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Introduction: CRC with liver metastases is a major contributor to cancer-related mortality. Despite advancements in liver resection techniques, patient survival remains a concern due to high recurrence rates. This study seeks to uncover prognostic biomarkers that predict overall survival in patients undergoing curative hepatic resection for CRC liver metastases., Methods: Prospectively collected serum samples from a cohort of 49 patients who received curative hepatic resection for CRC liver metastases were studied. The patients are part of a cohort, previously analyzed for perioperative complications (see methods). Various preoperative serum markers, clinical characteristics, and factors were analyzed. Univariate and multivariate Cox regression analyses were conducted to determine associations between these variables and disease-free survival as well as overall survival., Results: For disease-free survival, univariate analysis highlighted the correlation between poor outcomes and advanced primary tumor stage, high ASA score, and synchronous liver metastases. Multivariate analysis identified nodal-positive primary tumors and synchronous metastases as independent risk factors for disease-free survival. Regarding overall survival, univariate analysis demonstrated significant links between poor survival and high preoperative IL-8 levels, elevated neutrophil-lymphocyte ratio (NLR), and presence of metastases in other organs. Multivariate analysis confirmed preoperative IL-8 and having three or more liver metastases as independent risk factors for overall survival. The impact of IL-8 on survival was particularly noteworthy, surpassing the influence of established clinical factors., Conclusion: This study establishes preoperative IL-8 levels as a potential prognostic biomarker for overall survival in patients undergoing curative liver resection for CRC liver metastases. This study underscores the importance of incorporating IL-8 and other biomarkers into clinical decision-making, facilitating improved patient stratification and tailored treatment approaches. Further research and validation studies are needed to solidify the clinical utility of IL-8 as a prognostic marker., (© 2024. The Author(s).)

As the state of resection margins is an important prognostic factor after extirpation of colorectal liver metastases, surgeons aim to obtain negative margins, sometimes elaborated by resections of the positive resection plane after intraoperative frozen sections. However, this is time consuming and results sometimes remain unclear during surgery. Label-free multimodal multiphoton microscopy (MPM) is an optical technique that retrieves morpho-chemical information avoiding all staining and that can potentially be performed in real-time. Here, we investigated colorectal liver metastases and hepatic tissue using a combination of three endogenous nonlinear signals, namely: coherent anti-Stokes Raman scattering (CARS) to visualize lipids, two-photon excited fluorescence (TPEF) to visualize cellular patterns, and second harmonic generation (SHG) to visualize collagen fibers. We acquired and analyzed over forty thousand MPM images of metastatic and normal liver tissue of 106 patients. The morphological information with biochemical specificity produced by MPM allowed discriminating normal liver from metastatic tissue and discerning the tumor borders on cryosections as well as formalin-fixed bulk tissue. Furthermore, automated tissue type classification with a correct rate close to 95% was possible using a simple approach based on discriminant analysis of texture parameters. Therefore, MPM has the potential to increase the precision of resection margins in hepatic surgery of metastases without prolonging surgical intervention., (© 2023. The Author(s).)

Background: Colorectal cancer (CRC) is one of the most common types of cancer in Western civilization and responsible for a high number of yearly deaths. Long-term outcome is influenced by many factors, potentially including socioeconomic aspects like income, education, and employment. Furthermore, annual surgical case volume plays a major role in achieving good oncological results. In our retrospective study, we evaluated the effect of socioeconomic deprivation and hospital volume on overall survival (OS) in the federal state of Saxony, Germany., Methods: All patients with CRC who underwent surgery in Saxony, Germany between 2010 and 2020 and were living in Saxony at the time of diagnosis were included in our retrospective analysis. Uni- and multivariate analyses were conducted considering age, sex, tumor localization, UICC tumor stage, surgical approach (open/laparoscopic), number of resected lymph nodes, adjuvant chemotherapy, year of surgery, and hospital case volume. In addition, our model was adjusted for social disparity using the German Index of Socioeconomic Deprivation (GISD)., Results: A total of 24,085 patients were analyzed (15,883 with colon cancer and 8,202 with rectal cancer). Age, sex, UICC tumor stage and tumor localization were distributed as expected for CRC. Median overall survival time was 87.9 months for colon cancer and 110.0 months for rectal cancer. Univariate analysis revealed laparoscopic surgery (colon and rectum P < 0.001), high case volume (rectum: P = 0.002) and low levels of socioeconomic deprivation (colon and rectum P < 0.001) to be significantly associated with better survival. In multivariate analyses, the associations of laparoscopic surgery (colon: HR = 0.76, P < 0.001; rectum: HR = 0.87, P < 0.01), and mid-low to mid-high socioeconomic deprivation (colon: HR = 1.18-1.22, P < 0.001; rectum: HR = 1.18-1.36, P < 0.001-0.01) remained statistically significant. Higher hospital case volume was associated with better survival only in rectal cancer (HR = 0.89; P < 0.01)., Conclusion: In Saxony, Germany, better long-term survival after CRC surgery was associated with low socioeconomic deprivation, laparoscopic surgery and partly with high hospital case volume. Thus, there is a need to reduce social differences in access to high-quality treatment and prevention and increase hospital patient volume., (© 2023. The Author(s).)

