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Reducing recurrence following radical resection of colon cancer without overtreatment or undertreatment remains a challenge. Postoperative adjuvant chemotherapy (Adj) is currently administered based solely on pathologic TNM stage. However, prognosis can vary significantly among patients with the same disease stage. Therefore, novel classification systems in addition to the TNM are necessary to inform decision-making regarding postoperative treatment strategies, especially stage II and III disease, and minimize overtreatment and undertreatment with Adj. We developed a prognostic prediction system for colorectal cancer using a combined convolutional neural network and support vector machine approach to extract features from hematoxylin and eosin staining images. We combined the TNM and our artificial intelligence (AI)-based classification system into a modified TNM-AI classification system with high discriminative power for recurrence-free survival. Furthermore, the cancer cell population recognized by this system as low risk of recurrence exhibited the mutational signature SBS87 as a genetic phenotype. The novel AI-based classification system developed here is expected to play an important role in prognostic prediction and personalized treatment selection in oncology., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers of their prognosis and chemotherapeutic responsiveness is necessary. This study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab therapy, in mCRC., Methods: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer in the TCGA database. Preoperative serum PRTN3 levels were measured using an enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay., Results: Serum PRTN3 levels were an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio, 2.082; 95% confidence interval, 1.118-3.647; P=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data sets showed poorer overall survival in patients with PRTN3 expression than that in patients without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first-line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poor PFS (hazard ratio, 3.027; 95% confidence interval, 1.175-7.793; P=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. The tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab therapy., Conclusions: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy, especially for bevacizumab therapy, in patients with mCRC., (© 2024. The Author(s).)

Background: The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels., Methods: Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases., Results: Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3 + and CD4 + T cells as well as FOXP3 + cells. Mass cytometry analysis showed that IL-6 + cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4 + FOXP3 high CD45RA - effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10 + cells in MDSCs and that of IL-10 + or CTLA-4 + cells in eTregs correlated with IL-6 levels., Conclusion: Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME., (© 2023. Society of Surgical Oncology.)

