The relationships between systemic cytokine profiles and inflammatory markers in colorectal cancer and the prognostic significance of these parameters.

BACKGROUND: Immunomodulatory cytokines and systemic inflammatory markers are important during cancer development and progression. This study investigated the association and prognostic impact of systemic cytokine profiles and inflammatory markers in colorectal cancer (CRC). METHODS: Interleukin (IL)-1ß, IL-6, IL-8, IL-9, IL-10, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) serum levels were measured using multiplex bead assays in CRC patients. Data on systemic inflammatory markers, such as the modified Glasgow prognostic score (mGPS), the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), prognostic nutritional index (PNI) and fibrinogen, were collected. Survival analysis was performed to identify factors associated with progression-free survival (PFS) and overall survival (OS). RESULTS: There were moderate-to-strong correlations within serum cytokines, as well as within systemic inflammatory markers, whereas the associations between serum cytokines and systemic inflammatory markers were generally weak. IL-8 and the LMR were independent significant prognostic factors for PFS and OS. The low IL-8 and high LMR group had the best survival (both PFS and OS) of all groups. CONCLUSIONS: Systemic cytokine profiles and inflammatory markers have relatively weak intergroup correlations. A composite classification of systemic cytokine profiles and inflammatory markers has an enhanced prognostic value in CRC.

Serpin B5 is a CEA-interacting biomarker for colorectal cancer.

Serpin B5 is a candidate tumour suppressor, but its oncogenic activity has also been reported. Its function may be affected by protein interactions. The aim of this study was to assess the relationship between serpin B5 and carcinoembryonic antigen (CEA) expression in colorectal cancer (CRC). We also analysed the clinicopathological significance of serpin B5 expression in patients with CRC. Downregulation of serpin B5 was identified in a CEA-suppressed LoVo cell line using two-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionisation-mass spectrometry (MALDI-MS). The specific interaction and co-localisation of serpin B5 with CEA were confirmed by co-immunoprecipitation and confocal microscopy. Western blot analysis and ELISAs revealed significant positive correlations between levels of serpin B5 and CEA in human colon cancer cell lines and in the blood of patients with CRC. Tissue expression of serpin B5 in 377 patients with CRC was significantly associated with serum CEA, histological grade, stage, lymph node metastasis, lymphatic and perineural invasion, and infiltrative border. Strong expression of serpin B5 was also associated with a reduced DFS (p = 0.001) and OS (p = 0.017). Together, these findings describe a relationship between serpin B5 and CEA expression in CRC. Strong expression of serpin B5 was associated with a worse prognosis in patients with CRC and its expression may correlate with CEA levels in CRC.

Expression of phosphoenolpyruvate carboxykinase linked to chemoradiation susceptibility of human colon cancer cells.

BACKGROUND: Resistance to 5-fluorouracil (5-FU) in patients with colorectal cancer prevents effective treatment and leads to unnecessary and burdensome chemotherapy. Therefore, prediction of 5-FU resistance is imperative. METHODS: To identify the proteins linked to 5-FU resistance, two-dimensional gel electrophoresis-based proteomics was performed using the human colon cancer cell line SNU-C4R with induced 5-FU resistance. Proteins showing altered expression in SNU-C4R were identified by matrix-associated laser desorption/ionization-time-of-flight analysis, and their roles in susceptibility to 5-FU or radiation were evaluated in various cell lines by transfection of specific siRNA or creation of overexpression constructs. Changes in cellular signaling and expression of mitochondrial apoptotic factors were investigated by Western Blot analysis. A mitochondrial membrane potential probe (JC-1 dye) and a flow cytometry system were employed to determine the mitochondrial membrane potential. Finally, protein levels were determined by Western Blot analysis in tissues from 122 patients with rectal cancer to clarify whether each identified protein is a useful predictor of a chemoradiation response. RESULTS: We identified mitochondrial phosphoenolpyruvate carboxykinase (mPEPCK) as a candidate predictor of 5-FU resistance. PEPCK was downregulated in SNU-C4R compared with its parent cell line SNU-C4. Overexpression of mPEPCK did not significantly alter the susceptibility to either 5-FU or radiation. Suppression of mPEPCK led to a decrease in both the cellular level of phosphoenolpyruvate and the susceptibility to 5-FU and radiation. Furthermore, the cellular levels of phosphoenolpyruvate (an end product of PEPCK and a substrate of pyruvate kinase), phosphorylated AKT, and phosphorylated 4EBP1 were decreased significantly secondary to the mPEPCK suppression in SNU-C4. However, mPEPCK siRNA transfection induced changes in neither the mitochondrial membrane potential nor the expression levels of mitochondrial apoptotic factors such as Bax, Bcl-2, and Bad. Downregulation of total PEPCK was observed in tissues from patients with rectal cancer who displayed poor responses to preoperative 5-FU-based radiation therapy. CONCLUSION: Our overall results demonstrate that mPEPCK is a useful predictor of a response to chemoradiotherapy in patients with rectal cancer.

