Mismatch repair status in sporadic colorectal cancer: immunohistochemistry and microsatellite instability analyses.

BACKGROUND AND AIM: The aim of the present study was to evaluate associations between mismatch repair (MMR) status and clinicopathological characteristics and prognosis using immunohistochemistry (IHC) and microsatellite instability (MSI) analyses in a prospective cohort of a large number of accumulated samples. METHODS: Tumor tissue samples obtained during curative surgery (n = 2028) were analyzed using both MLH1/MSH2 IHC and MSI assays. Clinicopathological parameters and survival outcomes were compared according to IHC and MSI results. The median follow-up period was 43 months (range: 1-85 months). RESULTS: IHC identified 207 tumor samples (10.2%) with a loss of either MLH1 or MSH2 expression. The MSI analysis identified 203 tumor samples (10%) with high-frequency MSI (MSI-H). Patients with MMR defects were younger, and had tumors characterized by right-colon predilection; large-size, infrequent lymph node metastasis; poorly-differentiated or mucinous histology, and synchronous adenomas (P < 0.001-0.008). Patients with MSI-H status had higher 4-year disease-free survival rates than patients with microsatellite stable status (90.8% vs 80.6%, P = 0.001). A multivariate analysis showed that MSI-H status was a good prognostic factor for recurrence (hazard ratio: 0.48, 95% confidence interval: 0.30-0.83, P = 0.007). CONCLUSIONS: Patients with MMR defects had distinct clinicopathological characteristics, including a lower risk of recurrence. IHC and MSI analyses provided complementary information regarding specific clinicopathological parameters and prognosis.

SLAMF7 and TREM1 Mediate Immunogenic Cell Death in Colorectal Cancer Cells: Focus on Microsatellite Stability.

BACKGROUND/AIM: The aim of this study was to identify the association between SLAMF7 and TREM1 and anti-PD-1 drugs, and to determine whether they are molecular targets or predictors of responses to immunotherapy through induction of immunogenic cell death. MATERIALS AND METHODS: CRC cell lines over-expressing SLAMF7 and TREM1 were used to examine immunogenic and biological traits (e.g., proliferation and invasiveness) associated with factors related to anti-cancer immunity. In addition, multiplex immunofluorescence was used to examine immune cells in microsatellite instability-high (MSI-H) CRC and microsatellite stable (MSS) CRC. RESULTS: Proliferation rate and invasiveness of TREM1-over-expressing CRC cells were significantly greater than those of control cells (p<0.001 and 0.031, respectively), whereas SLAMF7-over-expressing CRC cells showed the opposite traits (p=0.005 and 0.002, respectively). SLAMF7-over-expressing DLD-1 cells harboring MSI-H showed increased apoptosis when treated with anti-PD-1 drugs, unlike SLAMF7-over-expressing SW480 cells harboring MSS. SLAMF7-over-expressing DLD1 and SW480 cells showed a marked increase in expression of the major cytokine mediator HMGB1 when exposed to anti-PD-1 drugs. Co-administration of anti-PD-1 drugs and TREM1 inhibitors induced apoptosis only in MSI-H HCT116 cells; HMGB1 was over-expressed regardless of microsatellite status. CONCLUSION: Expression of TREM1 and SLAMF7 is closely associated with immunogenic cell death, and TREM1 inhibitors may be an effective adjuvant that enhances anti-PD-1-mediated immunogenic cell death in MSS CRC.

Clinically Applicable Serum Biomarkers Among 14 Candidates Associated With Recurrence of Stage II and III Colorectal Cancer.

