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Introduction: Proteases play an important role in tumor progression. The predictive efficacy of proteases PRSS3 and PRSS22 mRNA levels for predicting relapse in surgically treated colon cancer (CC) patients was assessed.

Methods: mRNA expression was quantified in 371 half lymph nodes (LNs) from 121 CC patients, 77 control LNs (13 patients), 66 primary colon tumors, and 30 normal colon tissues of these patients. Patients were also stratified according to their CEA mRNA level. The occurrence of relapse following curative surgery was evaluated using the Cox regression and Kaplan-Meier survival model analyses. Protein expression was examined through immunohistochemistry.

Results: PRSS22 was superior to PRSS3 in identifying patients at risk of recurrence. Thus, high PRSS22 levels in LNs identified 76.5% of those who recurred, while PRSS3 only identified 17.6% of these patients and these were in TNM stages III and IV. The Kaplan-Meier analysis indicated that CC patients exhibiting elevated PRSS22 levels in lymph nodes experienced a reduction in survival time, averaging 37 months over the follow-up period (p = 0.009) and a 3-fold increased hazard risk (1.3–6.0; p = 0.01). In the group with low PRSS22 levels, only one patient experienced relapse at the 12-year follow-up when CEA mRNA analysis was included. A fraction of CEA-positive tumor cells expressed PRSS22 protein.

Conclusion: The importance of the secreted serine protease, S1 family member PRSS22 in tumor progression is highlighted. It shows promise as a biomarker for CC prognosis and as a target to prevent tumor spread by inhibiting its enzymatic activity.

Introduction: The prognostic value of determining mRNA levels of two markers for regulatory T cells, IL-10 and FoxP3, in lymph nodes (LNs) and primary tumors of colon cancer (CC) patients receiving curative surgery was investigated.

Methods: mRNA levels were determined by real-time qRT-PCR in 370 LNs from 120 CC patients representing all four TNM stages, 66 primary tumors, 30 normal colon tissue samples and appropriate cell lines. Protein expression was analyzed by immunohistochemistry. Patients were followed for 12 years.

Results: High levels of IL-10 mRNA in LNs were associated with poor prognosis with shorter mean survival time of 10 and 32 months (p = 0.001 and p = 0.004) at 5- and 12-year follow-up with hazard ratios of 12.4 and 6.3, respectively. No association between IL-10 level and prognosis was seen in the primary tumor. High levels of FoxP3 mRNA were associated with good prognosis, both in LNs and primary tumor. The difference in survival time was, however, small. Analysis of IL-10 mRNA in combination with LGR6 or CXCL17 mRNA in LNs generated patients with different risk of recurrence – low-, high- and very high risk. Immunohistochemistry identified IL-10 and FoxP3 positive cells located at the outer rim of tumor aggregates.

Conclusion: Level determinations of IL-10 mRNA in LNs are useful for prediction of outcome for CC patients after curative surgery. Low levels indicate that the patients do not require further treatment, while IL-10 in combination with LGR6 or CXCL17 can be used to identify patients at very high risk of recurrence.

Introduction: The aim was to investigate whether the stem cell marker LGR6 has prognostic value in colon cancer, alone or in combination with the prognostic biomarkers CEA and CXCL16.

Methods: LGR6 mRNA levels were determined in 370 half lymph nodes of 121 colon cancer patients. Ability to predict relapse after curative surgery was estimated by Kaplan-Meier survival model and Cox regression analyses.

Results: Patients with high LGR6 levels [LGR6(+)] had a decreased mean survival time of 11 months at 5-year follow-up and 47 months at 12-year follow-up, respectively, with hazard ratios of 3.2 and 2.8. LGR6 mRNA analysis added prognostic value to CEA and CXCL16 mRNA analysis. In the poor prognosis groups CEA(+) and CXCL16(+), further division was achieved by LGR6 analysis. LGR6(+) patients had a very poor prognosis. LGR6 also identified a small number of CEA(-), TNM stage I patients who relapsed suggesting stem cell origin of these tumors. LGR6 and LGR5 levels correlated strongly in lymph nodes of stage I and IV patients but not in stage II patients, suggesting that these stem cell markers are differentially regulated.

Conclusion: This study highlights LGR6 as a useful prognostic biomarker independently and in combination with CEA, CXCL16 or LGR5 identifying different risk groups.