Association of gut microbiome with immune microenvironment in surgically treated colorectal cancer patients.

This study explored the relationship between faecal microbiota distribution and local or systemic immune response in patients with colorectal cancer (CRC). The study population included 114 surgically treated CRC patients. Faeces were analysed using 16S rRNA gene sequencing. The immune score in tumour microenvironment was evaluated using CD3 and CD8 immunohistochemistry. Genetic alterations, microsatellite instability status and five systemic inflammatory markers were also analysed. Thirty of 114 (26.3%) CRC patients were categorised as the 'immune type' with a high density of T-cells. The immune type CRC cases showed lower angiolymphatic invasion and longer overall survival. Of the 123 selected bacterial species, Bacteroides fragilis and Collinsella aerofaciens were prevalent in immune CRC cases, whereas Odoribacter splanchnicus and Phascolarctobacterium succinatutens were prevalent in non-immune CRC patients. Bacteroides fragilis was associated with shorter disease free survival in univariable and multivariable survival analyses. Regarding systemic immunity, a high prevalence of C. aerofaciens was associated with a high modified Glasgow prognostic score. This study revealed a potential relationship among the gut microbiome, immune microenvironment, and disease progression in patients with CRC. Our findings suggest that abundant B. fragilis in patients with CRC is associated with a 'cold immune' tumour microenvironment.

Up-regulated expression of l-caldesmon associated with malignancy of colorectal cancer.

BACKGROUND: Caldesmon (CaD), a major actin-associated protein, is found in smooth muscle and non-muscle cells. Smooth muscle caldesmon, h-CaD, is a multifunctional protein, and non-muscle cell caldesmon, l-CaD, plays a role in cytoskeletal architecture and dynamics. h-CaD is thought to be an useful marker for smooth muscle tumors, but the role(s) of l-CaD has not been examined in tumors. METHODS: Primary colon cancer and liver metastasis tissues were obtained from colon cancer patients. Prior to chemoradiotherapy (CRT), normal and cancerous tissues were obtained from rectal cancer patients. Whole-tissue protein extracts were analyzed by 2-DE-based proteomics. Expression and phosphorylation level of main cellular signaling proteins were determined by western blot analysis. Cell proliferation after CaD siRNA transfection was monitored by MTT assay. RESULTS: The expression level of l-CaD was significantly increased in primary colon cancer and liver metastasis tissues compared to the level in the corresponding normal tissues. In cancerous tissues obtained from the patients showing poor response to CRT (Dworak grade 4), the expression of l-CaD was increased compared to that of good response group (Dworak grade 1). In line with, l-CaD positive human colon cancer cell lines were more resistant to 5-fluorouracil (5-FU) and radiation treatment compared to l-CaD negative cell lines. Artificial suppression of l-CaD increased susceptibility of colon cancer cells to 5-FU, and caused an increase of p21 and c-PARP, and a decrease of NF-kB and p-mTOR expression. CONCLUSION: Up-regulated expression of l-CaD may have a role for increasing metastatic property and decreasing CRT susceptibility in colorectal cancer cells.

Association of CHFR Promoter Methylation with Treatment Outcomes of Irinotecan-Based Chemotherapy in Metastatic Colorectal Cancer.