BACKGROUND/AIM: We evaluated the predictive value of candidate serum biomarkers for recurrence in stage II and III colorectal cancer (CRC) after curative surgery. PATIENTS AND METHODS: A total of 33 and 120 patients with CRC with or without recurrence at 5 years after curative surgery were included in the training set and the validation set, respectively. Possible serum biomarkers were examined for associations with CRC recurrence using receiver operating characteristics (ROC) curve analysis. RESULTS: In the training set, the expression levels of the 14 biomarkers were compared according to recurrence. Among them, five biomarkers that had significantly different expression levels were validated in 60 patients with recurrence at 5 years after curative surgery and 60 patients without. Multivariate analysis showed that natural log-transformed values of carcinoembryonic antigen (CEA), cyclin-dependent kinase regulatory subunit 2 (CKS2), 2'-5'-oligoadenylate synthetase 2 (OAS2), and autophagy-related gene 5 (ATG5) in preoperative serum were significantly related to recurrence. ROC analysis showed that these biomarkers were able to discriminate patients with recurrence from those without (area under the curve=0.828, 95% confidence interval=0.755-0.990). CONCLUSION: Preoperative serum levels of CEA, CKS2, OAS2 and ATG5 were independent risk factors for recurrence. A combination of serum CEA, CKS2, OAS2 and ATG5 predicted tumor recurrence well in patients with stage II and III CRC.

Analysis of genomic pathogenesis according to the revised Bethesda guidelines and additional criteria.

PURPOSE: As few genotype-phenotype correlations are available for nonsyndromic hereditary colorectal cancer (CRC), we implemented genomic analysis on the basis of the revised Bethesda guideline (RBG) and extended (12 items) to verify possible subtypes. METHODS: Patients with sporadic CRC (n = 249) were enrolled, stratified according to the revised Bethesda guidelines (RBG+ and RBG- groups) plus additional criteria. Exome/transcriptome analyses (n = 98) and cell-based functional assays were conducted. RESULTS: We detected 469 somatic and 830 germline gene mutations differing significantly between the positive and negative groups, associated with 12 RBG items/additional criteria. Twenty-one genes had significantly higher mutation rates in left, relative to right, colon cancer, while USP40, HCFC1, and HSPG2 mutation rates were higher in rectal than colon cancer. FAT4 mutation rates were lower in early-onset CRC, in contrast to increased rates in microsatellite instability (MSI)-positive tumors, potentially defining an early-onset microsatellite-stable subtype. The mutation rates of COL6A5 and MGAM2 were significantly and SETD5 was assumably, associated CRC pedigree with concurrent gastric cancer (GC). The predicted deleterious/damaging germline variants, SH2D4A rs35647122, was associated with synchronous/metachronous CRC with related tumors, while NUP160 rs381660 and KRTAP27-1 rs2244485 were potentially associated with a GC pedigree and less strictly defined hereditary CRC, respectively. SH2D4A and NUP160 acted as oncogenic facilitators. CONCLUSION: Our limited genomic analysis for RBG and additional items suggested that specific somatic alterations in the respective items may enlighten relevant pathogenesis along with the knowledge of germline mutations. Further validation is needed to indicate appropriate surveillance in suspected individuals.

Upregulation of SPRR3 promotes colorectal tumorigenesis.

Hereditary colorectal cancer develops through a series of well-defined genetic and histological changes. However, elucidation of the canonical pathway based on hereditary colorectal cancer has not provided a clear explanation of the molecular mechanisms of sporadic colorectal cancer. To identify the alterative pathways involved in sporadic colorectal tumorigenesis, we performed gene expression analysis in patients with sporadic colorectal tumors. A comparison analysis of gene expression profiles revealed a pattern of upregulation of small proline rich repeat protein 3 (SPRR3) in tumor samples. SPRR3 has previously been reported to be downregulated in esophageal cancer. However, in the present study, we observed that SPRR3 was strongly upregulated in 31 of 35 samples of sporadic colorectal tumors (88%). We also determined that overexpression of SPRR3 not only accelerates colorectal cancer cell proliferation but also is associated with lymphovascular invasion in colorectal cancer. Moreover, AKT was activated and p53 levels were decreased in cells that overexpressed SPRR3. In contrast to the pattern seen in esophageal cancer, these results suggest that increased expression of SPRR3 is involved in colorectal tumorigenesis.

In vitro chemosensitivity of gastric adenocarcinomas to histone deacetylase inhibitors, compared to established drugs.