One fourth of colorectal cancer patients having curative surgery will relapse of which the majority will die. Lymph node (LN) metastasis is the single most important prognostic factor and a key factor when deciding on postoperative treatment. Presently, LN metastases are identified by histopathological examination, a subjective method analyzing only a small LN volume and giving no information on tumor aggressiveness. To better identify patients at risk of relapse we constructed a qRT-PCR test, ColoNode, that determines levels of CEACAM5, KLK6, SLC35D3, MUC2 and POSTN mRNAs. Combined these biomarkers estimate the tumor cell load and aggressiveness allocating patients to risk categories with low (0, −1), medium (1), high (2) and very high (3) risk of recurrence. Here we present result of a prospective, national multicenter study including 196 colon cancer patients from 8 hospitals. On average, 21 LNs/patient, totally 4698 LNs, were examined by both histopathology and ColoNode. At 3-year follow-up, 36 patients had died from colon cancer or lived with recurrence. ColoNode identified all patients that were identified by histopathology and in addition 9 patients who were undetected by histopathology. Thus, 25% of the patients who recurred were identified by ColoNode only. Multivariate Cox regression analysis proved ColoNode (1, 2, 3 vs 0, −1) as a highly significant risk factor with HR 4.24 [95% confidence interval, 1.42-12.69, P =.01], while pTN-stage (III vs I/II) lost its univariate significance. In conclusion, ColoNode surpassed histopathology by identifying a significantly larger number of patients with future relapse and will be a valuable tool for decisions on postoperative treatment.

Introduction: Approximately 25% of colon cancer (CC) patients having curative surgery will relapse. Therefore, it is crucial to identify patients with increased recurrence risk to offer them adjuvant chemotherapy. Three markers with prominent expression in fibroblasts: forkhead box Q1 (FOXQ1), matrix metalloproteinase-11 (MMP11), and thrombospondin-2 (THBS2), and the fibroblast expressed chemokine CXCL12 were selected for studies because of the critical role of fibroblasts in the microenvironment of the tumor.

Methods: The expression levels of the biomarkers were assessed in primary CC tumors, lymph nodes of CC patients and controls, and CC cell lines at mRNA and protein levels by real-time qRT-PCR and immunohistochemistry, respectively.

Results: FOXQ1, MMP11, and THBS2 mRNAs were expressed at significantly higher levels in primary tumors compared to normal colon (P=0.002, P<0.0001, and P<0.0001, respectively). In contrast, CXCL12 mRNA levels were higher in normal colon tissue. FOXQ1, MMP11, and THBS2 levels were also expressed at significantly higher levels in metastasis-positive lymph nodes compared to both metastasis-negative- and control nodes (P<0.0001/P=0.002, P<0.0001/P<0.0001, and P<0.0001/P<0.0001, respectively). Immuno-morphometry revealed that 30–40% of the tumor cells expressed FOXQ1, MMP11, and THBS2. FOXQ1 and THBS2 were barely detected in normal colon epithelium (P<0.0001), while MMP11 was expressed in normal colon epithelium at high levels.

Discussion: We conclude that CC tumor cells show ectopic expression of FOXQ1 and THBS2 possibly making these tumor cells independent of fibroblast cell support. The high expression levels of these two biomarkers in metastatic lymph nodes suggest that they are potential indicators of patients at risk for recurrence.

The significance of cancer stem cells (CSCs) in initiation and progression of colon cancer (CC) has been established. In this study, we investigated the utility of measuring mRNA expression levels of CSC markers EpCAM, LGR5 and LGR4 for predicting survival outcome in surgically treated CC patients. Expression levels were determined in 5 CC cell lines, 66 primary CC tumors and 382 regional lymph nodes of 121 CC patients. Prognostic relevance was determined using Kaplan‐Meier survival and Cox regression analyses. CC patients with lymph nodes expressing high levels of EpCAM, LGR5 or LGR4 (higher than a clinical cutoff of 0.07, 0.06 and 2.558 mRNA cop-ies/18S rRNA unit, respectively) had a decreased mean survival time of 32 months for EpCAM and 42 months for both LGR5 and LGR4 at a 12‐year follow‐up (p = 0.022, p = 0.005 and p = 0.011, respec-tively). Additional patients at risk for recurrence were detected when LGR5 was combined with the biomarkers CXCL17 or CEA plus CXCL16. In conclusion, the study underscores LGR5 as a particularly useful prognostic biomarker and illustrates the strength of combining biomarkers detecting different subpopulations of cancer cells and/or cells in the tumor microenvironment for predicting recurrence.