Aberrant promoter methylation plays a vital role in colorectal carcinogenesis. However, its role in treatment responses is unclear, especially for metastatic disease. Here, we investigated the association between promoter methylation and treatment outcomes of irinotecan-based chemotherapy in 102 patients with metastatic colorectal cancer. Promoter methylation was examined by methylation-specific polymerase chain reaction for three loci (CHFR, WRN, and SULF2) associated with chemotherapy response and five CpG island methylator phenotype (CIMP)-specific markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1). Association between CHFR methylation and in vitro sensitivity to irinotecan was also evaluated. Promoter methylation of CHFR, WRN, and SULF2 was identified in 16 (15.7%), 24 (23.5%), and 33 (32.4%) patients, respectively. CIMP status was positive in 22 (21.6%) patients. CHFR methylation was associated with a significantly longer time to progression (TTP) (median: 8.77 vs. 4.43 months, P = .019), with trends favoring higher overall survival (OS) (median: 22.83 vs. 20.17 months, P = .300) and response rates (31.3% vs. 17.4%, P = .300). For patients with unmethylated CHFR, TTP (median: 5.60 vs. 3.53, P = .020) and OS (median: 20.57 vs. 9.23, P = .006) were significantly different according to CIMP status. Colorectal cancer cell lines with CHFR methylation demonstrated increased sensitivity to irinotecan. Both CHFR overexpression and combination with 5-aza-2'-deoxycytidine reversed irinotecan sensitivity in CHFR-methylated cell lines, whereas CHFR knockdown in unmethylated cells restored sensitivity to irinotecan. These data suggest that CHFR methylation may be associated with favorable treatment outcomes of irinotecan-based chemotherapy in patients with metastatic colorectal cancer.

Deleted in malignant brain tumor 1 is a novel prognostic marker in colorectal cancer.

The gene deleted in malignant brain tumor 1 (DMBT1) encoding a large scavenger receptor cysteine-rich protein was originally identified based on its deletion in a brain tumor cell line. The DMBT1 protein is involved in mucosal immune defense, epithelial differentiation and tumor suppression. In the present study, the clinicopathologic significance of DMBT1 protein expression in stool and tissue samples of colorectal cancer (CRC) patients was evaluated. Western blot analysis of fecal DMBT1 was performed for patients with CRC (n=177), colorectal adenoma (n=61), inflammatory bowel diseases (IBDs; n=54) and healthy individuals as the control group (n=151). Immunohistochemical analysis of tissue expression of DMBT1 was performed in 385 primary CRC tissues. Fecal DMBT1 expression was higher in CRC and IBD patients than in healthy controls or adenoma patients (P<0.0001), but not significantly different between IBD and CRC or between adenoma and healthy control groups. In CRC patients, fecal DMBT1 expression was not associated with the tumor stage or site. The sensitivity of fecal DMBT1 analysis for CRC was 50%, while the specificity and positive predictive value were 86.8 and 81.3%, respectively. Immunohistochemical expression patterns of DMBT1 in CRC tissues varied from loss to overexpression. Loss of expression was observed in 4.7% (18 out of 385 cases) and significantly associated with lymph node metastasis (P=0.016), distant metastasis (P=0.013), advanced stage (P=0.026), and higher histologic grade (P=0.033). In addition, DMBT1 loss was an independent poor prognostic factor for cancer-associated death (hazard ratio, 2.272; P=0.015) and disease recurrence (hazard ratio, 2.689; P=0.009). In conclusion, fecal DMBT1 has limited value as a diagnostic biomarker, while the tissue expression of DMBT1 may serve as an efficient prognostic marker for CRC. Furthermore, DMBT1 may have a role in the progression of CRC.

A New Cell Block Method for Multiple Immunohistochemical Analysis of Circulating Tumor Cells in Patients with Liver Cancer.