Background: Guidance regarding adjuvant treatment decisions in stage II colorectal cancer (CRC) remains uncertain due to lack of predictive clinical or molecular markers. Recently, postoperative circulating tumour (ct)DNA has been demonstrated to be a strong prognostic marker in early colon cancer., Patients and Methods: CIRCULATE enrols patients with stage II microsatellite stable CRC in Germany (AIO) and Austria (ABCSG). Within the AIO, screening is supported by ColoPredict Plus 2.0, a molecular registry, and screening platform for interventional trials. Patient-specific mutations are centrally analysed by next generation sequencing in the resected primary tumour. A postoperative plasma sample is subsequently screened for the specific mutation(s). ctDNA positive (ctDNApos) patients are randomised (2:1) chemotherapy (capecitabine, oxaliplatin added an investigator's choice) or to follow-up (control group). ctDNA negative (ctDNAneg) patients are randomised (1:4) to be followed-up within CIRCULATE (control group) or outside the trial. Patients in the control group remain blinded to the ctDNA results. The primary objective is to compare disease free survival (DFS) of ctDNApos patients with chemotherapy or control. To demonstrate a treatment effect with a hazard ratio of 0.617 (3-year DFS rates 42.5% vs. 25%), 231 ctDNApos and estimated 2079 ctDNAneg patients are randomised. Secondary aims include to compare overall survival and DFS in the ctDNApos and ctDNAneg patient cohorts and ctDNA kinetics., Conclusion: The CIRCULATE trial may establish ctDNA for adjuvant treatment decision in stage II colon cancer - and with the secondary objectives - support a ctDNA guided follow up in colon cancer stage II and beyond., Competing Interests: Declaration of Competing Interest GF received honoraries for lectures from Amgen, Bayer, BMS, Falk Foundation, Merck, MSD, Roche, Servier, Shire, honoraries for advisory boards from Amgen, Bayer, BMS, Merck, MSD, Pierre Fabre, Roche, Sanofi-Aventis, Servier, Shire and research funding from Merck. ARS received honoraries for lectures fromAmgen, AstraZeneca, Aurikamed, BMS, Celgene, iomedico, Lilly, Merck Serono, MCI Global, med publico, MSD, Pfizer, promedicis, Roche, Sanofi-Aventis, Servier , honoraries for advisory boards from Amgen, AstraZeneca, Baxalta, BMS, Celgene, Merck Serono, MSD, Onkowissen.de, Pierre Fabre, Pfizer, Roche, Servier, Onkowissen.de , travel expenses from AstraZeneca, Celgene, Ipsen, MCI Global, Merck Serono, MSD, onkowissen.de, Pierre Fabre, Roche, Servier and research funding for clinical studies from Roche and Ipsen, and compensation for clinical trials / research from Amgen, Alexion, Astra Zeneca, Celgene, Ipsen, Lilly, Roche, Servier, AIO Studien gGmbH, Agricola, PPD Global Limited UK, Mologen Berlin, Universität München, Universität Erlangen, Universität Köln, Pharma Consulting Group AB Schweden, Syneed medidata GmbH, Rafael-Pharmaceutics; BioNTech. LW has stocks from Guardant Health, received honoraries for lectures from Roche;Bristol-Myers Squibb; Lilly; Novocure; Merck Serono; Amgen; Pierre Fabre; Servier; MSD, honoraries for advisory boards from Bristol-Myers Squibb; Roche; Lilly; Novocure; Merck Serono; Takeda; MSD; Amgen; PharmaMar; Bayer, received research funding from Roche and Novocure and travel expenses from Ipsen; Pfizer; Merck Serono; Astra Zeneca. NvB received honoraries for lectures from Forum für Medizinische Fortbildung (FomF)CT has partly ownership at AgenDix GmbH and received honoraries for advisory boards from JAZZ, Novartis, honoraries for lectures from Novartis, Astellas, JAZZ, Janssen; Thermo and research funding from Novartis, Bayer and JAZZ. AT received honoraris for lecture from Amgen, AstraZeneca, BMS, Celgene, iomedico, Lilly, Merck Serono, MCI Global, med publico, MSD, Pfizer, promedicis, Roche, Sanofi-Aventis, Servier and support for clinical studies / research from Amgen, Alexion, Astra Zeneca, Celgene, Ipsen, Lilly, Roche, Servier, AIO Studien gGmbH, BioNTech. JW, CL, ALK,SW,DEA, and MK declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8 + T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8 + T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8 + T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8 + T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC., Competing Interests: Declaration of interests G.Z. is a named inventor on a patent (EP2955232A1: Method for diagnosing adenomas and/or colorectal cancer (CRC) based on analyzing the gut microbiome)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1 EV ) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1 EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1 EV -mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases., (© 2022. The Author(s).)

Lynch syndrome (LS), Lynch-like syndrome (LLS) and familial colorectal cancer type X (FCCX) are different entities of familial cancer predisposition leading to an increased risk of colorectal cancer (CRC). The aim of this prospective study was to characterise and to compare the risks for adenoma and CRC in these three risk groups. Data was taken from the registry of the German Consortium for Familial Intestinal Cancer. Patients were prospectively followed up in an intensified colonoscopic surveillance programme that included annual examinations. Cumulative risks for adenoma and CRC were calculated separately for LS, LLS and FCCX, and then for males and females. Multivariate Cox regression was used to analyse the independent contributions of risk group, mismatch repair gene (within LS), sex and previous adenoma. The study population comprised 1448 individuals (103 FCCX, 481 LLS and 864 LS). The risks were similar for colorectal adenomas, but different for first and metachronous CRC between the three risk groups. CRC risk was highest in LS, followed by LLS and lowest in FCCX. Male sex and a prevalent adenoma in the index colonoscopy were associated with a higher risk for incident adenoma and CRC. In patients with LS, CRC risks were particularly higher in female MSH2 than MLH1 carriers. Our study may support the development of risk-adapted surveillance policies in LS, LLS and FCCX., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Importance: Tumor relapse after partial hepatectomy for colorectal liver metastasis (CRLM) remains an unsolved issue. Intraoperative manipulation of the liver during conventional hepatectomy might enhance hematogenous tumor cell spread. The anterior approach is an alternative approach that may reduce intraoperative tumor cell dissemination., Objective: To determine the efficacy and safety of the anterior approach compared with conventional hepatectomy in patients undergoing resection for CRLM., Design, Setting, and Participants: This randomized clinical study evaluated the efficacy and safety of the anterior approach compared with conventional hepatectomy in adult patients with CRLM who were scheduled for hepatectomy from February 1, 2003, to March 31, 2012, at a tertiary-care hospital. A total of 80 patients with CRLM were randomized to the anterior approach and conventional hepatectomy groups in a 1:1 ratio. Bone marrow and blood samples were analyzed for disseminated tumor cells and circulating tumor cells (CTC) using cytokeratin 20 reverse transcriptase-polymerase chain reaction analysis. Data were analyzed from April 1 to December 1, 2018, using intention to treat., Interventions: Anterior approach vs conventional hepatectomy., Main Outcomes and Measures: The primary end point was intraoperative CTC detection in central blood samples after liver resection. Secondary end points included postoperative morbidity, mortality, and long-term survival., Results: Among the 80 patients included in the analysis (48 men [60%]; mean [SD] age, 61 [10] years), baseline characteristics, including preoperative CTC detection, were comparable between both groups. There was no statistically significant difference in intraoperative CTC detection between patients in the conventional hepatectomy (5 of 21 [24%]) and anterior approach (6 of 22 [27%]) groups (P = .54). Except for a longer operating time in the anterior approach group (mean [SD], 171 [53] vs 221 [53] minutes; P < .001), there were no significant differences in intraoperative and postoperative outcomes between both study groups. Although detection of CTC was associated with poor overall (median, 46 [95% CI, 40-52] vs 81 [95% CI, 54-107] months; P = .03) and disease-free (median, 40 [95% CI, 34-46] vs 60 [95% CI, 46-74] months; P = .04) survival, there was no significant difference in overall (median, 73 [95% CI, 42-104] vs 55 [95% CI, 35-75] months; P = .43) and disease-free (median, 48 [95% CI, 40-56] vs 40 [95% CI, 28-52] months; P = .88) survival between the conventional hepatectomy and anterior approach groups. Also, there was no significant difference in patterns of recurrence between both groups., Conclusions and Relevance: This randomized clinical trial found that the anterior approach was not superior to conventional hepatectomy in reducing intraoperative tumor cell dissemination in patients undergoing resection of CRLM., Trial Registration: isrctn.org Identifier: ISRCTN45066244.

