Robert R. J. Coebergh van den Braak

A Systematic Analysis of Oncogenic Gene Fusions in Primary Colon Cancer

Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of BRAF, NTRK3, and RET gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X-ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node-negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy. Cancer Res; 77(14); 3814-22. ©2017 AACR.

Confirmation of a metastasis-specific microRNA signature in primary colon cancer

The identification of patients with high-risk stage II colon cancer who may benefit from adjuvant therapy may allow the clinical approach to be tailored for these patients based on an understanding of tumour biology. MicroRNAs have been proposed as markers of the prognosis or treatment response in colorectal cancer. Recently, a 2-microRNA signature (let-7i and miR-10b) was proposed to identify colorectal cancer patients at risk of developing distant metastasis. We assessed the prognostic value of this signature and additional candidate microRNAs in an independent, clinically well-defined, prospectively collected cohort of primary colon cancer patients including stage I-II colon cancer without and stage III colon cancer with adjuvant treatment. The 2-microRNA signature specifically predicted hepatic recurrence in the stage I-II group, but not the overall ability to develop distant metastasis. The addition of miR-30b to the 2-microRNA signature allowed the prediction of both distant metastasis and hepatic recurrence in patients with stage I-II colon cancer who did not receive adjuvant chemotherapy. Available gene expression data allowed us to associate miR-30b expression with axon guidance and let-7i expression with cell adhesion, migration, and motility.

Completeness of pathology reports in stage II colorectal cancer

Abstract Introduction: The completeness of the pathological examination of resected colon cancer specimens is important for further clinical management. We reviewed the pathological reports of 356 patients regarding the five factors (pT-stage, tumor differentiation grade, lymphovascular invasion, tumor perforation and lymph node metastasis status) that are used to identify high-risk stage II colon cancers, as well as their impact on overall survival (OS). Methods: All patients with stage II colon cancer who were included in the first five years of the MATCH study (1 July 2007 to 1 July 2012) were selected (n = 356). The hazard ratios of relevant risk factors were calculated using Cox Proportional Hazards analyses. Results: In as many as 69.1% of the pathology reports, the desired information on one or more risk factors was considered incomplete. In multivariable analysis, age (HR: 1.07, 95%CI 1.04–1.10, p < .001), moderately- (HR: 0.35, 95%CI 0.18–0.70, p = .003) and well (HR 0.11, 95%CI 0.01–0.89, p = .038) differentiated tumors were significantly associated with OS. Conclusions: Pathology reports should better describe the five high-risk factors, in order to enable proper patient selection for further treatment. Chemotherapy may be offered to stage II patients only in select instances, yet a definitive indication is still unavailable.

Gene length corrected trimmed mean of M-values (GeTMM) processing of RNA-seq data performs similarly in intersample analyses while improving intrasample comparisons

BackgroundCurrent normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data.ResultsWe observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality.ConclusionsWe show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain.

High mRNA expression of splice variant SYK short correlates with hepatic disease progression in chemonaive lymph node negative colon cancer patients

Objective Overall and splice specific expression of Spleen Tyrosine Kinase (SYK) has been posed as a marker predicting both poor and favorable outcome in various epithelial malignancies. However, its role in colorectal cancer is largely unknown. The aim of this study was to explore the prognostic role of SYK in three cohorts of colon cancer patients. Methods Total messenger RNA (mRNA) expression of SYK, SYK(T), and mRNA expression of its two splice variants SYK short (S) and SYK long (L) were measured using quantitative reverse transcriptase (RT-qPCR) in 240 primary colon cancer patients (n = 160 patients with chemonaive lymph node negative [LNN] and n = 80 patients with adjuvant treated lymph node positive [LNP] colon cancer) and related to microsatellite instability (MSI), known colorectal cancer mutations, and disease-free (DFS), hepatic metastasis-free (HFS) and overall survival (OS). Two independent cohorts of patients with respectively 48 and 118 chemonaive LNN colon cancer were used for validation. Results Expression of SYK and its splice variants was significantly lower in tumors with MSI, and in KRAS wild type, BRAF mutant and PTEN mutant tumors. In a multivariate Cox regression analysis, as a continuous variable, increasing SYK(S) mRNA expression was associated with worse HFS (Hazard Ratio[HR] = 1.83; 95% Confidence Interval[CI] = 1.08–3.12; p = 0.026) in the LNN group, indicating a prognostic role for SYK(S) mRNA in patients with chemonaive LNN colon cancer. However, only a non-significant trend between SYK(S) and HFS in one of the two validation cohorts was observed (HR = 4.68; 95%CI = 0.75–29.15; p = 0.098). Conclusion In our cohort, we discovered SYK(S) as a significant prognostic marker for HFS for patients with untreated LNN colon cancer. This association could however not be confirmed in two independent smaller cohorts, suggesting that further extensive validation is needed to confirm the prognostic value of SYK(S) expression in chemonaive LNN colon cancer.

