Colorectal Liver Metastases Tissue Research Articles

KLK10 derived from tumor endothelial cells accelerates colon cancer cell proliferation and hematogenous liver metastasis formation.

Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation invivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation invivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.

Probe-based confocal laser endomicroscopy for real-time evaluation of colorectal liver metastasis in resected surgical specimens

Probe-based confocal laser endomicroscopy (pCLE) enables real-time examination of tissue structure. This study investigated pCLE with or without fluorescein sodium for the intraoperative diagnosis of colorectal liver metastasis (CLM) and detection of surgical margins. Thirty-four specimens of CLM and adjacent noncancerous tissue were obtained from 21 patients and examined by pCLE between May 2017 and March 2018. Images were obtained both without and with fluorescein sodium applied to the cut surface and compared with hematoxylin and eosin-stained tissue. Fluorescence intensity (FI) was measured by luminance-analysis software. Without external fluorophores, pCLE visualized 91.2% of CLM tissues as an irregular structure with low autofluorescence and 90.5% of noncancerous liver tissues as a regular structure with high autofluorescence. The median FI was significantly lower in cancer than in benign tissue in patients without chemotherapy [70.4 (51.6–110) vs. 48.3 (39.0–59.4), p = 0.002] and with chemotherapy [67.9 (54.6–89.2) vs. 48.6 (28.8–82.1), p < 0.001]. The border was clearly visible; pCLE with fluorescein sodium clearly showed their morphologies. In summary, our study demonstrated real-time pCLE distinguished CLM and noncancerous tissue by differences in structure and FI regardless of prehepatectomy chemotherapy. Fluorescein spray facilitated clear visualization of differences in the morphology.

Type IV Collagen in Human Colorectal Liver Metastases-Cellular Origin and a Circulating Biomarker.

Simple SummaryPatients with colorectal liver metastases (CLM) can be cured through surgery if metastases are detected early in disease progression. Today, CLM diagnosis relies heavily on diagnostic imaging, and cheap, non-invasive, and efficiently measurable biomarkers are needed. Circulating type IV collagen (COL IV) is a potential biomarker for detecting CLM. Patients with CLM show elevated circulating levels of COL IV and increased tissue expression of COL IV in CLM tissue, which could result from enhanced production and degradation of COL IV. This study aimed to establish the cellular source behind enhanced COL IV levels, which is helpful in the evaluation of the biomarker potential of COL IV. We show that fibroblasts express COL IV both in vitro and in the stromal tissue of CLM. We also found that CLM tissue expresses COL IV-degrading proteases. Lastly, CLM patients have higher circulating COL IV levels than healthy controls.Circulating type IV collagen (cCOL IV) is a potential biomarker for patients with colorectal liver metastases (CLM) who present with elevated levels of COL IV in both CLM tissue and circulation. This study aimed to establish the cellular origin of elevated levels of COL IV and analyze circulating COL IV in CLM patients. The cellular source was established through in situ hybridization, immunohistochemical staining, and morphological evaluation. Cellular expression in vitro was assessed by immunofluorescence. Tissue expression of COL IV-degrading matrix metalloproteinases (MMPs)-2, -7, -9, and -13 was studied with immunohistochemical staining. Plasma levels of COL IV in CLM patients and healthy controls were analyzed with ELISA. This study shows that cancer-associated fibroblasts (CAFs) express COL IV in the stroma of CLM and that COL IV is expressed in vitro by fibroblasts but not by tumor cells. MMP-2, -7, -9, and -13 are expressed in CLM tissue, mainly by hepatocytes and immune cells, and circulating COL IV is significantly elevated in CLM patients compared with healthy controls. Our study shows that stromal cells, not tumor cells, produce COL IV in CLM, and that circulating COL IV is elevated in patients with CLM.

