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Background: Blood is the most extensively studied body fluid and, because it contains circulating tumour cells (CTCs) and circulating tumour-derived cell-free DNA (ctDNA), it may represent a liquid biopsy for cancer. Methods for enrichment and detection of CTCs and ctDNA, their clinical applications and future opportunities in gastrointestinal cancers were the focus of this review., Methods: The PubMed database was searched for literature up to 24 June 2017, with a focus on the past 10 years. Identified articles were further scrutinized for relevant references. Articles were those in English relating to colorectal, gastric and pancreatic cancer., Results: Both CTCs and ctDNA are in low abundance compared with other cellular components of blood, but effective enrichment and highly sensitive techniques are available for their detection. Potential clinical applications of these liquid biopsies include screening, prognostic stratification, therapy administration, monitoring of treatment effect or resistance, and surveillance. Liquid biopsies provide opportunities to reduce the need for invasive tissue sampling, especially in the context of intratumoral heterogeneity and the need for tumour genotyping., Conclusion: Liquid biopsies have applications in gastrointestinal cancers to improve clinical decision-making., (© 2018 BJS Society Ltd Published by John Wiley & Sons Ltd.)

Background: Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer., Methods: Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns., Results: Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells., Conclusion: The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Background: Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients., Methods: We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the "Secondary Adjuvant Taxotere Treatment" (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive., Results: The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers., Conclusion: The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data., Trial Registration: ClinicalTrials.gov: NCT00248703.

Purpose: To investigate the prognostic value of occult metastases detected by quantitative measurements of candidate biomarkers in sentinel lymph nodes (SLNs) from patients curatively resected for colon cancer., Methods: Resection specimens from consecutive patients undergoing surgery for localized colon cancer were subjected to ex vivo SLN mapping. SLNs were examined for the presence of metastases by routine hematoxylin-erythrosin-safranin staining and by cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA quantification. The patients were stratified according to KRAS and BRAF mutation status and microsatellite instability status in their primary tumors. Survival end points were analyzed by Kaplan-Meier survival estimates and log-rank tests., Results: A total of 817 SLNs were identified in 206 (97 %) of the 213 included patients. Routine histological examination of SLNs and other regional lymph nodes identified 63 patients with positive nodes (pN+), of which 42 (67 %) were positive in one or more SLNs (sensitivity 67 %, false-negative rate 33 %). On the basis of the CK20 and MUC2 mRNA levels in SLNs, occult metastases were suggested in 86 (60 %) and 52 (36 %) of the 143 otherwise LN-negative (pN0) patients, respectively. Survival analysis with a median 3.6-year follow-up revealed that MUC2 mRNA quantification had significant prognostic value in SLNs from all patients; however, occult SLN metastasis detection did not., Conclusions: Occult SLN metastases detected by CK20 and MUC2 mRNA quantification had limited prognostic value.

To identify micro RNA (miRNA) biomarker candidates for early detection of breast cancer and detection of minimal residual breast cancer, we performed miRNA expression profiling in pooled RNA samples from breast tumors, and from bone marrow mononuclear cells, peripheral blood mononuclear cells and plasma from healthy controls. We found substantially higher levels of five miRNAs in the breast tumors compared to the normal samples. However, validation of these miRNA levels, and seven other candidates selected from the literature, in individual samples from healthy controls and patients with non-metastatic breast cancer did not suggest further examination of their biomarker potential.

Purpose: To investigate the prognostic relevance of disseminated tumor cells (DTCs) in bone marrow (BM) assessed by a multimarker mRNA panel consisting of TWIST1, cytokeratin 19 (CK19) and human mammaglobin A (hMAM) mRNA, in patients with early breast cancer., Patients and Methods: TWIST1 (gene name: TWIST1), CK19 (gene name: KRT19), and hMAM (gene name: SCGB2A2) mRNA was quantitated in BM samples from 191 operable breast cancer patients by real-time reverse transcriptase polymerase chain reaction (RT-PCR)., Results: Using the highest relative mRNA concentration of TWIST1 in the control population as a cut-off, 5 of the 191 breast cancer patients showed elevated TWIST1 mRNA levels in their BM by real-time RT-PCR. Two of these patients experienced a systemic relapse during a median follow-up of 98 months. Combining these results with previous hMAM and CK19 mRNA quantifications in the same BM samples, 12 (40%) of the 30 patients with BM positive for at least 1 marker (multimarker positive) experienced a systemic relapse as compared with 18 (11%) of the 161 patients with multimarker-negative BMs. The patients with multimarker-positive BM had significantly shorter systemic recurrence-free survival (P < .001, log-rank test), breast cancer-specific survival (P < .001), and overall survival (P = .03). The prognostic relevance of BM multimarker detection appeared to be independent of adjuvant treatment, although the difference was not statistically significant in the subgroup of patients who received adjuvant chemotherapy. Multivariate analysis demonstrated the BM multimarker panel status to be a strong independent predictor of clinical outcome., Conclusion: Our results demonstrated the prognostic relevance of BM DTCs assessed by a multimarker mRNA panel consisting of TWIST1, CK19, and hMAM in operable breast cancer patients.

Purpose: The utility of three different epithelial mRNA markers to detect clinically significant, disseminated tumour cells in bone marrow (BM) was explored., Methods: Mammaglobin A (hMAM), trefoil factor 1 (TFF-1) and prostate derived Ets factor (PDEF) mRNA were quantitated by real-time RT-PCR in BM samples from 192 breast cancer patients undergoing surgery (control group: 26 healthy women)., Results: During a median follow-up of 72 months, four of the five hMAM BM-positive and three of the seven TFF-1 BM-positive patients experienced a systemic relapse. Kaplan-Meier survival analyses demonstrated significantly shorter recurrence-free-, breast-cancer-specific- and overall survival for both hMAM and TFF-1 BM-positive patients. In contrast, PDEF mRNA quantitation did not reveal any significant differences in the survival analyses. Multivariate Cox regression demonstrated hMAM mRNA BM expression to be an independent predictor of both overall- (hazard ratio = 5.896), breast-cancer-specific- (hazard ratio = 10.208) and systemic-recurrence-free survival (hazard ratio = 14.304). TFF-1 status was related to hMAM status (P < 0.001)., Conclusion: Breast cancer patients with pre-operative elevated BM levels of hMAM and/or TFF-1 mRNA seem to constitute a small group of patients with a very poor prognosis.

Purpose: The presence of regional lymph node metastases is one of the most important prognostic factors in colon cancer. Nevertheless, up to 30% of the lymph node negative patients experience disease recurrence. Possibly, this patient group may be identified by more sensitive techniques than routine histopathological examination of the lymph nodes., Methods: In the present study, we have evaluated the detection of colon cancer lymph node metastases by real-time RT-PCR quantitation of the epithelial-specific cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNAs., Results: Both assays were able to detect dilutions of tumor cells down to one tumor cell in 10(6) normal lymphocytes. CK20 and MUC2 mRNA were quantitated in 52 normal lymph nodes from 12 patients undergoing surgery for benign bowel diseases and in 144 primary colon tumors. The median tumor level of both markers were more than 10(4)-fold higher than the highest level in normal lymph nodes, indicating that the markers had a potential for metastasis detection in a clinical context. We applied the assays to 61 lymph nodes with known metastases detected by routine staining. Elevated CK20 or MUC2 mRNA levels were detected in 57 (95%) of the 61 LNs., Conclusions: Thus, CK20 and MUC2 quantitation by real-time RT-PCR seems to be a promising, sensitive tool to detect metastases in regional lymph nodes from colon cancer patients.