Multicenter fresh frozen tissue sampling in colorectal cancer: does the quality meet the standards for state of the art biomarker research?

The growing interest in the molecular subclassification of colorectal cancers is increasingly facilitated by large multicenter biobanking initiatives. The quality of tissue sampling is pivotal for successful translational research. This study shows the quality of fresh frozen tissue sampling within a multicenter cohort study for colorectal cancer (CRC) patients. Each of the seven participating hospitals randomly contributed ten tissue samples, which were collected following Standard Operating Procedures (SOP) using established techniques. To indicate if the amount of intact RNA is sufficient for molecular discovery research and prove SOP compliance, the RNA integrity number (RIN) was determined. Samples with a RIN < 6 were measured a second time and when consistently low a third time. The highest RIN was used for further analysis. 91% of the tissue samples had a RIN ≥ 6 (91%). The remaining six samples had a RIN between 5 and 6 (4.5%) or lower than 5 (4.5%). The median overall RIN was 7.3 (range 2.9–9.0). The median RIN of samples in the university hospital homing the biobank was 7.7 and the median RIN for the teaching hospitals was 7.3, ranging from 6.5 to 7.8. No differences were found in the outcome of different hospitals (p = 0.39). This study shows that the collection of high quality fresh frozen samples of colorectal cancers is feasible in a multicenter design with complete SOP adherence. Thus, usin

mRNA expression profiles in circulating tumor cells of metastatic colorectal cancer patients

The molecular characterization of circulating tumor cells (CTCs) is a promising tool for the repeated and non-invasive evaluation of predictive and prognostic factors. Challenges associated with CTC characterization using the only FDA approved method for CTC enumeration, the CellSearch technique, include the presence of an excess of leukocytes in CTC-enriched blood fractions. Here we aimed to identify colorectal tumor-specific gene expression levels in the blood of patients with and without detectable CTCs according to CellSearch criteria. Materials and methods: Blood of 30 healthy donors (HDs) and 142 metastatic colorectal cancer (mCRC) patients was subjected to CellSearch CTC enumeration and isolation. In all samples, 95 mRNAs were measured by reverse transcriptase quantitative PCR (RT-qPCR). HD blood samples and patient samples with three or more CTCs were compared to identify CTC-specific mRNAs. Patient samples without detectable CTCs were separately analyzed. Results: Thirty-four CTC-specific mRNAs were higher expressed in patients with ≥3 CTCs compared with HDs (Mann-Whitney U-test P<0.05). Among patients without detectable CTCs, a HD-unlike subgroup was identified which could be distinguished from HDs by the expression of epithelial genes such as KRT19, KRT20 and AGR2. Also, in an independent patient set, a similar HD-unlike group could be identified among the patients without detectable CTCs according to the CellSearch system. Conclusion: Extensive molecular characterization of colorectal CTCs is feasible and a subgroup of patients without detectable CTCs according to CellSearch criteria bears circulating tumor load, which may have clinical consequences. This CTC-specific gene panel for mCRC patients may enable the exploration of CTC characterization as a novel means to further individualize cancer treatment

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

Background: Current 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. Results: We 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. Conclusions: We 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

Is the Clinical Risk Score for Patients with Colorectal Liver Metastases Still Useable in the Era of Effective Neoadjuvant Chemotherapy?

Background: Several clinical risk scores (CRSs) for the outcome of patients with colorectal liver metastases have been validated, but not in patients undergoing neoadjuvant chemotherapy. Therefore, this study evaluates the predictive value of these CRSs in this specific group. Methods: Between January 2000 and December 2008, all patients undergoing a metastasectomy were analyzed and divided into two groups: 193 patients did not receive neoadjuvant chemotherapy (group A), and 159 patients received neoadjuvant chemotherapy (group B). In group B, the CRSs were calculated before and after administration of neoadjuvant chemotherapy. Results were evaluated by using the CRSs proposed by Nordlinger et al., Fong et al., Nagashima et al., and Konopke et al. Results: In groups A and B, the overall median survival was 43 and 47 months, respectively (P = 0.648). In group A, all CRSs used were of statistically significant predictive value. Before administration of neoadjuvant chemotherapy, only the Nordlinger score was of predictive value. After administration of neoadjuvant chemotherapy, all CRSs were of predictive value again, except for the Konopke score. Conclusions: Traditional CRSs are not a reliable prognostic tool when used in patients before treatment with neoadjuvant chemotherapy. However, CRSs assessed after the administration of neoadjuvant chemotherapy are useful to predict prognosis

Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer

Background: CTCs are a promising alternative for metastatic tissue biopsies for use in precision medicine approaches. We investigated to what extent the molecular characteristics of circula

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 (l et-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 m iR-30b expression with axon guidance and l et-7i expression with cell adhesion, migration, and motility

Anatomical Versus Nonanatomical Resection of Colorectal Liver Metastases: Is There a Difference in Surgical and Oncological Outcome?

Background: The increased use of neoadjuvant chemotherapy and minimally invasive therapies for recurrence in patients with colorectal liver metastases (CLM) makes a surgical strategy

Outcome of Microscopic Incomplete Resection (R1) of Colorectal Liver Metastases in the Era of Neoadjuvant Chemotherapy

Background: Data from patients with colorectal liver metastases (CRLM) who received neoadjuvant chemotherapy before resection were reviewed and evaluated to see whether neoadjuvant chemotherapy influences the predictive outcome of R1 resections (margin is 0 mm) in patients with CRLM. Methods: Between January 2000 and December 2008, all consecutive patients undergoing liver resection for CRLM were analyzed. Patients were divided into those who did and did not receive neoadjuvant chemotherapy. The outcome after R0 (tumor-free margin >0 mm) and R1 (tumor-free margin 0 mm) resection was compared. Results: A total of 264 were eligible for analysis. Median follow-up was 34 months. Patients without chemotherapy showed a significant difference in median disease-free survival (DFS) after R0 or R1 resection: 17 [95% confidence interval (CI) 10-24] months versus 8 (95% CI 4-12) months (P < 0.001), whereas in

Completeness of pathology reports in stage II colorectal cancer

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