Abstract 1346: T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases

Abstract Background: Colorectal liver metastasis (CLM) is a leading cause of cancer-related mortality in the industrialized world, and only a small fraction of patients are eligible for curative resections. While immune checkpoint inhibition (ICI) has resulted in major breakthroughs in some cancers, the response in microsatellite stable (MSS) colorectal cancer has been disappointing. High density of infiltrating T cells has been associated with improved overall survival and response to ICI. We previously found that T cell infiltration varied considerably in a cohort of MSS CLM cases, which could reflect varying sensitivity to ICI treatment. The aim of this study was to quantify and characterize T cell infiltration in CLM using T cell receptor (TCR) repertoire sequencing (TCRR-seq). Methods: Resection specimens were collected from 85 patients with CLM included in the Oslo CoMet trial (NCT01516710). Of these, 37 and 13 patients had received neoadjuvant chemotherapy (NACT) within a short or long interval, respectively, prior to resection, while 35 had not (no-NACT group). TCRR-seq libraries were prepared from DNA extracted from CLM tissue using the hsTCRB kit from Adaptive Biotechnologies, which allows both quantitative and qualitative analysis of TCR repertoires. The T cell fraction relative to the total number of cells was estimated for each CLM sample. Repertoire clonality was analyzed using Hill diversity profiles, a mathematical framework for determining species diversity derived from the ecological literature. Network analyses were conducted to analyze convergence of clonal T cell receptor amino acid sequences in each repertoire. Results: The mean CLM T cell fraction was 8.1% (0.4-31.3%), and tumors with high T cell fraction had more monoclonal repertoires (p=1.86E-6). Interestingly, CLM from patients in the short interval group had increased T cell infiltration compared to the no-NACT and long interval groups (mean 10.5% and 6.3%, respectively; p=0.04), and these repertoires were also more monoclonal (p=0.003). Network analysis revealed that clonally expanded T cells tended to exhibit little overall receptor similarity to other T cells in the repertoire. Clonally expanded T cells were overall also private (unique) to each patient. Conclusion: A short interval between exposure to cytotoxic chemotherapy and surgical resection was associated with increased T cell infiltration in CLM samples, and the increase was associated with clonal expansion. The findings suggest that chemotherapy could be a driver of clonal expansion in this setting, possibly by induction of immunogenic cell death. Furthermore, the CLM neo-antigen landscape appears to be diverse and private to the individual patient. Citation Format: Eirik Høye, Vegar Johansen Dagenborg, Annette Torgunrud, Christin Lund-Andersen, Åsmund Avdem Fretland, Susanne Lorenz, Bjørn Edwin, Eivind Hovig, Anne Hansen Ree, Kjersti Flatmark. T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1346.

Clinicopathological and prognostic significance of HER2 status in surgically resected colorectal liver metastases.

The clinicopathological and prognostic significance of human epidermal growth factor receptor 2 (HER2) status in surgically resected colorectal liver metastases (CRLM) remains uncertain. HER2 expression was evaluated by immunohistochemical (IHC) in two CRLM tissue microarrays (TMAs). For samples with an IHC score of 2+ or 3+, fluorescence in situ hybridization (FISH) was performed to assess HER2 amplification. The association of HER2 amplification with clinicopathological parameters and prognosis was assessed using Fisher's exact test and Kaplan-Meier method, respectively. HER2 expression was consistent between primary tumor and liver metastases in 66.9% (85/127) cases (r = 0.643, p = 0.001). After FISH validation, HER2 amplification was identified in 6.25% (13/208) patients. HER2 amplification was significantly associated with age (p = 0.017), bilobar involvement (p = 0.005) and left-sided RAS/RAF wild-type status (p = 0.002). In the overall cohort, HER2 amplification was correlated with significantly worse relapse-free survival (RFS). Further stratification revealed that among left-sided RAS/RAF wild-type cases, HER2 amplification was significantly associated with worse overall survival (OS) (30.2 vs. 50.9 months, p = 0.040) and RFS (5.77 vs. 19.97 months, p = 0.017). HER2 amplification is more enriched in CRLMs with younger age, left-sided RAS/RAF wild-type, and bilobar involvement. Moreover, HER2 amplification predicts a poorer prognosis especially in left-sided RAS/RAF wild-type CRLMs.

Down-Regulation of Collagen Hydroxylation in Colorectal Liver Metastasis.