PURPOSE: We developed a new method of detecting circulating tumor cells (CTCs) in liver cancer patients by constructing cell blocks from peripheral blood cells, including CTCs, followed by multiple immunohistochemical analysis. MATERIALS AND METHODS: Cell blockswere constructed from the nucleated cell pellets of peripheral blood afterremoval of red blood cells. The blood cell blocks were obtained from 29 patients with liver cancer, and from healthy donor blood spikedwith seven cell lines. The cell blocks and corresponding tumor tissues were immunostained with antibodies to seven markers: cytokeratin (CK), epithelial cell adhesion molecule (EpCAM), epithelial membrane antigen (EMA), CK18, α-fetoprotein (AFP), Glypican 3, and HepPar1. RESULTS: The average recovery rate of spiked SW620 cells from blood cell blocks was 91%. CTCs were detected in 14 out of 29 patients (48.3%); 11/23 hepatocellular carcinomas (HCC), 1/2 cholangiocarcinomas (CC), 1/1 combined HCC-CC, and 1/3 metastatic cancers. CTCs from 14 patients were positive for EpCAM (57.1%), EMA (42.9%), AFP (21.4%), CK18 (14.3%), Gypican3 and CK (7.1%, each), and HepPar1 (0%). Patients with HCC expressed EpCAM, EMA, CK18, and AFP in tissue and/or CTCs, whereas CK, HepPar1, and Glypican3 were expressed only in tissue. Only EMA was significantly associated with the expressions in CTC and tissue. CTC detection was associated with higher T stage and portal vein invasion in HCC patients. CONCLUSION: This cell block method allows cytologic detection and multiple immunohistochemical analysis of CTCs. Our results show that tissue biomarkers of HCC may not be useful for the detection of CTC. EpCAM could be a candidate marker for CTCs in patients with HCC.

A predictive model combining fecal calgranulin B and fecal occult blood tests can improve the diagnosis of colorectal cancer.

AIM: Current fecal screening tools for colorectal cancer (CRC), such as fecal occult blood tests (FOBT), are limited by their low sensitivity. Calgranulin B (CALB) was previously reported as a candidate fecal marker for CRC. This study investigated whether a combination of the FOBT and fecal CALB has increased sensitivity and specificity for a diagnosis of CRC. MATERIALS AND METHODS: Patients with CRC (n = 175), and healthy individuals (controls; n = 151) were enrolled into the development (81 cases and 51 controls) and validation (94 cases and 100 controls) sets. Stool samples were collected before bowel preparation. CALB levels were determined by western blotting. FOBT and fecal CALB results were used to develop a predictive model based on logistic regression analysis. The benefit of adding CALB to a model with only FOBT was evaluated as an increased area under the receiver operating curve (AUC), partial AUC, and reclassification improvement (RI) in cases and controls, and net reclassification improvement (NRI). RESULTS: Mean CALB level was significantly higher in CRC patients than in controls (P<0.001). CALB was not associated with tumor stage or cancer site, but positivity on the FOBT was significantly higher in advanced than in earlier tumor stages. At a specificity of 90%, the cross-validated AUC and sensitivity were 89.81% and 82.72%, respectively, in the development set, and 92.74% and 79.79%, respectively, in the validation set. The incremental benefit of adding CALB to the model, as shown by the increase in AUC, had a p-value of 0.0499. RI in cases and controls and NRI all revealed that adding CALB significantly improved the prediction model. CONCLUSION: A predictive model using a combination of FOBT and CALB may have greater sensitivity and specificity and AUC for predicting CRC than models using a single marker.

Dipeptidyl peptidase 10, a novel prognostic marker in colorectal cancer.

PURPOSE: The dipeptidyl peptidase IV (DPPIV) gene family exhibits multiple functions and is involved in the pathogenesis of various diseases. It has attracted pharmaceutical interest in the areas of metabolic disorders as well as cancer. However, clinicopathologic significance of DPPIV family in colorectal cancer is not fully understood. MATERIALS AND METHODS: The clinical relevance of DPPIV and DPP10 expression was determined by immunohistochemical staining, and by assessing its clinicopathologic correlation in 383 colorectal cancer patients with known clinical outcomes. RESULTS: DPPIV was not expressed in normal colon mucosa, but it showed luminal expression in 52 of the 383 colorectal cancers (13.5%). DPPIV expression in tumors was associated with right-sided location of the colon (p=0.010) and more advanced tumor stage (p=0.045). DPP10 was expressed in normal colonic mucosa, but its expression varied in primary colorectal cancer tissues. Loss of DPP10 expression was found in 11 colorectal cancers (CRCs) (2.9%), and multivariate analysis showed that loss of DPP10 expression was an independent factor for poor patient prognosis (p=0.008). CONCLUSION: DPP10 may play a role in disease progression of colorectal cancer and loss of DPP10 expression in primary CRC is significantly associated with poor survival outcomes.