Background: Individuals with pathogenic germline variants in DNA mismatch repair (MMR) genes are at increased risk of developing colorectal, endometrial and other cancers (Lynch syndrome, LS). While previous studies have extensively described cancer risks in LS, cancer risks in individuals from families without detectable MMR gene defects despite MMR deficiency (Lynch-like syndrome, LLS), and in individuals from families fulfilling the Amsterdam-II criteria without any signs of MMR deficiency (familial colorectal cancer type X, FCCX) are less well studied. The aim of this prospective study was to characterise the risk for different cancer types in LS, LLS, and FCCX, and to compare these with the cancer risks in the general population., Methods: Data was taken from the registry of the German Consortium for Familial Intestinal Cancer, where individuals were followed up prospectively within the framework of an intensified surveillance programme at recommended annual examination intervals. A total of 1120 LS, 594 LLS, and 116 FCCX individuals were analysed. From this total sample, eight different cohorts were defined, in which age-dependent cumulative risks and standardised incidence ratios were calculated regarding the first incident occurrence of any, colorectal, stomach, small bowel, urothelial, female breast, ovarian, and endometrial cancer, separately for LS, LLS, and FCCX., Results: The number of individuals at risk for first incident cancer ranged from 322 to 1102 in LS, 120 to 586 in LLS, and 40 to 116 in FCCX, depending on the cancer type of interest. For most cancer types, higher risks were observed in LS compared to LLS, FCCX, and the general population. Risks for any, colorectal, stomach, urothelial, and endometrial cancer were significantly higher in LLS compared to the general population. No significantly increased risks could be detected in FCCX compared to LLS patients, and the general population. Colorectal and endometrial cancer risks tended to be higher in LLS than in FCCX., Conclusions: The characterisation of cancer risks in patients with LLS and FCCX is important to develop appropriate surveillance programmes for these specific intermediate risk groups. Larger prospective studies are needed to obtain more precise risk estimates.

Purpose: Intraoperative detection of intrahepatic lesions can be demanding. The use of preoperative contrast-enhanced magnetic resonance imaging (MRI) or computer tomography (CT) combined with intraoperative ultrasound of the liver is state of the art. Near totally regressed colorectal liver metastases (CRLM) after neoadjuvant chemotherapy or nodules in severely altered liver tissue as steatosis or cirrhosis are often hard to detect during the operative procedure. Especially differentiation between benign atypical nodules and malignant tumors can be very difficult. The intraoperative use of contrast-enhanced ultrasound or intraoperative navigation are helpful tools. However, both methods show relevant limitations. The use of intraoperative MRI (ioMRI) can overcome this problem. Relevant structures can be marked within the operative site or immediate control of complete tumor resection can be achieved. This might allow immediate surgical optimization in case of failure., Methods: We report the intraoperative application of ioMRI in a case of a 61-year-old male patient suffering from rectal cancer with 10 synchronous bilobar CRLM who was treated stepwise by multimodal treatment and staged hepatectomy. Intraoperative contrast-enhanced MRI of the liver was used during completion procedure of an extended right hemihepatectomy performed as "Associating Liver Partition and Portal vein Ligation for Staged hepatectomy (ALPPS)"., Results: ioMRI provided excellent images and showed absence of liver metastases in the liver remnant. Procedure of ioMRI was safe, fast and feasible., Conclusion: To the best of our knowledge, we describe the first case of intraoperative application of a contrast-enhanced MRI during open liver surgery at the University Hospital of Dresden.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

Introduction : Metastasis is the main cause of cancer-associated death in colorectal cancer (CRC). The presence of circulating tumor cells (CTC) in the blood is associated with an increased risk of recurrence and poor prognosis. The clinical significance of CTCs as a novel biomarker has been extensively studied in the last decade. It has been shown that CTC detection applies to early cancer detection. The presence of CTCs is associated with metastatic spread and poor survival and is also useful as a marker for therapy response. Areas covered : We summarize the role of CTC in CRC, their clinical significance, current methods for CTC detection and challenges as well as future perspectives of CTC research. Expert commentary : The clinical significance of CTC in CRC patients is well established. Although insightful, the available marker-based approaches hampered our understanding of the CTCs and their biology, as such approaches do not take into account the heterogeneity of these cell populations. New technologies should expand the marker-based detection to multi biomarker-based approaches together with recent technological advances in microfluidics for single cell enrichment and analysis.

Background: The free flap transfer of a latissimus dorsi flap (LDF) for the closure of sacral wound defects after pelvic exenteration and radiation therapy offers a successful tool of the plastic surgeon. This case report shows the successful coverage using an upstream arterio-venous (AV) loop in combination with an LDF., Case Presentation: We describe the case of a patient who underwent a pelvic exenteration and radiation therapy due to a local recurrence of rectal cancer. The initially used VRAM flap could not ensure a satisfactorily wound closure. An interdisciplinary approach first yielded an AV loop using both greater saphenous veins and was connected to the arteria and vena femoris followed by a free LDF transfer, which was performed 11 days later. The result was an excellent reconstructive and plastic coverage of the sacral wound defect with a well-perfused LDF. The long-term result showed a perfectly integrated flap in the sacral region., Conclusion: We recommend the free LDF for the coverage of large wound defects in irradiated areas after the failure of VRAM flap. If an AV loop is necessary within the flap transfer, we recommend conducting two procedures to guarantee the perfusion of the AV loop.

The aim of this study was to determine the prognostic value of circulating angiogenic cytokines in non-metastatic colorectal cancer (CRC) patients. Preoperative serum samples of a training (TC) (n = 219) and a validation cohort (VC) (n = 168) were analyzed via ELISA to determine PlGF, EGF, VEGF, Ang1, PDGF-A, PDGF-B, IL-8 and bFGF levels. In addition, survival was correlated with PlGF and EGF expression measured by microarray and RNAseq in two publicly available, independent cohorts (n = 550 and n = 463, respectively). Prognostic values for overall (OS) and disease-free survival (DFS) were determined using uni- and multivariate Cox proportional hazard analyses. Elevated PlGF is predictive for impaired OS (TC: HR 1.056; p = 0.046; VC: HR 1.093; p = 0.001) and DFS (TC: HR 1.052; p = 0.029; VC: HR 1.091; p = 0.009). Conversely, elevated EGF is associated with favorable DFS (TC: HR 0.998; p = 0.045; VC: HR 0.998; p = 0.018) but not OS (TC: p = 0.201; VC: p = 0.453). None of the other angiogenic cytokines correlated with prognosis. The prognostic value of PlGF (OS + DFS) and EGF (DFS) was confirmed in both independent retrospective cohorts. Serum PlGF and EGF may serve as prognostic markers in non-metastatic CRC.

Objective: The aim of this study was to evaluate outcomes of metastases at various time intervals after colorectal cancer (CRC) diagnosis., Background: Earlier studies have indicated a short time interval between CRC diagnosis and distant metastases to be associated with poor prognosis. The majority of studies assessed outcome from CRC diagnosis or metastasis resection rather than from metastasis diagnosis and might be subject to immortal time bias., Methods: Patients in the population-based DACHS study were stratified: metastases at/within 1 month (immediate), 2 to 6 months (early), 7 to 12 months (intermediate), and >12 months (late) after CRC diagnosis. The primary endpoint was overall survival (OS) from metastasis diagnosis. Cox proportional hazards regression models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CI). HRs were adjusted for important confounders and immortal time., Results: A total of 1027 patients were included. T4 (P < 0.0001) and node-positive tumors (P < 0.0001) were more frequent in the immediate group. Lung metastases (P < 0.0001) and single-site metastases (P < 0.0001) were more prevalent in the late group. In multivariable analysis, immediate metastases were not associated with poor OS compared to metastases at later time points (late vs immediate: HR 1.21; 95% CI, 0.98-1.48). Subgroup analyses revealed poor OS of late versus immediate metastases for females (1.45; 1.08-1.96), proximal colon cancer (1.54; 1.09-2.16), and N0 (1.46; 1.00-2.12) or N1 disease (1.88; 1.17-3.05)., Conclusions: Immediate or early metastases are not associated with unfavorable outcome compared to late metastases. These findings challenge the current notion of poor outcome for CRC with immediate or early metastases.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired. As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