BACKGROUND/AIMS: This study was performed to determine the efficacy of histone deacetylase inhibitors in gastric cancer, together with other established regimens. METHODOLOGY: The chemosensitivities of 93 gastric cancer patients to established drugs, and three histone deacetylase inhibitors (SAHA, PXD101, and a novel candidate, CG-2) were evaluated using the histoculture drug response assay. RESULTS: Tumor growth inhibition rates were the highest with cisplatin, followed by PXD101, taxol, docetaxel, and TS-1, in descending order. The response rates were 41.9-68.8%, and 37.6-47.3%, respectively, at an inhibition rate cutoff value of 30%. Synergistic activity was evident with most combinations of established drugs and histone deacetylase inhibitors. Diffuse- or mixed-type carcinomas on Lauren classification were closely associated with increased chemosensitivity to TS-1 (p = 0.044). Node-positive and "other than tubular type" tumors on WHO classification were chemosensitive to cisplatin (p = 0.011 and 0.014, respectively). CG-2 chemosensitivity was markedly associated with low preoperative CA724 level (< or = 4 U/ml) (p = 0.046). CONCLUSIONS: This in vitro chemosensitivity assay validates the comparable chemo-response of gastric cancers to histone deacetylase inhibitors and established drugs, indicating considerable therapeutic efficacy of these agents. Additionally, a number of clinicopathological parameters are significantly associated with specific regimens.

Growth and invasion of sporadic colorectal adenocarcinomas in terms of genetic change.

Integrative genetic changes were examined in relation to tumor growth and progression of sporadic colorectal cancers. Ninety-two sporadic colorectal cancer patients and 12 human colorectal cancer cell lines were evaluated. Genetic changes in representative steps of colorectal tumorigenesis were determined. Biological characteristics, i.e., clinicopathologic parameters, expression of invasion-associated molecules, and in vitro invasion and migration, in association with these changes were further analyzed. Adenomatous polyposis coli (APC) and/or Wnt-activated alterations occurred in 66% patients, whereas mismatch repair (MMR) defects and/or RAF-mediated alterations were identified in 47% patients. The crossover rate between these two alterations was 26%. Differential mRNA expression of ARK5 was closely associated with that of MMP2, MMP9, and S100A4 (P< or =0.044-0.001). Additionally, enhanced ARK5 mRNA expression was more frequent in tumors displaying RAF-mediated alterations and crossover pathways (P=0.01 and 0.03, respectively). Upregulation of CEA mRNA was more common in the advanced stages (P=0.034), while VEGF expression was greater in poorly differentiated or mucinous tumors (P=0.042). The high expressions of MMP2 and MMP9 were closely associated with invasion and migration of colorectal tumors and cell lines. Our results conclusively show that specific pathways of colorectal tumorigenesis are closely associated with characteristic tumor growth and invasion.

Biological Characteristics and Clinical Significance of ITGB1 and RHOC in Patients With Recurrent Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC. MATERIALS AND METHODS: To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied. RESULTS: ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression. CONCLUSION: ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.

Clinical assessment and identification of immuno-oncology markers concerning the 19-gene based risk classifier in stage IV colorectal cancer.

BACKGROUND: Genomic profiling of tumors has contributed to the understanding of colorectal cancer (CRC), facilitating diagnosis, prognosis and selection of treatments, including targeted regimens. A report suggested that a 19-gene-based risk classifier (TCA19) was a prognostic tool for patients with stage III CRC. The survival outcomes in patients with stage IV CRC are still poor and appropriate selection of targeted therapies and immunotherapies is challenging. AIM: To assess clinical implication of TCA19 in patients with stage IV CRC, and to identify TCA19 with involvement in immune-oncology. METHODS: A retrospective review of the medical records of 60 patients with stage IV CRC was conducted, assessing clinicopathological variables and progression-free survival (PFS). TCA19 gene expression was determined by quantitative polymerase chain reaction (qPCR) in matched normal and tumor tissues taken from the study cohort. Expression of potential immune-oncology regulatory proteins and targets was examined by immunohistochemistry (IHC), western blot, immunofluorescence staining in tissues from a validation cohort of 10 patients, and in CRC cell lines co-cultured with monocyte in vitro. RESULTS: In the patients with TCA19 score higher than the median, the PFS rates of eight patients who received the targeted regimens were significantly higher than the PFS rates of four patients who received 5-fluorouracil-based regimen (P = 0.041). In multivariate analysis, expression of signaling lymphocytic activation molecule family, member 7 (SLAMF7) and triggering receptor expressed on myeloid cells 1 (TREM1) was associated with PFS in the 60-patient cohort. After checking another 10 validate set, the expression of the IHC, the level of real-time qPCR, and the level of western blot were lower for SLAMF7 and higher for TREM7 in primary and metastatic tumors than in normal tissues. In CRC cells expressing SLAMF7 that were co-cultured with a monocytic cell line, levels of CD 68 and CD 73 were significantly lower at day 5 of co-culture than at day 0. CONCLUSION: The TCA19 score might be prognostic for target-regimen-specific PFS in stage IV CRC. Down-regulation of SLAMF7 and up-regulation of TREM1 occur in primary and metastatic tumor tissues.