Collagen is significantly upregulated in colorectal liver metastasis (CRLM) compared to liver tissue. Expression levels of specific collagen types in CRLM resemble those in colorectal cancer (CRC) and colon tissue. We investigated whether the collagen hydroxylation pattern from the primary tumor also migrates with the metastatic tumor. The degree of collagen alpha-1(I) hydroxylation in colon, CRC, liver, and CRLM tissue of the same individuals (n = 14) was studied with mass spectrometry. The degree of hydroxylation was investigated in 36 collagen alpha-1(I) peptides, covering 54% of the triple helical region. The degree of hydroxylation in liver tissue was similar to that in colon tissue. The overall degree of hydroxylation was significantly lower (9 ± 14%) in CRC tissue and also significantly lower (12 ± 22%) in CRLM tissue compared to colon. Furthermore, eleven peptides with a specific number of hydroxylations are significantly different between CRLM and liver tissue; these peptides could be candidates for the detection of CRLM. For one of these eleven peptides, a matching naturally occurring peptide in urine has been identified as being significantly different between patients suffering from CRLM and healthy controls. The hydroxylation pattern in CRLM resembles partly the pattern in liver, primary colorectal cancer and colon.

Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis.

It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance.

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases.

BackgroundCancer cell migration, tumor angiogenesis, and activated hepatic stellate cells (a-HSCs) promote the development of colorectal liver metastases (CLM). Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2) has been associated with CLM, although the underlying molecular mechanisms remain unclear.MethodsIn the current study, we evaluated the relationship between WAVE2 and CLM in 103 CLM patients who underwent liver resection. Immunohistochemistry (IHC) staining was performed to determine the association between WAVE2 protein expression and hepatic micro-metastasis in human CLM tissues. WAVE2 knockout was performed in hepatic stellate cells (HSC) to explore the function and signaling pathways of WAVE2 in colorectal cancer progression.ResultsSignificantly higher levels of WAVE2 were detected in portal-associated relative to sinusoid-associated micro-metastasis. A strong correlation was identified between WAVE2 levels and microvessel density (MVD) in hepatic metastasis. Similarly, expression of WAVE2 was closely associated with activation of HSCs. Mechanistically, WAVE2 regulated the progression of human CLM acts by regulating the growth factor β (TGF-β) and Hippo pathways via effector yes-associated protein (YAP1).ConclusionOverall, our results demonstrated that WAVE2 participates in CLM tumor microenvironment, and can be a potential latent therapeutic target for CLM.

Identification of 4-Hydroxyproline at the Xaa Position in Collagen by Mass Spectrometry.

Collagen has a triple helix form, structured by a [-Gly-Xaa-Yaa-] repetition, where Xaa and Yaa are amino acids. This repeating unit can be post-translationally modified by enzymes, where proline is often hydroxylated into hydroxyproline (Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro, substrate for 4-prolyl hydroxylase) and 3-hydroxyproline (3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is lacking at the Yaa position, then Pro at the Xaa position should remain unmodified. Nevertheless, in literature 41 positions have been described where Hyp occurs at the Xaa position (?xHyp) lacking an adjacent 4Hyp. We report four additional positions in liver and colorectal liver metastasis tissue (CRLM). We studied the sequence commonalities between the 45 known positions of ?xHyp. Alanine and glutamine were frequently present adjacent to ?xHyp. We showed that proline, position 584 in COL1A2, had a lower rate of modification in CRLM than in healthy liver. The isomeric identity of ?xHyp, that is, 3- and/or 4Hyp, remains unknown. We present a proof of principle identification of ?xHyp. This identification is based on liquid chromatography retention time differences and mass spectrometry using ETD-HCD fragmentation, complemented by ab initio calculations. Both techniques identify ?xHyp at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).

High hepatic expression of PDK4 improves survival upon multimodal treatment of colorectal liver metastases

BackgroundPatients with borderline resectable colorectal liver metastases (CRLM) frequently receive neoadjuvant chemotherapy (NC) to reduce tumour burden, thus making surgical resection feasible. Even though NC can induce severe liver injury, most studies investigating tissue-based prognostic markers focus on tumour tissue. Here, we assessed the prognostic significance of pyruvate-dehydrogenase-kinase isoenzyme 4 (PDK4) within liver tissue of patients undergoing surgical resection due to CRLM.MethodsTranscript levels of hypoxia-adaptive genes (such as PDK isoenzymes) were assessed in the tissue of healthy liver, corresponding CRLM, healthy colon mucosa and corresponding tumour. Uni- and multivariate analyses were performed. Responses to chemotherapy upon up- or down-regulation of PDK4 were studied in vitro.ResultsPDK4 expression within healthy liver tissue was associated with increased overall survival and liver function following surgical resection of CRLM. This association was enhanced in patients with NC. PDK4 expression in CRLM tissue did not correlate with overall survival. Up-regulation of PDK4 increased the resistance of hepatocytes and colon cancer cells against chemotherapy-induced toxicity, whereas knockdown of PDK4 enhanced chemotherapy-associated cell damage.ConclusionOur findings suggest that up-regulated PDK4 expression reduces hepatic chemotherapy-induced oxidative stress and is associated with improved postoperative liver function in patients undergoing multimodal treatment and resection of CRLM.