Abstract CT067: The Prospective Dutch ColoRectal Cancer Cohort (PLCRC): a prospective nationwide observational cohort study providing the infrastructure for registry based trials

Background: Colorectal cancer (CRC) was once considered a single entity, but now appears to exist of multiple, often molecular defined, subclasses. These subclasses have implications for treatment, and hamper the feasibility of prospective randomized clinical trials which require large sample sizes. To anticipate further developments and to obtain more detailed insight in the outcome of daily practice, the Prospective Dutch CRC cohort (PLCRC) was initiated. In this prospective observational multicenter cohort, patients with all stages of colorectal cancer are included, and clinical data, tissue, blood samples and patient-reported outcome measures are collected. The cohort serves as an infrastructure for registry based trials in the Netherlands (CRC incidence approximately 16.000 patients per year). Methods: Patients ≥18 years with histologically proven stage I-IV CRC are eligible to participate. The informed consent includes consent for systematic collection of long-term clinical data and optionally 1) standardized collection of tissue and blood samples; 2) being informed when clinically relevant DNA mutations are detected; 3) patient-reported outcome questionnaires; and 4) invitation for future interventional studies according to (amongst others) the cohort multiple randomized controlled trial design (cmRCT). Results: In 2016 the number of participating hospitals increased from 6 to 17. The number of included patients roughly doubled to more than 1100 patients. The PLCRC infrastructure is used in the execution of fifteen studies, which amongst others focus on the prognostic value of circulating tumor DNA, the quality of life during treatment with new compounds and the influence of nutrition on treatment outcome. The studies use the option to repeatedly withdraw blood, send out PROMs and/or randomize patients according to the cmRCT design. The study populations in the substudies vary from newly diagnosed stage 2 colon cancer to non-liver limited metastatic CRC. In 2017, at least 20 hospitals which are currently in the initiation process will be added with which a 50% coverage of the Dutch hospitals will be reached. Conclusions: PLCRC provides long-term clinical data, tissue, blood samples and patient-reported outcome measures of a large cohort of patients with colorectal cancer. The cohort will be representative of the colorectal cancer population in the Netherlands. Multiple studies are ongoing making use of the infrastructure provided. These studies will make it possible to optimize treatment for specific small subgroups. Citation Format: Geraldine Vink, Robert Coebergh van den Braak, Haiko Bloemendal, Veerle Coupe, Marloes Elferink, Frans Erdkamp, Helma van Grevenstein, Jan-Willem de Groot, Jan Ijzermans, Martijn Intven, Maartje Los, Mirre de Noo, Martijn van Oijen, Cornelis Punt, Ron Rietbroek, Wilfried Roeloffzen, Anandi Schiphorst, Huig Schipper, Hein Stockmann, Manuel Tjin-a-Ton, Ankie van der Velden, Marlies Verhaar, Wouter Vles, David Zimmerman, Gerrit Meijer, Miriam Koopman. The Prospective Dutch ColoRectal Cancer Cohort (PLCRC): a prospective nationwide observational cohort study providing the infrastructure for registry based trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT067. doi:10.1158/1538-7445.AM2017-CT067