Background: There is urgent need for improved staging in patients with metastatic colorectal cancer (mCRC). In this study, we evaluated the prognostic value of circulating endothelial cells (CEC) in comparison with circulating tumor cells (CTC) in patients with mCRC amenable for potentially curative surgery., Methods: A total of 140 patients were enrolled prospectively. CTC and CEC were measured with the CellSearch System (Veridex, NJ, USA). Cut-off values were determined using ROC analyses. Prognostic factors were identified by Cox proportional hazards models., Results: ROC analyses revealed ≥ 21 CEC as cut-off levels for detection, which was present in 68 (49%). CEC detection was associated with female gender (p = 0.03) only, whereas CTC detection was associated with presence of the primary tumor (p = 0.007), metastasis size (p < 0.001), bilobar liver metastases (p = 0.02), CEA (p < 0.001) and CA 19-9 levels (p < 0.001). On multivariate analysis only CEC detection (HR 1.81; p = 0.03) and preoperative CA19-9 levels (HR 2.28, p = 0.005) were revealed as independent predictors of poor survival., Conclusions: CEC are of stronger prognostic value than CTC. Further studies are required to validate these results and to evaluate CEC as predictive biomarker for systemic therapy alone as well as in combination with other markers such as CA19-9.

Background: Colorectal cancer (CRC) is the third most common cancer in western countries and is driven by the Wnt signaling pathway. LIM-domain-binding protein 1 (LDB1) interacts with the Wnt signaling pathway and has been connected to malignant diseases. We therefore aimed to evaluate the role of LDB1 in CRC., Results: Overexpression of LDB1 in CRC is associated with strikingly reduced overall and metastasis free survival in all three independent patient cohorts. The expression of LDB1 positively correlates with genes involved in the Wnt signaling pathway (CTNNB1, AXIN2, MYC and CCND1). Overexpression of LDB1 in CRC cell lines induced Wnt pathway upregulation as well as increased invasivity and proliferation. Upon separate analysis, the role of LDB1 proved to be more prominent in proximal CRC, whereas distal CRC seems to be less influenced by LDB1., Materials and Methods: The expression of LDB1 was measured via RT-qPCR in 59 clinical tumor and normal mucosa samples and correlated to clinical end-points. The role of LDB1 was examined in two additional large patient cohorts from publicly available microarray and RNAseq datasets. Functional characterization was done by lentiviral overexpression of LDB1 in CRC cell lines and TOP/FOP, proliferation and scratch assays., Conclusions: LDB1 has a strong role in CRC progression, confirmed in three large, independent patient cohorts. The in vitro data confirm an influence of LDB1 on the Wnt signaling pathway and tumor cell proliferation. LDB1 seems to have a more prominent role in proximal CRC, which confirms the different biology of proximal and distal CRC.

MicroRNAs are small non-coding RNAs with a length of 18-25 nucleotides. They can regulate tumor invasion and metastasis by changing the expression and translation of their target mRNAs. Their expression is substantially altered in colorectal cancer cells as well as in the adjacent tumor-associated stroma. Both of these compartments have a mutual influence on tumor progression. In the development of metastases, cancer cells initially interact with the host tissue. Therefore, compartment-specific expression signatures of these three locations-tumor, associated stroma, and host tissue-can provide new insights into the complex tumor biology of colorectal cancer. Frozen tissue samples of colorectal liver ( n = 25) and lung metastases ( n = 24) were laser microdissected to separate tumor cells and the adjacent tumor-associated stroma cells. Additionally, normal lung and liver tissue was collected from the same patients. We performed a microarray analysis in four randomly selected liver metastases and four randomly selected lung metastases, analyzing a total of 939 human miRNAs. miRNAs with a significant change >2-fold between the tumor, tumor stroma, and host tissue were analyzed in all samples using RT-qPCR (11 miRNAs) and correlated with the clinical data. We found a differential expression of several miRNAs between the tumor, the tumor-associated stroma, and the host tissue compartment. When comparing liver and lung metastases, miR-194 showed a 1.5-fold; miR-125, miR-127, and miR-192 showed a 2.5-fold; miR-19 and miR-215 a 3-fold; miR-145, miR-199-3, and miR-429 a 5-fold; miR-21 a 7-fold; and, finally, miR-199-5 a 12.5-fold downregulation in liver metastases compared to lung metastases. Furthermore miR-19, miR-125, miR-127, miR-192, miR-194, miR-199-5, and miR-215 showed a significant upregulation in the normal liver tissue compared to the normal lung tissue. Univariate analysis identified an association of poor survival with the expression of miR-125 ( p = 0.05), miR-127 ( p = 0.001), miR-145 ( p = 0.005), miR-192 ( p = 0.015), miR-194 (0.003), miR-199-5 ( p = 0.008), miR-215 ( p < 0.001), and miR-429 ( p = 0.03) in the host liver tissue of the liver metastases. Colorectal liver and lung metastases have a unique miRNA expression profile. miRNA expression in the host tissue of colorectal liver metastases seems to be able to influence tumor progression and survival. These findings can be used in the development of tailored therapies., Competing Interests: The authors declare no conflict of interest.

Purpose: CRC remains the third most common cancer worldwide with a high 5-year mortality rate in advanced cases. Combined with chemotherapy, targeted therapy is an additional treatment option. However as CRC still escapes targeted therapy the vigorous search for new targets is warranted to increase patients´ overall survival., Results: In this study we describe a new role for Gas6/protein S-TAM receptor interaction in CRC. Gas6, expressed by tumor-infiltrating M2-like macrophages, enhances malignant properties of tumor cells including proliferation, invasion and colony formation. Upon chemotherapy macrophages increase Gas6 synthesis, which significantly attenuates the cytotoxic effect of 5-FU chemotherapy on tumor cells. The anti-coagulant protein S has similar effects as Gas6.In CRC patient samples Tyro3 was overexpressed within the tumor. In-vitro inhibition of Tyro3 and Mer reduces tumor cell proliferation and sensitizes tumor cells to chemotherapy. Moreover high expression of Tyro3 and Mer in tumor tissue significantly shortens CRC patients´ survival., Experimental Design: Various in vitro models were used to investigate the role of Gas6 and its TAM receptors in human CRC cells, by stimulation (rhGas6) and knockdown (siRNA) of Axl, Tyro3 and Mer. In terms of a translational research, we additionally performed an expression analysis in human CRC tissue and analyzed the medical record of these patients., Conclusions: Tyro3 and Mer represent novel therapeutic targets in CRC and warrant further preclinical and clinical investigation in the future., Competing Interests: The authors have no conflicts of interests.

