1).ConclusionsThe probability of POP was higher in women with COL3A1 2209G>A. This polymorphism showed to be a relevant risk factor for POP.

MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls.

•  Introduction •  RNA isolation from formalin‐fixed paraffin‐embedded (FFPE) tissue •  RNA quality control •  cDNA synthesis •  Real Time Quantitative PCR •  Reference genes •  Data analysis •  Conclusions

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study

BACKGROUND The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrells C-statistics was used to assess model performance. FINDINGS Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0·97 for colon tumour; r=0·97 for invasive margin; p<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system. INTERPRETATION The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer. FUNDING French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results?

The use of real‐time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost‐effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample‐to‐sample and run‐to‐run variation, we have evaluated the adequacy of normalisation procedures in qPCR‐based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

Chromosomal copy number aberrations in colorectal metastases resemble their primary counterparts and differences are typically non-recurrent

The metastatic process is complex and remains a major obstacle in the management of colorectal cancer. To gain a better insight into the pathology of metastasis, we investigated genomic aberrations in a large cohort of matched colorectal cancer primaries and distant metastases from various sites by high resolution array comparative genomic hybridization. In total, 62 primary colorectal cancers, and 68 matched metastases (22 liver, 11 lung, 12 ovary, 12 omentum, and 11 distant lymph nodes) were analyzed. Public datasets were used for validation purposes. Metastases resemble their matched primary tumors in the majority of the patients. This validates the significant overlap in chromosomal aberrations between primary tumors and corresponding metastases observed previously. We observed 15 statistically significant different regions between the primary tumors and their matched metastases, of which only one recurrent event in metastases was observed. We conclude, based on detailed analysis and large independent datasets, that chromosomal copy number aberrations in colorectal metastases resemble their primary counterparts, and differences are typically non-recurrent.

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.

Abstract 534: MicroRNA-143 is a putative predictive biomarker for 5-FU-based chemotherapy in patients with metastatic colorectal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Approximately half of the colorectal cancer (CRC) patients develop metastatic disease, either at diagnosis or during follow-up. 5-FU-based chemotherapy forms the backbone of treatment in these patients. However, the response to this therapy varies between individuals. Therefore, an important challenge in CRC research is to identify biomarkers that are predictive of this response. MicroRNAs are short RNAs that post-transcriptionally regulate the gene expression of many genes. MicroRNA levels are frequently altered in cancer, and can affect expression of genes that modulate tumorigenesis. Objective: In this study, we explore the potential of microRNAs, and the microRNA producing protein Dicer, as biomarkers that can predict chemo-sensitivity in patients with metastatic colorectal cancer. Methods: We analyzed the levels of a selected panel of 22 miRNAs and the Dicer protein in primary CRC tumors from patients enrolled in the CAIRO study (a phase III study of the Dutch Colorectal Cancer Group). MiRNA levels were determined with Taqman microRNA assays and Dicer level with immunohistochemistry. Correlation between the expression status of microRNAs or Dicer in primary tumors and the progression free survival (PFS) were investigated. In addition, we evaluated correlations between Dicer and miRNA levels. Results: Variable Dicer intensities were observed among the analyzed samples, but there was no significant association between Dicer levels and PFS. In addition, no clear correlation between Dicer and miRNA levels could be identified. We especially detected higher expression of miR-31 and miR-21 in tumors compared to matched normal tissue, whereas miR-137 and miR-215 were lower expressed. Interestingly, miR-143 expression was reduced in a subset of the primary CRC tumors, and this differential expression showed a relationship with PFS. The group with reduced miR-143 expression showed higher PFS. The median PFS for patients with reduced, neutral and increased miR-143 expression was 261, 167 and 142 days, respectively (p-value log-rank test=0.018). Conclusion: The expression level of miR-143 may have clinical utility as a biomarker for response to 5-FU-based chemotherapy. These findings warrant further studies to investigate the relationship between miR-143 expression and 5-FU. Citation Format: Femke Simmer, Jeroen Dijkstra, Sabine Venderbosch, Claudius Faber, Leonie Mekenkamp, Miriam Koopman, Ton de Haan, Cornelis Punt, Iris Nagtegaal. MicroRNA-143 is a putative predictive biomarker for 5-FU-based chemotherapy in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 534. doi:10.1158/1538-7445.AM2014-534