Background/aim: Chemotherapy combined with anti-EGFR or anti-VEGF monoclonal antibodies (mAb) is widely used to treat patients with metastatic colorectal cancer (mCRC). Here, we investigated the effects of these antibodies on T-cell infiltration and T-cell receptor (TCR) repertoire variation in CRC liver metastases., Materials and Methods: Ten patients with mCRC received chemotherapy in combination with anti-EGFR (n=6) or anti-VEGF (n=4) mAb. T-cell infiltration was examined for CD3 and CD8 by carrying out immunohistochemistry on biopsy or surgical specimens from liver metastases before and after treatment. TCR repertoire analysis was carried out on specimens with post-treatment CD3 + T-cell infiltration., Results: T-cell infiltrations were approximately 83% (5/6) and 50% (2/4), following treatment with anti-EGFR or anti-VEGF mAb, respectively. TCR repertoire analysis revealed higher clonality and lower diversity of TCR alpha and beta (TRA and TRB) in the anti-VEGF mAb group than that in the anti-EGFR group mAb. Furthermore, the percentage of the common TCR clones between infiltrating T cells and T cells in peripheral blood was significantly lower in the anti-VEGF mAb group compared to that in the anti-EGFR mAb group., Conclusion: The population of T cells infiltrating liver metastases in the anti-VEGF mAb group differed from that in the anti-EGFR mAb group., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Irinotecan is a useful anticancer drug for colorectal cancer treatment. UGT1A1*28 and *6 gene polymorphisms are known risk factors for irinotecan-associated toxicity. However, severe adverse effects due to irinotecan have been observed even in patients who do not harbor UGT1A1*28 or *6. We investigated gene polymorphisms in the whole exome to identify useful biomarkers for irinotecan toxicity other than UGT1A., Methods: A total of 178 patients with metastatic colorectal cancer (mCRC) and 87 patients with pancreatic cancer were treated with FOLFIRI, FOLFOX, FOLFOXIRI, modified FOLFIRINOX, or gemcitabine plus nab-paclitaxel. Genome-wide screening was performed using whole-exome sequencing (WES), and validation analysis was performed using qPCR with a hydrolysis probe., Results: Using WES after a doublet chemotherapy regimen comprising irinotecan and 5-fluorouracil (n = 15), seven single nucleotide polymorphisms (SNPs) were identified as candidate biomarkers for irinotecan-associated toxicity of neutropenia. Among the seven SNPs, an SNP in R3H domain and coiled-coil containing 1 (R3HCC1; c.919G > A, rs2272761) showed a significant association with neutropenia (>grade 3) after doublet chemotherapy. Patients receiving irinotecan including triplet chemotherapy, FOLFOXIRI for mCRC (n = 23) or modified FOLFIRINOX for pancreatic cancer (n = 40), also showed significant linear trends between R3HCC1 polymorphism and neutropenia (p = 0.017 and 0.046, respectively). No significant association was observed in patients treated with irinotecan-free regimens, FOLFOX for mCRC (n = 66), and gemcitabine plus nab-paclitaxel for pancreatic cancer (n = 47)., Conclusion: Thus, an SNP in the R3HCC1 gene may be a useful biomarker for the toxicity of irinotecan-containing chemotherapy for mCRC and pancreatic cancer., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background/aim: Colorectal cancer is the third most common cancer globally, and the poor prognosis of patients with metastatic colorectal cancer (mCRC) warrants urgent attention. We previously obtained 10 candidate serum biomarkers for mCRC. Our aim with this study was to determine the prognostic performance of the pre-treatment serum C-C motif chemokine ligand 7 (CCL7) concentration in patients with mCRC., Patients and Methods: Protein concentrations of CCL7 were examined using ELISA and immunohistochemistry for serum (n=110) and surgical specimens (n=85), respectively, of patients with mCRC. The relationship between protein concentration and prognosis was examined using Cox regression analysis, receiver operator characteristic curve analysis and the Kaplan-Meier method., Results: The overall survival (OS) of patients with high concentrations of serum CCL7 was significantly poorer than that of patients with low concentrations. Patients with a high CCL7 concentration in the stroma had significantly poorer outcomes than those with a low concentration. The concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 were significantly higher in the high-CCL7 group, compared to those in the low-CCL7 group. Univariate and multivariate analysis revealed that serum CCL7 concentration was a significant prognostic factor for mCRC. The combination of serum CCL and CEA concentrations was also useful in this regard (area under the curve=0.71)., Conclusion: The combined pre-treatment serum levels of CCL7 and CEA are useful prognostic biomarkers for mCRC., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Although faecal DNA testing of Fusobacterium nucleatum ( Fn ) is expected to be useful for colorectal neoplasia detection, there is no standardized quantification method of Fn . We performed this study to establish a possible standardized method., Methods: In this study, 322 participants including 71 subjects without colorectal neoplasia (control group), 31 patients with non-advanced colorectal adenoma, 93 patients with advanced colorectal adenoma, and 127 patients with colorectal cancer were enrolled. Faecal Fn were quantified by droplet digital PCR (ddPCR) using two PCR primer-probe sets reported previously that are tentatively named Fn1 and Fn2. Fn1 has been used in ddPCR by us and Fn2 has been widely used in quantitative real-time PCR., Results: The Fn copy number using Fn1 was five times higher than that using Fn2, with a linear relationship shown between them. Receiver operating characteristic curve analysis showed the area under the curve (AUC) to be almost the same between Fn1 and Fn2 in discriminating between the control group and the colorectal cancer group (AUC = 0.81 and 0.81, respectively), and between the control/non-advanced colorectal adenoma group and the advanced colorectal adenoma/colorectal cancer group (AUC = 0.74 and 0.74, respectively)., Conclusions: As the diagnostic performance was quite similar between Fn1 and Fn2, ddPCR-based Fn testing using Fn1 and Fn2 could be a possible standardized method for a colorectal neoplasia screening test, considering that Fn levels quantified by Fn1 are about five times higher than those by Fn2.