Objective: Cancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli, we used peripheral blood monocytes in order to characterise a distinct gene expression profile and to evaluate its potential as a candidate diagnostic biomarker in patients with colorectal cancer (CRC), a still unmet clinical need., Design: We performed a case-control study including 360 peripheral blood monocyte samples from four European oncological centres and defined a gene expression profile specific to CRC. The robustness of the genetic profile and disease specificity were assessed in an independent setting., Results: This screen returned 43 putative diagnostic markers, which we refined and validated in the confirmative multicentric analysis to 23 genes with outstanding diagnostic accuracy (area under the curve (AUC)=0.99 (0.99 to 1.00), Se=100.0% (100.0% to 100.0%), Sp=92.9% (78.6% to 100.0%) in multiple-gene receiver operating characteristic analysis). The diagnostic accuracy was robustly maintained in prospectively collected independent samples (AUC=0.95 (0.85 to 1.00), Se=92.6% (81.5% to 100.0%), Sp=92.3% (76.9% to 100.0%). This monocyte signature was expressed at early disease onset, remained robust over the course of disease progression, and was specific for the monocytic fraction of mononuclear cells. The gene modulation was induced specifically by soluble factors derived from transformed colon epithelium in comparison to normal colon or other cancer histotypes. Moreover, expression changes were plastic and reversible, as they were abrogated upon withdrawal of these tumour-released factors. Consistently, the modified set of genes reverted to normal expression upon curative treatment and was specific for CRC., Conclusions: Our study is the first to demonstrate monocyte plasticity in response to tumour-released soluble factors. The identified distinct signature in tumour-educated monocytes might be used as a candidate biomarker in CRC diagnosis and harbours the potential for disease follow-up and therapeutic monitoring., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The prognosis of colorectal cancer (CRC) is closely linked to the occurrence of distant metastases, which putatively develop from circulating tumor cells (CTCs) shed into circulation by the tumor. As far more CTCs are shed than eventually metastases develop, only a small subfraction of CTCs harbor full tumorigenic potential. The aim of this study was to further characterize CRC-derived CTCs to eventually identify the clinically relevant subfraction of CTCs.We established an orthotopic mouse model of CRC which reliably develops metastases and CTCs. We were able to culture the resulting CTCs in vitro, and demonstrated their tumor-forming capacity when re-injected into mice. The CTCs were then subjected to qPCR expression profiling, revealing downregulation of epithelial and proliferation markers. Genes associated with cell-cell adhesion (claudin-7, CD166) were significantly downregulated, indicating a more metastatic phenotype of CTCs compared to bulk tumor cells derived from hepatic metastases. The stem cell markers DLG7 and BMI1 were significantly upregulated in CTC, indicating a stem cell-like phenotype and increased capacity of tumor formation and self-renewal. In concert with their in vitro and in vivo tumorigenicity, these findings indicate stem cell properties of mouse-derived CTCs.In conclusion, we developed an orthotopic mouse model of CRC recapitulating the process of CRC dissemination. CTCs derived from this model exhibit stem-cell like characteristics and are able to form colonies in vitro and tumors in vivo. Our results provide new insight into the biology of CRC-derived CTCs and may provide new therapeutic targets in the metastatic cascade of CRC., Competing Interests: The authors disclose no potential conflicts of interest.

Background: Little is known about the safety of the anti-VEGF antibody bevacizumab in patients undergoing resection for colorectal liver metastases (CLM). This meta-analysis evaluates the impact of bevacizumab on parenchymal damage and functional recovery in patients undergoing resection for CLM., Methods: The Medline, Embase and Cochrane Library were systematically searched for studies on preoperative chemotherapy with and without bevacizumab prior to resection of CLM. Studies that reported histological and/or clinical outcomes were eligible for inclusion. Meta-analyses were performed using a random effects model., Results: A total of 18 studies with a total sample size of 2430 patients (1050 patients with bevacizumab) were found. Meta-analyses showed a significant reduction in sinusoidal obstruction syndrome (SOS) (Odds ratio 0.50 [95% confidence interval 0.37, 0.67]; p < 0.001; I(2) = 0%) and hepatic fibrosis (0.61 [0.4, 0.86]; p = 0.004; I(2) = 7%) after preoperative chemotherapy with bevacizumab. The reduced incidence of posthepatectomy liver failure in patients with bevacizumab treatment just failed to reach statistical significance (0.61 [0.34, 1.07]; p = 0.08 I(2) = 6%). While there was no difference in perioperative morbidity and mortality, the incidence of wound complications was significantly increased in patients who received bevacizumab (1.81 [1.12, 2.91]; p = 0.02 I(2) = 4%)., Conclusions: The combination of bevacizumab with cytotoxic chemotherapy is safe but increases the incidence of wound complications after resection of CLM. The reduction of SOS and hepatic fibrosis warrant further investigation and may explain the inverse association of bevacizumab administration and posthepatectomy liver failure.

Objective: To investigate whether liver metastases contribute to metastatic spread of colorectal cancer (CRC) by shedding intact tumor cells., Background: Metastases represent the primary cause of death in CRC. Understanding the metastatic activity of metastases and which patients are at high risk for tumor cell dissemination may, therefore, have significant influence on cancer care in the future., Methods: Circulating tumor cells (CTCs) were detected in the hepatic inflow (portal venous blood [PVB]) and outflow compartment (hepatic venous blood [HVB]) of a training (n = 55) and validation (n = 50) set using the CellSearch system. Isolated CTC from the HVB were subjected to gene expression analyses by quantitative polymerase chain reaction., Results: CTC detection rate (37.2% vs 19.6%; P = 0.04) and count (mean: 12.7, SEM: ± 5.9 vs 1.9; ± 1.2; P = 0.01) were significantly higher in HVB compared to PVB. The increased CTC detection rate (54% vs 11.4%; P < 0.001) and CTC count (14.7 ± 5.1 vs 1.1 ± 0.6; P < 0.001) in the HVB compared to the PVB compartment was confirmed in the validation cohort. Expression of epithelial markers and genes involved in cell-to-cell and cell-to-matrix adhesion was reduced in CTC compared to tumor cells in liver metastases. Metastasis size greater than 5 cm was associated with CTC shedding from established liver metastases in the training and validation cohorts., Conclusions: Colorectal liver metastases shed intact tumor cells with an invasive phenotype. Metastasis size serves as a surrogate marker for metastatic activity of colorectal liver metastases.

Epithelial-to-mesenchymal transition (EMT) contributes significantly to tumor progression and metastasis. The assessment of EMT-associated transcription factors could be a promising approach to identify biomarkers and potential therapeutic targets in colorectal cancer. In our study, we focused on the transcription factor "Sine oculis homeobox" (SIX) 1, which is a member of the superfamily of the homeobox genes and has been described to promote EMT in different types of tumors. Immunohistochemistry against SIX1 was performed on colorectal mucosa, adenomas, carcinomas-in situ and primary adenocarcinomas. An expression score was developed and subsequently assessed for its prognostic value in two independent cohorts. Cohort 1 consisted of 128 patients with stage I-III colorectal cancer; cohort 2 included 817 patients with stage I-III colorectal cancer who had participated in the DACHS study. HCT-116 cells were transfected with SIX1 plasmids and subjected to migration and colony formation assays. The expression of SIX1 increases gradually from mucosa to colorectal adenocarcinomas (p > 0.0001). Univariate and multivariate analyses reveal that high expression of SIX1 is associated with decreased overall survival (cohort 1: HR: 4.01, CI: 1.20-14.07, p = 0.025; cohort 2: HR: 1.43, CI: 1.014-2.02, p = 0.047). Overexpression of SIX1 induces a more mesenchymal-like phenotype in HCT-116 cells and enhances tumor migration. High expression of SIX1 is an independent prognostic marker in colorectal cancer. It might be a promising biomarker to stratify patients into different risk groups. Moreover, targeting SIX1 might be a novel therapeutic approach in patients with colorectal cancer., (© 2015 UICC.)