G protein-coupled receptor 55 (GPR55) probably plays a role in innate immunity and tumor immunosurveillance through its effect on immune cells, such as T cells and NK cells. In this study, the prognostic value of GPR55 in colon cancer (CC) was investigated. mRNA expression levels of GPR55 were determined in 382 regional lymph nodes of 121 CC patients with 12 years observation time after curative surgery. The same clinical material had previously been analyzed for expression levels of CEA, CXCL16, CXCL17, GPR35 V2/3 and LGR5 mRNAs. Clinical cutoffs of 0.1365 copies/18S rRNA unit for GPR55 and 0.1481 for the GPR55/CEA ratio were applied to differentiate between the high-and low-GPR55 expression groups. Kaplan–Meier survival analysis and Cox regression risk analysis were used to determine prognostic value. Improved discrimination between the two groups was achieved by combining GPR55 with CEA, CXCL16 or CXCL17 compared with GPR55 alone. The best result was obtained using the GPR55/CEA ratio, with an increased mean survival time of 14 and 33 months at 5 and 12 years observation time, respectively (p = 0.0003 and p = 0.003) for the high-GPR55/CEA group. The explanation for the observed improvement is most likely that GPR55 is a marker for T cells and B cells in lymph nodes, whereas CEA, CXCL16 and CXCL17, are markers for tumor cells of epithelial origin.

BACKGROUND: Lymph node metastasis is the single most important prognostic risk factor for recurrence in patients with colon cancer who have undergone curative surgery. The routine method for detecting disseminated tumor cells in lymph nodes is microscopic examination of one or a few hematoxylin and eosin-stained tissue sections by a trained pathologist. This method, however, is insensitive mainly because less than 1% of the lymph node volume is examined, leading to misclassification.

OBJECTIVE: This study aimed to investigate whether analysis of a selected group of biomarker mRNAs improves detection and characterization of lymph node metastases/micrometastases compared with the routine method.

DESIGN: This study is a side-by-side comparison of biomarker mRNA analysis and histopathology of 185 lymph nodes from patients with colon cancer representing stages I to IV, and an investigation of the importance of lymph node tissue volume for tumor cell detection.

SETTINGS: This is a collaborative study between a high-volume central hospital and a preclinical university institution.

PATIENTS: Fifty-seven patients who had undergone tumor resection for colon cancer were included.

MAIN OUTCOME MEASURES: The primary outcomes measured were mRNA copies per 18S rRNA copy of CEACAM5, KLK6, SLC35D3, POSTN, and MUC2 by multiplex assay and metastases/micrometastases detected by histopathology.

RESULTS: The number of tumor cell-positive lymph nodes was 1.33-fold higher based on CEACAM5 mRNA levels compared with histopathological examination. Increasing the tissue volume analyzed for CEACAM5 levels from an 80-µm section to half a lymph node increased the number of positive nodes from 34 of 107 to 80 of 107 (p < 0.0001). Similarly, the number of positive nodes for the aggressiveness marker KLK6 increased from 9 of 107 to 24 of 107.

LIMITATIONS: Only a limited number of individual lymph nodes per patient was available for analysis.

CONCLUSIONS: mRNA analysis of CEACAM5, KLK6, and SLC35D3 improves the detection of tumor cells in lymph nodes from patients surgically treated for colon cancer, and, together with POSTN and MUC2, it further allows characterization of the tumor cells with respect to aggressiveness and the tumor cell environment. See Video Abstract at https://links.lww.com/DCR/B650.

The genus Lachnoanaerobaculum comprises obligately anaerobic, Gram-stain-positive chemoorganotrophic, saccharolytic, and nonproteolytic bacilli. Cells are spore forming, rod shaped, and filamentous, 5 to greater than 20 μm in length, some cells with curving and swelling. All species of the genus grow with glucose as the sole carbon source. All species produce H2S, NH3, butyric acid, acetic acid, and lactic acid as metabolic end products. The predominant cellular fatty acids are C14:0, C16:0, and C18:1 ω7c DMA. The G + C contents of genomic DNA of the species are 35.0–37.8 mol%. Phylogenetic relationships of the species of Lachnoanaerobaculum, based on comparative 16S rRNA gene sequence analyses, indicate that they cluster within the phylum Firmicutes, within the family Lachnospiraceae, and exhibit a clear delineation to the other genera of the family, with a relatively close relationship to Johnsonella species. The first described strain of Lachnoanaerobaculum umeaense, the type species of the genus, was isolated from the jejunal mucosa of a child with coeliac disease. Strains of the species of Lachnoanaerobaculum are typically found in intestinal microbiota and oral microbiota of the human microbiome, isolated from the human gut, saliva, blood, amniotic fluid, and, predominantly, from the oral cavity.

OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated.

METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM.

RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P <  0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(-) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P <  0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells.

CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.