KRAS mutation status and clinical outcome of preoperative chemoradiation with cetuximab in locally advanced rectal cancer: a pooled analysis of 2 phase II trials.

PURPOSE: Cetuximab-containing chemotherapy is known to be effective for KRAS wild-type metastatic colorectal cancer; however, it is not clear whether cetuximab-based preoperative chemoradiation confers an additional benefit compared with chemoradiation without cetuximab in patients with locally advanced rectal cancer. METHODS AND MATERIALS: We analyzed EGFR, KRAS, BRAF, and PIK3CA mutation status with direct sequencing and epidermal growth factor receptor (EGFR) and Phosphatase and tensin homolog (PTEN) expression status with immunohistochemistry in tumor samples of 82 patients with locally advanced rectal cancer who were enrolled in the IRIX trial (preoperative chemoradiation with irinotecan and capecitabine; n=44) or the ERBIRIX trial (preoperative chemoradiation with irinotecan and capecitabine plus cetuximab; n=38). Both trials were similarly designed except for the administration of cetuximab; radiation therapy was administered at a dose of 50.4 Gy/28 fractions and irinotecan and capecitabine were given at doses of 40 mg/m2 weekly and 1650 mg/m2/day, respectively, for 5 days per week. In the ERBIRIX trial, cetuximab was additionally given with a loading dose of 400 mg/m2 on 1 week before radiation, and 250 mg/m2 weekly thereafter. RESULTS: Baseline characteristics before chemoradiation were similar between the 2 trial cohorts. A KRAS mutation in codon 12, 13, and 61 was noted in 15 (34%) patients in the IRIX cohort and 5 (13%) in the ERBIRIX cohort (P=.028). Among 62 KRAS wild-type cancer patients, major pathologic response rate, disease-free survival and pathologic stage did not differ significantly between the 2 cohorts. No mutations were detected in BRAF exon 11 and 15, PIK3CA exon 9 and 20, or EGFR exon 18-24 in any of the 82 patients, and PTEN and EGFR expression were not predictive of clinical outcome. CONCLUSIONS: In patients with KRAS wild-type locally advanced rectal cancer, the addition of cetuximab to the chemoradiation with irinotecan plus capecitabine regimen was not associated with improved clinical outcome compared with chemoradiation without cetuximab.

Association of serum lycopene and brachial-ankle pulse wave velocity with metabolic syndrome.

Metabolic syndrome (MetS) is known to inversely correlate with antioxidant status. Recently, it has been reported that MetS is associated with arterial stiffness, a composite risk factor for early atherosclerosis. In addition, our recent study for healthy women showed an inverse relationship between arterial stiffness and circulating lycopene. Therefore, this study aimed to investigate the interrelationship between arterial stiffness, antioxidant status, and the risk of MetS. Korean men (N = 299) were subgrouped according to the number of MetS risk factors (RF 0, RF 1-2, RF ≥ 3). Anthropometric parameters, brachial-ankle pulse wave velocity (baPWV; a marker of arterial stiffness), antioxidants (lycopene, ß-carotene, α-tocopherol), lipid profiles, glucose, insulin, and oxidative stress (low-density lipoprotein [LDL] particle size, oxidized LDL) were measured. Corresponding to the number of MetS RF, baPWV (1306 ± 17, 1364 ± 16, and 1420 ± 33 cm/s; P < .001) and insulin resistance (1.5 ± 0.1, 1.9 ± 0.1, and 2.7 ± 0.2; P < .001) gradually increased after adjustment for age, body mass index, smoking, and drinking, whereas serum lycopene among antioxidants and LDL particle size gradually decreased (0.036 ± 0.001, 0.031 ± 0.001, and 0.028 ± 0.001 mmol/L; P = .004 and 23.9 ± 0.1, 23.7 ± 0.1, and 23.3 ± 0.1 nm; P < .001, respectively). Brachial-ankle pulse wave velocity inversely correlated with serum lycopene after adjustment for the above confounders, blood pressure, insulin resistance, and oxidative stress (r = -0.136, P < .05). Oxidative stress markers also significantly correlated with baPWV as well as serum lycopene. Study subjects were divided into 2 groups by the median level of serum lycopene. When serum lycopene was lower than median level (≤ 0.0294 mmol/L), baPWV was significantly higher in MetS subjects than non-MetS subjects (1436 ± 41 vs 1367 ± 23 cm/s) after adjustment for age, body mass index, smoking, drinking, and oxidative stress (P = .041). However, when serum lycopene levels were high, no statistically significant difference was observed between the 2 subject groups (1386 ± 36 vs 1326 ± 13 cm/s). In conclusion, our result shows the interrelationship between circulating lycopene, baPWV, and MetS. In addition, much enhanced baPWV in MetS may be associated with lower lycopene concentration.