Up-regulation of collagen proteins in colorectal liver metastasis compared with normal liver tissue

Changes to extracellular matrix (ECM) structures are linked to tumor cell proliferation and metastasis. We previously reported that naturally occurring peptides of collagen type I are elevated in urine of patients with colorectal liver metastasis (CRLM). In the present study, we took an MS-based proteomic approach to identify specific collagen types that are up-regulated in CRLM tissues compared with healthy, adjacent liver tissues from the same patients. We found that 19 of 22 collagen-α chains are significantly up-regulated (p < 0.05) in CRLM tissues compared with the healthy tissues. At least four collagen-α chains were absent or had low expression in healthy colon and adjacent tissues, but were highly abundant in both colorectal cancer (CRC) and CRLM tissues. This expression pattern was also observed for six noncollagen colon-specific proteins, two of which (CDH17 and PPP1R1B/DARP-32) had not previously been linked to CRLM. Furthermore, we observed CRLM-associated up-regulation of 16 proteins (of 20 associated proteins identified) known to be required for collagen synthesis, indicating increased collagen production in CRLM. Immunohistochemistry validated that collagen type XII is significantly up-regulated in CRLM. The results of this study indicate that most collagen isoforms are up-regulated in CRLM compared with healthy tissues, most likely as a result of an increased collagen production in the metastatic cells. Our findings provide further insight into morphological changes in the ECM in CRLM and help explain the finding of tumor metastasis-associated proteins and peptides in urine, suggesting their utility as metastasis biomarkers.

Serum levels of soluble urokinase plasminogen activator receptor (suPAR) predict outcome after resection of colorectal liver metastases.

BackgroundIn colorectal cancer (CRC), the liver is the most common site of metastasis. Surgical resection represents the standard therapy for patients with colorectal liver metastases (CRLM). However, 5-year survival rates after resection do not exceed 50%, and despite existing preoperative stratification algorithms it is still debated which patients benefit most from surgical treatment. The soluble urokinase plasminogen activator receptor (suPAR) has recently evolved as a promising biomarker for distinct clinical conditions. Here, we examined a potential role of suPAR as a biomarker in patients undergoing resection of CRLM.ResultsCorrelating with upregulated uPAR tissue expression in resected metastases, serum concentrations of suPAR were significantly elevated in CRLM patients compared to healthy controls. Importantly, patients with preoperative suPAR serum levels above the identified ideal cut-off value of 4.83 ng/ml showed a significantly reduced overall survival after resection of CRLM, both in right- and left-sided primary CRC. Moreover, multivariate Cox regression analysis revealed preoperative suPAR serum levels as a prognostic factor for mortality. Additionally, elevated preoperative suPAR but not creatinine levels were a predictor of acute kidney injury (AKI) after CRLM resection, correlating with a longer postoperative hospitalization.ConclusionSuPAR represents a promising novel biomarker in CRLM patients that might help to guide preoperative treatment decisions regarding patients’ outcome and to identify patients particularly susceptible to AKI.MethodsExpression levels of uPAR were analyzed in CRLM tissue using RT-PCR and immunohistochemistry. SuPAR serum levels were measured by ELISA in 104 CRC patients undergoing hepatic resection for CRLM and 50 healthy controls.

Soluble urokinase plasminogen activator receptor (suPAR) as a novel serum biomarker for patients undergoing resection of colorectal liver metastases.