A prognostic index based on an eleven gene signature to predict systemic recurrences in colorectal cancer.

Approximately half of colorectal cancer (CRC) patients experience disease recurrence and metastasis, and these individuals frequently fail to respond to treatment due to their clinical and biological diversity. Here, we aimed to identify a prognostic signature consisting of a small gene group for precisely predicting CRC heterogeneity. We performed transcriptomic profiling using RNA-seq data generated from the primary tissue samples of 130 CRC patients. A prognostic index (PI) based on recurrence-associated genes was developed and validated in two larger independent CRC patient cohorts (n = 795). The association between the PI and prognosis of CRC patients was evaluated using Kaplan-Meier plots, log-rank tests, a Cox regression analysis and a RT-PCR analysis. Transcriptomic profiling in 130 CRC patients identified two distinct subtypes associated with systemic recurrence. Pathway enrichment and RT-PCR analyses revealed an eleven gene signature incorporated into the PI system, which was a significant prognostic indicator of CRC. Multivariate and subset analyses showed that PI was an independent risk factor (HR = 1.812, 95% CI = 1.342-2.448, P < 0.001) with predictive value to identify low-risk stage II patients who responded the worst to adjuvant chemotherapy. Finally, a comparative analysis with previously reported Consensus Molecular Subgroup (CMS), high-risk patients classified by the PI revealed a distinct molecular property similar to CMS4, associated with a poor prognosis. This novel PI predictor based on an eleven gene signature likely represents a surrogate diagnostic tool for identifying high-risk CRC patients and for predicting the worst responding patients for adjuvant chemotherapy.

Gene expression profiling: canonical molecular changes and clinicopathological features in sporadic colorectal cancers.

AIM: To investigate alternative or subordinate pathways involved in colorectal tumorigenesis and tumor growth, possibly determining at-risk populations and predicting responses to treatment. METHODS: Using microarray gene-expression analysis, we analyzed patterns of gene expression relative to canonical molecular changes and clinicopathological features in 84 sporadic colorectal cancer patients, standardized by tumor location. Subsets of differentially expressed genes were confirmed by real-time reverse-transcript polymerase chain reaction (RT-PCR). RESULTS: The largest number of genes identified as being differentially expressed was by tumor location, and the next largest number by lymphovascular or neural invasion of tumor cells and by mismatch repair (MMR) defects. Amongst biological processes, the immune response was significantly implicated in entire molecular changes observed during colorectal tumorigenesis (P < 0.001). Amongst 47 differentially expressed genes, seven (PISD, NIBP, BAI2, STOML1, MRPL21, MRPL16, and MKKS) were newly found to correlate with tumorigenesis and tumor growth. Most location-associated molecular changes had distinct effects on gene expression, but the effects of the latter were sometimes contradictory. CONCLUSION: We show that several differentially expressed genes were associated with canonical molecular changes in sporadic colorectal cancers, possibly constituting alternative or subordinate pathways of tumorigenesis. As tumor location was the dominant factor influencing differential gene expression, location-specific analysis may identify location-associated pathways and enhance the accuracy of class prediction.

Opposite functions of GSN and OAS2 on colorectal cancer metastasis, mediating perineural and lymphovascular invasion, respectively.