Background: We recently reported the relapse-free survival (RFS) significance of the combination of CD4 + and forkhead box P3 + (FOXP3) T-cell densities identified by immunohistochemistry in patients with stage I, II, and III colorectal cancer (CRC) who underwent curative resections. This study was designed to determine the optimal combination of markers that predict recurrence in patients with T factors of T3/T4a stage II CRC by applying a novel Bayes decision rule., Methods: Using 137 cancer tissue specimens from T3/T4a stage II patients, 12 clinicopathologic and immune factors were analysed as predictive candidates for recurrence., Results: Our study showed that the combination of low CD4 + and low FOXP3 + T-cell densities resulted in extremely poor RFS., Conclusions: Adjuvant chemotherapy may be considered for patients with a combination of low CD4 + and low FOXP3 + T-cell densities. The discovery of this new prognostic indicator is important for the appropriate management of patients undergoing curative resection for T3/T4a stage II CRC., (© 2022. The Author(s).)

Background & Aims: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated., Methods: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data., Results: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes., Conclusions: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Despite its adverse effects, chemotherapy is generally used for the treatment of colorectal cancer (CRC). Development of supplement preparations targeting cancer stem cells (CSCs) that cause distant metastasis and drug resistance is required. Although curcumin is known to have anti-tumor, hepatoprotective, and hypoglycemic-like actions, its low water solubility, oral absorption, and bioavailability impede its therapeutic uses. Patient-derived organoid cultures can recapitulate heterogeneity, epithelial structures, and molecular imprints of their parental tissues. In the present study, anti-carcinogenic properties of amorphous curcumin (AC), a compound with improved solubility and bioavailability, were evaluated against human CRC organoids. Treatment with AC inhibited the cell viability of CRC organoids in a concentration-dependent manner. AC arrested the cell cycle of CRC organoids and induced apoptosis. AC inhibited phosphorylation of ERK. Expression of downstream signals of ERK, namely c-MYC and cyclin-D1, were inhibited. Expressions of CSC markers, CD44, LGR5, and CD133, were declined in the AC-treated CRC organoids. The combinational treatment of CRC organoids with AC and anti-cancer drugs, oxaliplatin, 5-FU, or irinotecan showed a synergistic activity. In vivo, AC decreased the tumor growth of CRC organoids in mice with the induction of necrotic lesions. In conclusion, AC diminished the cell viability of CRC organoids through the inhibition of proliferation-related signals and CSC marker expression in addition to arresting the cell cycle. Collectively, these data suggest the value of AC as a promising supplement that could be used in combination with anti-cancer drugs to prevent the recurrence and metastasis of CRC., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Tumor infiltration of CD3 and CD8-positive T cells has been reported as a good prognostic marker for patients with colorectal cancer(CRC). To clarify the significance of CD4 and FOXP3-positive T cells in CRC for prognosis of intratumoral infiltration., Methods: CD3, CD8, CD4 and FOXP3-positive T cells were immunostained(IHC)from tissue specimens of 342 CRC patients who underwent curative resection to quantify the number of infiltrating cells in the tumor. Microsatellite instability(MSI)was also evaluated in 322 samples and the clinicopathological factors and survival were analyzed., Results: Highly infiltrated groups of CD3, CD4 and FOXP3-positive T cells were associated with improved relapse-free survival(RFS). Highly infiltrated groups of CD8, CD4 and FOXP3-positive T cells were associated with improved disease- specific survival(DSS). Invasion depth, vascular infiltration, and CD4-positive T cell density were independent prognostic factors for DSS. CD4 and FOXP3-positive T cell infiltration was not associated with the high-frequency microsatellite instability group, in contrast to CD3 and CD8-positive T cell infiltration., Conclusions: Intratumoral CD4-positive T cell density and FOXP3-positive T cell densities were stronger prognostic indicators than other clinicopathological features. These results may facilitate the establishment of novel prognostic factors and therapeutic strategies for CRC.