Background: Radical resection is the treatment of choice for colorectal liver metastases (CLM). Unfortunately, only about 20 % of patients present with initially resectable disease, in most cases due to bilobar disease. In the last two decades, major achievements have been made to extend surgical indications to patients with bilobar CLM, such as two-stage hepatectomy with or without portal vein occlusion and associating liver partition and portal vein ligation for staged hepatectomy (ALPPS)., Purpose: The purpose of this review article was to summarize current surgical approaches and their safety and efficacy for patients with initially unresectable bilobar CLM., Conclusion: In selected patients, two-stage hepatectomy and ALPPS are efficient and safe to convert unresectable to resectable CLM. Further studies are required to evaluate long-term outcome of these procedures.

In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c+ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.

The composition of tumor-targeted T cell infiltrates is a major prognostic factor in colorectal cancer (CRC) outcome; however, the functional role of these populations in prolonging patient survival remains unclear. Here, we evaluated 190 patients with CRC for the presence of functionally active tumor-infiltrating lymphocytes (TILs), the tumor specificity of these TILs, and the correlation between patient TILs and long-term survival. Using intracytoplasmic cytokine staining in conjunction with HLA multimers loaded with tumor peptide and antigen-specific cytokine secretion assays, we determined that TNF-α expression delineates a population of tumor antigen-specific (TA-specific) cytotoxic T lymphocytes (CTLs) present within tumors from patients with CRC. Upregulation of TNF-α expression in TILs strongly correlated with an increase in the total amount of intratumoral TNF-α, which is indicative of tumor-specific CTL activity. Moreover, a retrospective multivariate analysis of 102 patients with CRC, which had multiple immune parameters evaluated, revealed that increased TNF-α concentration was an independent prognostic factor. Together, these results indicate that the prognostic impact of T cell infiltrates for CRC maybe largely based on subpopulations of active TA-specific T cells within the tumor, suggesting causal implication for these cells in patient survival. Additionally, these results support the use of intratumoral TNF-α, which is indicative of T cell function, as a prognostic parameter for CRC.

Purpose: The aims of this study are to compare the 30-day rate of bowel obstruction for stapled vs. handsewn closure of loop ileostomy, and to further assess efficacy and safety for each technique by secondary endpoints such as operative time, rates of anastomotic leakage, and other post-operative complications within 30 days., Methods: A systematic literature search (MEDLINE, The Cochrane Library, EMBASE and ISI Web of Science) was performed to identify randomized controlled trials (RCTs) comparing stapled and handsewn closure of loop ileostomy after low anterior resection. Random effects meta-analyses were calculated and presented as risk ratio (RR) and mean difference (MD) with corresponding 95 % confidence intervals., Results: Forty publications were retrieved and 4 RCTs (649 patients) were included. There was methodological and clinical heterogeneity of included trials, but statistical heterogeneity was low for most endpoints. Stapler use significantly reduced the rate of bowel obstruction compared to hand-sewn closure (RR 0.53 [0.32, 0.88]; P = 0.01). The operation time was significantly lower for stapling compared to hand suture (MD -15.5 min [-18.4, 12.6]; P < 0.001). All other secondary outcomes did not show significant differences., Conclusions: This meta-analysis shows superiority of stapled closure of loop ileostomy compared to handsewn closure in terms of bowel obstruction rate and mean operation time. Other relevant complications such as anastomotic leakage are equivalent. Even so, both techniques are options with opposing advantages and disadvantages.

T‑cell costimulation is necessary to induce a response of naïve T cells. Whether T‑cell costimulation can also cause reactivation of unreactive, possibly anergized memory T cells (MTCs) from late‑stage cancer patients is unknown. To investigate this question, we developed a bispecific anti‑CD28 fusion protein (bsHN‑CD28) which can easily be attached to the vaccine ATV‑NDV. This virus‑modified autologous tumor cell vaccine has already shown effectivity in colon cancer patients following resection of liver metastases. In this phase Ⅰ clinical study, 14 colorectal carcinoma (CRC) patients with late‑stage disease which could not be operated anymore with curative intent were treated with the vaccine ATV‑NDV to which bsHN‑CD28 was attached. No severe adverse events were recorded. All patients showed an immunological response of tumor‑reactive T cells, at least once during the course of five vaccinations. Also, we demonstrate a dose‑response relationship with the costimulatory molecule added to the vaccine. A partial response of metastases was documented in four patients. The study suggests that the three‑component vaccine is safe and can reactivate possibly anergized T cells from a chronic disease like advanced‑stage cancer.

Drug-induced gene-expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer, for which there is a pressing need to find novel antimetastatic compounds. We computationally predicted three novel and still unknown compounds against colorectal cancer: citalopram (an antidepressant), troglitazone (an antidiabetic), and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation, and migration and in a subcutaneous mouse model. We found evidence that the mode of action of these compounds may be through inhibition of TGFβ signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of colorectal cancer. This study proposes citalopram as a potential therapeutic option for patients with colorectal cancer, illustrating the potential of systems pharmacology., (©2014 American Association for Cancer Research.)

Presence of occult minimal residual disease in patients with colorectal cancer (CRC) has a strong prognostic impact on survival. Minimal residual disease plays a major role in disease relapse and formation of metastases in CRC. Analysis of circulating tumor cells (CTC) in the blood is increasingly used in clinical practice for disease monitoring of CRC patients. In this review article the role of CTC, disseminated tumor cells (DTC) in the bone marrow and micrometastases and isolated tumor cells (ITC) in the lymph nodes will be discussed, including literature published until September 2013. Occult disease is a strong prognostic marker for patient survival in CRC and defined by the presence of CTC in the blood, DTC in the bone marrow and/or micrometastases and ITC in the lymph nodes. Minimal residual disease could be used in the future to identify patient groups at risk, who might benefit from individualized treatment options.

Purpose: Tailored operative strategies have been proposed for patients with bilobar colorectal liver metastases (CLM). The aim of the study was to evaluate the long-term outcome, safety and efficacy, including cancer-specific survival, morbidity, and mortality, of three different surgical strategies for extensive bilateral CLM., Methods: This is a retrospective study of a prospective database of 356 consecutive patients, who underwent hepatic resection due to CLM between January 2003 and January 2009. Fifty-nine patients underwent three different therapeutic approaches: 22 patients with portal vein embolization (PVE) + staged resections, 11 patients with staged resections solely, and 26 patients with an extensive liver resection and simultaneous or subsequent radiofrequency ablation (RFA)., Results: The three groups were comparable regarding their general patient characteristics. The overall morbidity and mortality rates were 27.1 and 1.7 %, respectively. There were no significant differences in morbidity, mortality, or survival between the three groups. The median survival of all patients was 48 months, with a recurrence-free survival of 30 months., Conclusions: The clearance of bilobar CLM can be achieved by various strategies, all of them providing an acceptable mortality rate and survival for the patients. Therefore, patients with bilobar liver metastases should receive a procedure tailored for their individual extent of disease.

The prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer-derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer-derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer-derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694-704. ©2014 AACR.