The present study aimed to identify molecules associated with lymphovascular invasion (LVI) and perineural invasion (PNI) and to examine their biological behavior in colorectal cancer (CRC). LVI- and PNI-associated molecules were identified and verified using sequential processes including (1) identification of 117 recurrence-associated genes differentially expressed on RNA-seq analysis using primary cancer tissues from 130 CRC patients with and without systemic recurrence; (2) analysis of molecules associated with LVI and PNI; (3) assessment of biological properties by measuring proliferation, anoikis, invasion/migration, epithelial-mesenchymal transition and autophagy flux; and (4) verification of disease-free survival using public datasets. Gelsolin (GSN) and 2'-5'-oligoadenylate synthetase 2 (OAS2) were associated with PNI and LVI, respectively. Invasion potential was >2-fold greater in GSN-overexpressing LoVo cells than in control cells (p<0.001-0.005), whereas OAS2-overexpressing RKO cells showed reduced invasion (p<0.001-0.005). GSN downregulated E-cadherin, ß-catenin, claudin-1 and snail, and upregulated N-cadherin and ZEB1, whereas OAS2 overexpression had the opposite effects. Several autophagy-related proteins including ATG5-12, ATG6/BECN1, ATG7 and ATG101 were downregulated in GSN-overexpressing LoVo cells, whereas the opposite pattern was observed in OAS2-overexpressing RKO cells. Patients with low GSN expression had significantly higher 5-year recurrence-free survival (RFS) rates than those with GSN overexpression (73.6% vs. 64.7%, p = 0.038), whereas RFS was longer in patients with OAS2 overexpression than in those with underexpression (73.4% vs. 63.7%, p = 0.01). In conclusion, GSN and OAS2 were positively and negatively associated with recurrence, respectively, suggesting their potential value as predictors of recurrence or therapeutic targets in CRC patients.

Up-regulation of UVRAG by HDAC1 Inhibition Attenuates 5FU-induced Cell Death in HCT116 Colorectal Cancer Cells.

The ultraviolent irradiation resistance-associated gene (UVRAG), a component of the Beclin 1/autophagy-related 6 complex, regulates the autophagy initiation step and functions in the DNA-damage response. UVRAG is frequently mutated in various cancer types, and mutations of UVRAG increase sensitivity to chemotherapy by impairing DNA-damage repair. In this study, we addressed the epigenetic regulation of UVRAG in colorectal cancer cells. UVRAG expression was increased in cells treated with histone deacetylase (HDAC) inhibitors, such as valproic acid and suberoylanilide hydroxamic acid. Down-regulation of HDAC1 enhanced UVRAG expression in colorectal cancer cells. In addition, both chemical and genetic inhibition of HDAC1 reduced the activation of caspase-3 and cytotoxicity in 5-fluorouracil (5FU)-treated cancer cells. In contrast, UVRAG overexpression inhibited caspase activation and cell death in 5FU-treated cells. Taken together, our findings suggest that up-regulation of UVRAG by HDAC1 inhibition potentiates DNA-damage-mediated cell death in colorectal cancer cells.

Development and Applicability of Integrative Tumor Response Assays for Metastatic Colorectal Cancer.

AIM: The present study investigated how well the results of integrative tumor-response assay (ITRA) compared to those of clinical response to chemotherapy in patients with metastatic colorectal cancer (CRC). PATIENTS AND METHODS: A total of 129 patients with metastatic CRC were prospectively enrolled. ITRA consisted of two sequential histoculture drug-response assays (HDRAs). First-stage HDRAs were performed using 5-fluorouracil with leucovorin and oxaliplatin (FX), or with irinotecan (FR). Second-stage HDRAs (ITRA) were performed for cells surviving after the first-stage HDRA, using FX, FR, and their combinations with bevacizumab and cetuximab. RESULTS: Among 129 patients, 42 (32.6%) completed second-line chemotherapy, results that correlated with those of ITRA. The accuracy of ITRA for predicting response to second-line chemotherapy was 61.9% (26/42), with a sensitivity of 44.4% (8/18) and a specificity of 75% (18/24). CONCLUSION: Despite its relatively low accuracy, ITRA might be a useful technique for predicting therapeutic efficacy and selecting for appropriate first-line and second-line anticancer regimens for patients.