309 Background: In colorectal cancer (CRC), the liver is the most common site of metastasis. Surgical resection represents the standard potentially curative therapy for patients with colorectal liver metastases (CRLM). However, 5-year survival rates after resection do not exceed 50%, and despite existing preoperative stratification algorithms it is still not fully understood which patients benefit most from surgery. The soluble urokinase plasminogen activator receptor (suPAR) has recently evolved as a promising biomarker for distinct clinical conditions. Here, we examined a potential role of circulating suPAR as a biomarker in patients undergoing resection of CRLM. Methods: Expression levels of uPAR, the membrane-bound source of circulating suPAR, were analysed in tissue samples of CRLM using RT-PCR and IHC. SuPAR serum levels were measured by ELISA in 104 patients undergoing surgical resection of CRLM as well as 50 healthy controls. Results were correlated with clinical data. Results: In line with an upregulation of uPAR in the CRLM tissue, serum levels of suPAR were significantly elevated in patients with CRLM compared to healthy controls. Patients with preoperative suPAR serum levels above our defined ideal cut-off value of 4.83 ng/ml showed a strikingly reduced overall survival after resection of CRLM, which could be confirmed for right- and left-sided primary CRC. Importantly, none of these patients reached long-term survival compared to patients with preoperative suPAR serum concentrations below the cut-off value. Moreover, multivariate Cox-regression analysis revealed preoperative suPAR serum levels as an independent prognostic factor in this setting. Additionally, elevated preoperative suPAR but not creatinine levels were a predictor of acute kidney injury after CRLM resection, correlating with a longer postoperative hospitalization. Conclusions: Serum levels of suPAR represent a promising novel biomarker in patients with resectable CRLM that might help to guide preoperative treatment decisions with regards to patients’ outcome and the identification of patients particularly susceptible to postoperative acute kidney injury.

Abstract 3960: The role and therapeutic potential of miRNAs in colorectal liver metastasis

Abstract Colorectal cancer (CRC) is a leading cause of cancer related deaths worldwide. Liver metastasis occurs in 60% of CRC patients and usually responds poorly to treatment - contributing to mortality rate. MicroRNAs (miRNAs) are highly conserved, endogenously encoded small, non-coding RNA molecules regulating global gene expression. Their role in cancer pathogenesis, including CRC, has been well documented. Detailed miRNA expression analysis on a large cohort of CRC tumors is needed to identify clinically relevant miRNAs and explore their potential to target liver metastases. We analyzed miRNA expression data of 406 CRC tumors from publically available CRC genome sequencing project and identified 58 miRNAs, which were significantly downregulated. Using miRNA target prediction analysis, 10 miRNAs were selected for further studies as they were either known to target genes in cellular pathways associated with CRC liver metastases, or located within a chromosomal loci commonly lost in colorectal liver metastases. qPCR analysis of these 10 selected miRNAs in primary and colorectal liver metastasis tissues and in CRC cell lines demonstrated four miRNAs; miR-132, miR-378f, miR-605, and miR-1976 that are significantly down-regulated with &amp;lt;2 fold change (p&amp;gt;0.05). To investigate the anti-tumorigenic and metastatic properties of miR-132, -378f, -605, and -1976, we transfected 3 different CRC cell lines (SW620, HCT-116, and CT-26) with miR-mimics and subjected them to cell proliferation, apoptosis, soft agar, invasion, and migration assays. Ectopic expression of miR-378f, -605, and -1976 suppressed CRC cell proliferation, anchorage independent growth, migration, and invasion and increased apoptosis. Interestingly, CRC patients with high miR-378f and miR-1976 had better survival compared to low expressing patients (p&amp;lt;0.044). In conclusion, our in vitro findings suggest anti-tumorigenic/metastatic properties of miR-378f, -605, and -1976 in CRC. A further understanding of their function and in vivo therapeutic evaluation may help in developing novel therapeutic strategies for this malignancy. Citation Format: Smiti Snigdha Sahu, Sarah Nabinger, Jiang Guanglong, Allison Bates, Sangbin Lee, Tanaka Hiromi, Yunlong Liu, Janaiah Kota. The role and therapeutic potential of miRNAs in colorectal liver metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2015-3960

Aberrant expression of long noncoding RNAs in colorectal cancer with liver metastasis