Background: Despite introduction of effective chemotherapy protocols, it has remained uncertain, if patients with colorectal cancer (CRC) liver metastases should receive adjuvant therapy. Clinical or molecular predictors may help to select patients at high risk for disease recurrence and death who obtain a survival advantage by adjuvant chemotherapy., Methods: A total of 297 patients with potentially curative resection of CRC liver metastases were analyzed. These patients had no neoadjuvant therapy, no extrahepatic disease and negative resection margins. The primary endpoint was overall survival. Patients' risk status was evaluated using the Memorial Sloan-Kettering Cancer Center clinical risk score (MSKCC-CRS). Multivariable analyses were performed using Cox proportional hazard models., Results: A total of 137 (43%) patients had a MSKCC-CRS > 2. Adjuvant chemotherapy was administered to 116 (37%) patients. Patients who received adjuvant chemotherapy were of younger age (p = 0.03) with no significant difference in the presence of multiple metastases (p = 0.72) or bilobar metastases (p = 0.08). On multivariate analysis adjuvant chemotherapy was associated with improved survival in the entire cohort (Hazard ratio 0.69; 95% confidence interval 0.69-0.98). It improved survival markedly in high-risk patients with a MSKCC-CRS > 2 (HR 0.40; 95% CI 0.23-0.69), whereas it was of no benefit in patients with a MSKCC-CRS ≤ 2 (HR 0.90; 95% CI 0.57-1.43)., Conclusions: The MSKCC-CRS offers a tool to select patients for adjuvant therapy after resection of CRC liver metastases. Validation in independent patient cohorts is required.

Background: Advances in neoadjuvant therapy enabled novel strategies for treating resectable and initially unresectable colorectal cancer liver metastases. Although it is well known that chemotherapeutic agents cause certain types of liver parenchymal injury, the actual contribution of chemotherapy-associated hepatotoxicity to postoperative morbidity remains poorly defined. The aim of this study was to define all kinds of chemotherapy-associated liver injury and to examine its impact on postoperative morbidity., Patients and Methods: We included 119 patients who were treated between 2002 and 2010. Chemotherapy-associated changes of the liver were subclassified in 11 different categories and correlated with postoperative morbidity with the ultimate aim of generating a liver injury risk score., Results: On univariate analysis severity (P = .004) and localization of parenchymal inflammation (P = .04) were associated with morbidity. Steatosis did not correlate with postoperative outcome (P = .69), whereas steatohepatitis (as assessed by the nonalcoholic fatty liver disease activity score score) was related with morbidity (P = .03). On multivariate analysis, the severity of inflammation (95% confidence interval, 1.008-6.526; odds ratio, 2.56; P = .04) was significantly correlated with postoperative morbidity. The newly developed liver injury risk score was highly associated with postoperative complications (P = .006)., Conclusion: In this study, the induction of inflammation by conventional chemotherapy and its relevance for the development of clinical complications could be demonstrated. The proposed risk score for liver injury-related morbidity might help to better select patients eligible for an operation., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Background: We performed a systematic review and meta-analysis to investigate the prognostic value of tumor cells in blood (circulating tumor cells [CTCs]) or bone marrow (BM) (disseminated tumor cells) of patients with resectable colorectal liver metastases or widespread metastatic colorectal cancer (CRC)., Materials and Methods: The following databases were searched in May 2011: MEDLINE, EMBASE, Science Citation Index, BIOSIS, Cochrane Library. Studies that investigated the association between tumor cells in blood or BM and long-term outcome in patients with metastatic CRC were included. We extracted hazard ratios (HRs) and confidence intervals (CIs) from the included studies and performed random-effects meta-analyses for survival outcomes., Results: The literature search yielded 16 studies representing 1,491 patients. The results of 12 studies representing 1,329 patients were suitable for pooled analysis. The overall survival (HR, 2.47; 95 % CI 1.74-3.51) and progression-free survival (PFS) (HR, 2.07; 95 % CI 1.44-2.98) were worse in patients with CTCs. The subgroup of studies with more than 35 % CTC-positive patients was the only subgroup with a statistically significant worse PFS. All eight studies that performed multivariable analysis identified the detection of CTCs as an independent prognostic factor for survival., Conclusion: The detection of CTCs in peripheral blood of patients with resectable colorectal liver metastases or widespread metastatic CRC is associated with disease progression and poor survival.

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.

Background: The prognostic role of circulating tumor cells (CTCs) has been established for colorectal cancer (CRC). We investigated the qualitative and quantitative detection of CTC in the central (CVBC) and mesenteric (MVBC) venous blood compartments to elucidate the patterns of hematogenous tumor cell dissemination in patients with CRC., Methods: A total of 200 patients were enrolled prospectively. Blood samples were collected from the tumor-draining vein and via a central venous line. CTCs were detected and quantified by using the CellSearch system. Factors associated with CTC detection in both compartments were analyzed by using univariate and multivariate analyses., Results: CTC analyses were performed in the CVBC and MVBC in 200 and 80 patients, respectively. CTCs were found at a higher rate (P=0.01) and at a higher count (P=0.006) in the MVBC compared with the CVBC. On multivariate analyses, stage IV disease (odds ratio, 3.83; 95% confidence interval, 1.42-10.35) and increased preoperative carbohydrate antigen 19-9 level (odds ratio, 3.57; 1.30-9.79) were associated with CTC detection in the CVBC. CTCs were detected more frequently (P=0.05) and at higher numbers (P=0.05) in the CVBC of patients with low compared with mid or high rectal tumors., Conclusions: The qualitative and quantitative detection of CTCs is higher in the MVBC compared with the CVBC of patients with CRC.

The non-ABC transport protein RalBP1 has been shown to be overexpressed in various cancer cell lines and implicated in the process of metastasis formation, but its expression in tissue samples and prognostic significance has not been shown. In this study matched tumor-mucosa tissue samples from 78 CRC patients were investigated. The RalBP1 mRNA and protein levels were quantified by real-time quantitative PCR (qPCR) and ELISA. RalBP1 was found to be overexpressed in tumor at the mRNA level both overall (p = 0.027), and for stages I (p = 0.024), II (p = 0.038) and IV (p = 0.004). At the protein level, RalBP1 was only significantly overexpressed in stage IV patients (p = 0.018). Expression of RalBP1 mRNA and protein were inversely correlated (r = 0.4173; p = 0.0004). Multivariate Cox regression analysis including sex, age, stage, grade, and nodal status as covariates showed that overexpression of RalBP1 protein, but not mRNA, was an independent predictor of both decreased disease free survival (p = 0.016, RR = 6.892) and overall survival (p = 0.039, RR = 5.986). These results suggest that RalBP1 protein is an independent predictor of poor survival and early relapse for CRC patients. Owing to its multifunctional intermediary role in cell survival, chemotherapeutic resistance, and metastasis formation, RalBP1 represents a promising novel therapeutic target.