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells.

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.

PSMB8 as a Candidate Marker of Responsiveness to Preoperative Radiation Therapy in Rectal Cancer Patients.

PURPOSE: The ability to predict individual responsiveness to cancer therapy is urgently needed. This is particularly true for patients with locally advanced rectal cancer (LARC) because a large proportion are resistant to preoperative chemoradiation therapy (CRT). In this study, we sought to identify markers that could predict response by comparing the gene expression profiles of the tumors of patients who received preoperative CRT. METHODS AND MATERIALS: The basal gene expression profiles of tumors from 22 LARC patients who were responders (n=9) and nonresponders (n=13) to preoperative CRT were analyzed using RNA sequencing (RNA-Seq). To validate the RNA-Seq findings, real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed on tumor samples from an additional 40 LARC patients (n=20 responders; n=20 nonresponders). Candidate genes were stably overexpressed or knocked down in colorectal cancer (CRC) cell lines, and the effect on response to radiation was tested in vitro and also in vivo in a mouse xenograft model. RESULTS: Eight differentially expressed (>16-fold) genes (B3GALT4, HSPA1B, KRBOX1, PPBP, PPP1R18, PSMB8, SLC39A7, and TAP2) associated with the preoperative CRT response were identified (P<.0005). Among these genes, real-time RT-PCR showed that PSMB8 and SLC39A7 were upregulated in the responsive group of the additional 40 LARC patients. In CRC cell lines, PSMB8 overexpression significantly reduced colony formation and increased the apoptosis-inducing molecules cleaved caspase-3 and cleaved PARP after 6-Gy irradiation. PSMB8 knockdown increased colony formation and decreased caspase-3 activation and cleaved PARP levels after irradiation. SLC39A7 overexpression had no significant effects on irradiated CSC cells. After irradiation of the xenografted mice, tumors that arose from CRC cell line HCT116 overexpressing PSMB8 grew more slowly than did those from HCT116 with vector alone. CONCLUSION: These results suggest that PSMB8 is a predictive marker of preoperative radiosensitivity in LARC patients. Clinical validation in a larger cohort is now required.

The prognostic significance and treatment modality for elevated pre- and postoperative serum CEA in colorectal cancer patients.

PURPOSE: The purpose of this study was to evaluate the prognostic significance of serum CEA (s-CEA) changes in colorectal cancer (CRC) patients with sustained elevated postoperative s-CEA levels. METHODS: Between January 1999 and December 2008, 9,380 CRC patients underwent surgery. Curative resection was performed in 1,242 CRC patients with high preoperative s-CEA levels (>6 ng/mL). High s-CEA levels were normalized in 924 patients (74.4%) within 2 weeks from surgery, whereas high s-CEA levels were persistent in 318 patients (25.6%). Patients were divided into 2 groups according to their postoperative s-CEA levels: group 1 (37 patients with a 1-year postoperative s-CEA>6 ng/mL) and group 2 (281 patients with a 1-year postoperative s-CEA≤6 ng/mL). RESULTS: A postoperative recurrence was identified in 24 patients (64.9%) in group 1 and 65 patients (23.1%) in group 2 (P < 0.001). A curative resection after recurrence was performed in 22 patients (33.8%) from group 2, but no patients from group 1 (P = 0.001). The 5-year overall survival and time to recurrence were significantly lower in patients with recurrent cancer in group 1 (P < 0.001). CONCLUSION: Patients with persistent elevated postoperative s-CEA levels are at high risk for recurrence and a low survival rate. More intensive surveillance of patients with high postoperative s-CEA levels should be mandatory.

ZKSCAN3 Facilitates Liver Metastasis of Colorectal Cancer Associated with CEA-expressing Tumor.