Long noncoding RNA (lncRNA) plays a crucial role in the regulation of various cellular processes and human diseases. However, little is known about the role of lncRNAs in colorectal liver metastasis (CLM). In the present study, we aimed to determine whether lncRNAs are differentially expressed in CLM tissue and to further assess their clinical value. lncRNA arrays were employed to screen for differentially expressed lncRNAs in colorectal cancer (CRC) tissues with synchronous, metachronous, or nonliver metastasis. Based on bioinformatics data, a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was performed to identify target lncRNAs in an expanded set of CRC samples with various subtypes of liver metastasis. The relationships between the target lncRNAs and the clinical characteristics and patient prognosis were further analyzed. After determining the expression profile of lncRNAs (n = 1332) in CLM tissue, 40 differentially expressed lncRNAs that were potentially related to CLM were selected for further examination in an expanded set of clinical samples, and three novel target lncRNAs, termed lncRNA-CLMAT1-3, were verified. High lncRNA-CLMAT3 expression strongly correlated with liver metastasis (P = 0.03) and lymph node metastasis (P = 0.009). Moreover, patients displaying high lncRNA-CLMAT3 expression exhibited a shorter median overall survival duration than those displaying low lncRNA-CLMAT3 expression (30.7 vs. 35.2 months, P = 0.007). Multivariate analysis demonstrated that the lncRNA-CLMAT3 expression level is an independent prognostic factor (hazard ratio 2.05, P = 0.02) after adjusting for other known prognostic factors. lncRNA-CLMAT3 over-expression was significantly associated with CLM and was an independent predictor of poor survival for patients with CRC.

CCR6/CCL20 Chemokine Expression Profile in Distinct Colorectal Malignancies

Originally, chemokines and their G-protein-coupled receptors were described to regulate multiple physiological functions, particularly tissue architecture and compartment-specific migration of white blood cells. Now, it is established that the chemokine/chemokine receptor system is also used by cancer cells for migration and metastatic spread. Here, we examined the relative levels of CC-chemokine CCL20 and its corresponding receptor CCR6 in resection specimens from patients with different malignant and non-malignant colorectal diseases as well as in colorectal liver metastases (CRLM). CCL20/CCR6 mRNA and protein expression profiles were assessed by quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in resection specimens from patients with ulcerative colitis (UC, n=15), colorectal adenoma (CRA, n=15), colorectal adenocarcinoma (CRC, n=61) and colorectal liver metastases (CRLM, n=16). Corresponding non-diseased tissues served as control. In contrast to UC tissues, the CCL20/CCR6 system showed a distinct upregulation in CRA, CRC and CRLM related to corresponding non-affected tissues (P<0.05, respectively). Furthermore, CRA, CRC and CRLM tissue samples displayed significantly higher protein amounts of CCL20 in comparison with UC specimens (P<0.05, respectively). Our results strongly suggest an association between CCL20/CCR6 expression and the induction of CRA, CRC and the development of CRLM. Therefore, CCL20 and CCR6 may provide potential targets for novel treatment strategies of CRC.

Poor prognosis of KRAS or BRAF mutant colorectal liver metastasis without microsatellite instability

The discovery of practical biomarkers is important to realize personalized medicine for patients with malignant neoplasias, including colorectal cancer (CRC). The aim of this study was to determine reliable prognostic biomarkers by the analysis of patients with resectable colorectal liver metastases (CRLM). Genomic DNA was obtained from the CRLM tissues of a cohort of 126 patients with CRLM with curative hepatic resection. The KRAS/BRAF mutation spectrum and microsatellite instability (MSI) status were successfully analyzed in 100 of the 126 CRLM tissues and these findings were examined in relation to the patients' clinical outcomes. The cohort of 100 CRLM patients consisted of 46 with synchronous and 54 with metachronous liver metastasis. Overall survival and disease-free survival at 5 years were 57.4 and 24.9 %, respectively. MSI analysis revealed that none of the 100 CRLM specimens showed any evidence of MSI. By KRAS/BRAF mutation analysis, the analyzed CRLM patients were divided into 3 groups; KRAS-mutant (KRAS-Mt; n = 27), BRAF-mutant (BRAF-Mt; n = 3), and wild-types of both genes (Wild-type; n = 70). In the survival analysis, both KRAS-Mt and BRAF-Mt patients showed significantly poorer prognoses compared with Wild-type patients. Furthermore, although the population with the BRAF mutation was small, this mutation had a significant negative impact on disease-free survival. In this study, all tumors in the cohort of CRLM patients were non-MSI tumors, suggesting MSI cancer in primary CRC would rarely reveal metastatic potential. KRAS and BRAF mutations are suggested to be poor prognostic factors in CRLM. Genetic information has an essential role as a prognostic marker and could contribute to the decisions on treatment strategy for CRLM.

OC-056 Viral warfare: front line defence and arming the immune system: the use of an oncolytic virus as a vaccine against colorectal liver metastases

IntroductionLiver resection for colorectal liver metastases (CRLM) from colorectal cancer (CRC) has a 50% 5-year survival rate. However, recurrence rates are reported as 50% within 2 years. Thus, there is...