Purpose: More than 130 years ago, circulating tumour cells (CTCs) and disseminated tumour cells (DTCs) have been linked to metastasis. Since then, a myriad of studies attempted to characterise and elucidate the clinical impact of CTCs/DTCs, amongst others in colorectal cancer (CRC). Due to a flood of heterogeneous findings regarding CTCs/DTCs in CRC, this review aims to describe the known facts about CTC/DTC biology and clinical impact., Methods: To identify the basic scientific literature regarding the biology and clinical impact of CTCs/DTCs in CRC, we reviewed the literature in the PubMed database. We focused on publications written in English and published until January 2012. As search terms, we used "colorectal cancer (CRC)", "colon cancer (CC)", "CTC", "DTC", "bone marrow (BM)", "lymph node (LN)", "peripheral blood (PB)", "significance" and "prognosis"., Results: CTC detection and quantification under standardised conditions is feasible. Several studies in large patient settings have revealed prognostic impact of CTCs in CRC. CRC-derived DTC detection and analysis in BM exhibits a more heterogeneous picture but also shows clinical value. Furthermore, the presence of DTCs in LN has a strong prognostic impact in CRC., Conclusions: Clinical relevance and prognostic significance of CTCs/DTCs in CRC have been clearly demonstrated in many experimental studies. The major challenge in CTC/DTC research is now to harmonise the various identification and detection approaches and consequently to conduct large prospective multi-institutional trials to verify the use of CTCs/DTCs as a valid prognostic and predictive biomarker for clinical routine.

Purpose: Up to 25% of patients with node-negative colorectal cancer (CRC) on conventional histopathologic analysis ultimately die of recurrent disease. We performed a systematic review with meta-analyses to clarify whether molecular detection of isolated tumor cells or micrometastases in regional lymph nodes indicates high risk of disease recurrence and poor survival in node-negative CRC., Methods: The following databases were searched in August 2011 to identify studies on the prognostic significance of molecular tumor-cell detection in regional lymph nodes of node-negative CRC: MEDLINE, BIOSIS, Science Citation Index, EMBASE, CCMed, and publisher databases. We extracted hazard ratios (HRs) and associated 95% CIs from the identified studies and performed random-effects model meta-analyses on overall survival, disease-specific survival, and disease-free survival., Results: A total of 39 studies with a cumulative sample size of 4,087 patients were included. Immunohistochemistry, reverse transcriptase polymerase chain reaction, and both techniques were applied in 30, seven, and two studies, respectively. Thirteen studies were graded with low risk of bias. Meta-analyses revealed that molecular tumor-cell detection in regional lymph nodes was associated with poor overall survival (HR, 2.20; 95% CI, 1.43 to 3.40), disease-specific survival (HR, 3.37; 95% CI, 2.31 to 4.93), and disease-free survival (HR, 2.24; 95% CI, 1.57-3.20). Subgroup analyses showed the prognostic significance of molecular tumor-cell detection of being independent of the applied detection method, molecular target, and number of retrieved lymph nodes., Conclusion: Molecular detection of occult disease in regional lymph nodes is associated with an increased risk of disease recurrence and poor survival in patients with node-negative CRC.

Purpose: Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and dissemination. EMT occurs predominantly at the tumor edge where it is induced by cytokines, the extracellular matrix environment, or hypoxia. In the tumor cell, it is further mediated by several transcription factors and microRNAs. The aim of this study was to explore the expression of EMT-associated genes at the invasive front in colorectal cancer and to evaluate their prognostic significance., Experimental Design: We evaluated the expression of 13 EMT-associated genes at the invasion front of 30 colorectal liver metastases by quantitative real-time PCR. Immunostaining against zinc finger E-box-binding homeobox 2 (ZEB2) was carried out on 175 primary colorectal cancer specimens and 30 colorectal liver metastases and correlated to clinical and histopathologic data. DLD-1 cells were transfected with siRNA and subjected to migration and invasion assays., Results: Gene expression analysis and immunohistochemistry showed an upregulation of ZEB2 at the invasion front in primary colorectal cancer and liver metastases. Overexpression of ZEB2 at the invasion front correlated significantly with tumor stage in primary colorectal cancer. Moreover, univariate and multivariate analysis revealed overexpression of ZEB2 at the invasion front as an independent prognostic marker for cancer-specific survival. Downregulation of ZEB2 by siRNA decreased the migration and invasion capacity of DLD-1 cells in vitro., Conclusions: Overexpression of ZEB2 at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2 may be interesting as biomarker and potential target for treatment of colorectal cancer., (©2011 AACR.)

Background: Insufficient migration and activation of tumor specific effector T cells in the tumor is one of the main reasons for inadequate host anti-tumor immune response. External radiation seems to induce inflammation and activate the immune response. This phase I/II clinical trial aims to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with colorectal liver metastases., Methods/design: This is an investigator-initiated, prospective randomised, 4-armed, controlled Phase I/II trial. Patients undergoing elective hepatic resection due to colorectal cancer liver metastasis will be enrolled in the study. Patients will receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation targeted to their liver metastasis. Radiation will be applied by external beam radiotherapy using a 6 MV linear accelerator (Linac) with intensity modulated radiotherapy (IMRT) technique two days prior to surgical resection. All patients admitted to the Department of General-, Visceral-, and Transplantion Surgery, University of Heidelberg for elective hepatic resection are consecutively screened for eligibility into this trial, and written informed consent is obtained before inclusion. The primary objective is to assess the effect of active local external beam radiation dose on, tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include radiogenic treatment toxicity, postoperative morbidity and mortality, local tumor control and recurrence patterns, survival and quality of life. Furthermore, frequencies of systemic tumor reactive T cells in blood and bone marrow will be correlated with clinical outcome., Discussion: This is a randomized controlled patient blinded trial to assess the safety and efficiency of low dose radiotherapy on metastasis infiltrating T cells and thus potentially enhance the antitumor immune response., Trial Registration: ClinicalTrials.gov: NCT01191632.

Background: Circulating angiogenic factors (CAF) have been shown as therapeutic targets and prognostic biomarkers in metastatic colorectal cancer. However, their correlation with circulating tumor cells (CTC) is unknown, as is their role as prognostic biomarkers in patients amenable for curative resection., Methods: Preoperative blood samples were collected in patients undergoing potentially curative resection of colorectal liver metastases. Serum levels of eight CAF and CTC were analyzed by using ELISA and CK20 RT-PCR, respectively. Prognostic factors were identified by a Cox proportional hazards model., Results: A total of 107 patients were eligible for final analyses. Circulating levels of PlGF, EGF, and bFGF were increased, whereas PDGF-A and Ang-1 levels were decreased in patients compared with healthy control subjects. CTC were detected in 36 of 63 patients (57%) and were associated with significantly lower levels of EGF and bFGF. On univariate analyses, multiple metastases (p = 0.04), a MSKCC risk score >2 (p = 0.004), and detection of CTC (p = 0.05) were associated with disease recurrence. Multivariate analysis, including the panel of eight CAF, revealed a MSKCC score >2 [hazard ratio (HR), 2.01; 95% confidence interval (CI), 1.11-3.82; p = 0.02] and low levels of circulating PlGF (HR, 0.26; 95% CI, 0.08-0.81; p = 0.02) as independent predictors of poor recurrence-free survival., Conclusions: CAF may indicate patients who are at high risk for disease recurrence. The notion that CAF may identify patients who benefit from adjuvant therapy or antiangiogenic agents warrants further investigation.

Aims: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133 and AC133 cells., Methods: We analysed the gene expression profiles of EpCAM/CEA/AC133 and EpCAM/CEA/AC133 cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set., Results: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133 cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts., Conclusions: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.