AIM: Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3) is overexpressed in invasive colorectal cancer (CRC) cells and regulates the expression of several genes favoring tumor progression, including vascular endothelial growth factor (VEGF) and integrin ß4. We evaluated the association of ZKSCAN3 and colorectal cancer liver metastasis (CLM) to determine whether it is related to invasive signaling pathways. MATERIALS AND METHODS: The ratios of expression by primary tumor to normal tissue and metastatic tumor to normal tissue were compared between ZKSCAN3-overexpressing and underexpressing primary tumor groups. RESULTS: In terms of CLM, the ZKSCAN3 overexpression was positively correlated with carcinoembryonic antigen (CEA), VEGF, and AKT expression. The protein-expression analysis showed that ZKSCAN-specific siRNA knockdown reduced CEA expression in LoVo and LS174T CRC cells. Matrigel invasion by ZKSCAN3-overexpressing HCT116 cells was increased when examined on CEA-coated filters compared with phosphate-buffered saline-treated controls. Additionally, matrix metalloproteinase 9 (MMP9) expression was greater in cells with reference allele (GG) than substitution allele (CC) for ZKSCAN3 rs733743 (p=0.032). ZKSCAN3 protein expression of the high serum CEA group was increased in hepatic metastatic tissue compared with the primary tumor tissue, while in the group with normal serum CEA it decreased or was similar. Reference ZKSCAN3 alleles were correlated with male dominance, a family history of malignancy, high serum CEA concentration and stage IV CRC in 450 patients with sporadic CRC. In conclusion, ZKSCAN3 appears to promote colorectal tumor progression and invasion. ZKSCAN3 may facilitate hepatic metastasis of CRC associated with CEA particularly in cases with CEA-producing tumor.

Feasibility of novel PPP1R15A and proposed ANXA11 single nucleotide polymorphisms as predictive markers for bevacizumab regimen in metastatic colorectal cancer.

PURPOSE: Bevacizumab improves survival in patients with metastatic colorectal cancer (mCRC) under chemotherapy, but few predictive markers have been identified. METHODS: To investigate chemosensitive single nucleotide polymorphisms (SNPs) of mCRC, we performed exome sequencing and RNA sequencing in 19 patients. A clinical association analysis was performed with the other 116 patients who had received chemotherapy to bevacizumab regimens. In vivo biodistribution studies and [(18)F]FDG-PET imaging were performed on mice bearing human colorectal cancer (HCT116 and SW480) xenografts after injection of bevacizumab with 5-FU, leucovorin, and irinotecan (FOLFIRI). RESULTS: PPP1R15A rs557806 showed the most significant association with FRB-driven tumor IR in exome sequencing and the highest correlation (r = 0.74) with drug responses in RNA sequencing. Patients homozygous for the reference alleles (GG) of PPP1R15A rs557806 exhibited greater disease control rate and a tendency toward greater objective response rate (ORR) than those with homozygous or heterozygous substitution alleles (GC and CC; P = 0.027 and 0.073, respectively). In xenografted mice, HCT116 clones transfected with the G allele at PPP1R15A rs557806 were more sensitive to bevacizumab regimens than those with the C allele. Tumor volume of xenografts with the G allele was significantly lower than that of xenografts with the C allele (P = 0.004, day 13). [(18)F]FDG uptake decreased to 75 % in HCT116 xenograft-bearing mice with the G allele, whereas [(18)F]FDG uptake was 42 % in mice xenografts with the C allele (P = 0.032). ANXA11 rs1049550, a predictive biomarker of SNP described in our previous study, was validated using the xenograft model. Tumor volume and [(18)F]FDG uptake analyses showed that tumors in the SW480 xenografts expressing the substitution allele (T) at ANXA11 rs1049550 were more susceptible to FOLFIRI plus bevacizumab-induced suppression than those expressing the reference allele (C) (P = 0.001 and 0.026, respectively). CONCLUSION: ANXA11 rs1049550 and PPP1R15A rs557806 may improve the identification of mCRC patients sensitive to bevacizumab regimens, and further validation is required in large cohorts.

Complex Behavior of ALDH1A1 and IGFBP1 in Liver Metastasis from a Colorectal Cancer.

Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001-0.003) and suppressed invasiveness by ≥3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of ß-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.