Lipoprotein-associated phospholipase A2 activity is associated with coronary artery disease and markers of oxidative stress: a case-control study.

BACKGROUND: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a lipoprotein-bound enzyme that can release atherogenic isoprostanes from esterified phospholipids and that may be involved in inflammation and atherosclerosis. OBJECTIVE: This study investigates the association between Lp-PLA(2) activity and coronary artery disease (CAD) in relation to oxidative stress markers, in particular urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)). DESIGN: We conducted a case-control study in which the cross-sectional relation between Lp-PLA(2) activity, lipoproteins, and oxidative stress markers was determined in 799 patients with angiographically confirmed CAD and 925 healthy controls. RESULTS: Lp-PLA(2) activity was significantly (P < 0.001) higher in CAD cases than in controls (32.9 +/- 0.46 and 29.7 +/- 0.42 nmol . mL(-1) . min(-1), respectively). Both elevated Lp-PLA(2) activity and urinary excretion concentrations of 8-epi-PGF(2alpha) were associated with greater CAD risk (P for trend < 0.001). Odds ratios for the upper quartiles of Lp-PLA(2) activity and 8-epi-PGF(2alpha).excretion were 2.47 (95% CI: 1.79, 3.40) and 2.19 (1.52, 3.15), respectively, after adjustment for sex, age, BMI, blood pressure, smoking and alcohol consumption status, and LDL and HDL cholesterol. When we examined the additive effect of both markers for CAD risk, the relation between 8-epi-PGF(2alpha) and CAD was weakened above the second quartile of Lp-PLA(2) activity. Moreover, Lp-PLA(2) activity was positively correlated with urinary excretion concentrations of 8-epi-PGF(2alpha) in controls (r = 0.277, P < 0.001) and cases (r = 0.202, P < 0.001) and with the tail moment of lymphocyte DNA (r = 0.213, P < 0.001) in controls. CONCLUSION: This study shows an association of elevated Lp-PLA(2) activity with CAD risk in relation to oxidant stress and thus supports a proatherogenic role of Lp-PLA(2).

Evaluation of metabolic syndrome risk in Korean premenopausal women: not waist circumference but visceral fat.

BACKGROUND: In clinical practice, using the patient's waist circumference (WC) to evaluate visceral obesity may underestimate disorders with a metabolic origin. This study examined whether or not the WC derived from the cut-off point of the visceral fat area (VFA) can reflect the features of metabolic syndrome (MetS) in premenopausal women. METHODS AND RESULTS: Computed tomography-scanned VFA, MetS components and the concentrations of high-sensitivity C-reactive protein (CRP) and adiponectin were measured in a total of 349 premenopausal women. The VFA at the L1 and the L4 sites was a significant index (p<0.001) of incremental MetS risk. Receiver-operating characteristic curve analysis showed that 75 cm2 of VFA at L4 and 87.5 cm2 at L1 were the optimal thresholds for discrimination of MetS risk. Significant differences in all MetS components, as well as CRP (p<0.05) and adiponectin levels (p<0.005), were observed when subjects were subdivided by the L4 VFA cut-off point (<75/>or=75 cm2), whereas there was a significant difference only in the triglycerides level in the groups divided by WC (WC<88/>or=88 cm). Moreover, subjects with a lower WC-higher VFA showed a similar pattern in MetS components and lower adiponectin than those with a higher WC-higher VFA. CONCLUSIONS: This study clarified that VFA rather than WC is a major determinant of MetS risk in premenopausal women.