Background: Colorectal cancer is the third most common cancer worldwide. If biomarkers can be identified in liquid biopsy, diagnosis and treatment can be optimized even when cancerous tissues are not available. The purpose of this study was to identify proteins from liquid biopsy that would be useful as markers of poor prognosis., Methods: First, we comprehensively analyzed serum proteins to identify potential biomarkers and focused on serum lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). The relationship between LOX-1 and the prognosis of patients with colorectal cancer has not been reported. Next, we validated this marker using serum samples from 238 patients with colorectal cancer by ELISA and 100 tissue samples by immunohistochemical staining., Results: The optimal cut-off value of serum LOX-1 was 538.7 pg/mL according to time-dependent receiver operating characteristics curve analysis. The overall survival of patients with high levels of serum LOX-1 was significantly poorer than that of individuals with low levels of LOX-1 in the training and test datasets. In multivariate analysis for overall survival, serum LOX-1 was an independent prognostic factor identified in liquid biopsy (hazard ratio = 1.729, p = 0.027). The prognosis of patients with high LOX-1 expression in tumor tissues was significantly poorer than that of individuals with low expression (p =0.047 ). Additionally, inflammatory factors such as white blood cell count, C-reactive protein level, neutrophil/lymphocyte ratio, and monocyte/lymphocyte ratio were significantly higher in the group with high serum LOX-1 levels., Conclusions: Serum LOX-1 might be a useful biomarker of poor prognosis in colorectal cancer.

Introduction: We have reported previously that fecal DNA testing of TWIST1 methylation in combination with the fecal immunochemical test for hemoglobin (FIT) (combination test) is useful for colorectal neoplasia screening. In this study, using larger sample sizes, we studied the clinical performance of the combination test for the detection of colorectal neoplasia and, especially, advanced colorectal adenoma., Methods: We performed a prospective study in which FIT, fecal DNA testing of TWIST1 methylation, and colonoscopy were performed on 372 patients with colorectal neoplasia and 71 subjects without colorectal neoplasia. We assessed the individual clinical performance of each of FIT and fecal DNA testing of TWIST1 methylation and of the combination test for the detection of colorectal neoplasia including advanced adenoma based on morphologic subtypes., Results: The FIT alone had a sensitivity of 7.5% (3/40) for nonadvanced adenoma, 32.3% (41/127) for advanced adenoma, and 93.7% (192/205) for colorectal cancer and a specificity of 87.3% (62/71). The combination test had a sensitivity of 35.0% (14/40) for nonadvanced adenoma, 68.5% (87/127) for advanced adenoma, and 95.6% (196/205) for colorectal cancer and a specificity of 80.3% (57/71). For morphological subtypes of advanced adenoma, the sensitivity of FIT was only 28.2% (20/71) for polypoid type and 16.1% (5/31) for nonpolypoid type, whereas the combination test increased the sensitivities to 64.8% (46/71) and 71.0% (22/31), respectively., Discussion: The combination of the fecal DNA test with FIT seemed to be useful to detect colorectal neoplasia and, especially, advanced adenoma of the nonpolypoid type.

Background: Triweekly capecitabine plus irinotecan (CAPIRI) was not a replacement for fluorouracil, leucovorin, and irinotecan (FOLFIRI) in the treatment of metastatic colorectal cancer (mCRC) because of the potential for greater toxicity. Recently, it has reported that mCAPIRI is well tolerated and non-inferior to FOLFIRI. In this study, we conducted a multicenter phase II trial to assess the efficacy and safety of biweekly CAPIRI plus bevacizumab as second-line chemotherapy for mCRC with reduced toxicity and preserved efficacy., Methods: Patients with mCRC who had received prior chemotherapy, including oxaliplatin-based regimens, were eligible for this study. The treatment protocol administered capecitabine at 1000 mg/m 2 twice daily from the evening of day 1 to the morning of day 8, intravenous irinotecan at 150 mg/m 2 on day 1, and bevacizumab at 10 mg/kg on day 1 every 2 weeks. Primary endpoints for this study were progression-free survival (PFS) and safety. Secondary endpoints were overall survival (OS), time to treatment failure, response rate (RR), and disease control rate (DCR)., Results: Fifty-one patients were enrolled in this study. Median PFS was 5.5 months [95% confidence interval (CI) 4.23-7.40 months], and median OS was 13.5 months (95% CI 11.57-20.23 months). The RR was 14.6% (95% CI 6.5-28.4%), and the DCR was 66.7% (95% CI 51.5-79.2%). Hypertension was the most common Grade 3 adverse event (27.5%), followed by neutropenia (17.6%). Only two patients suffered from grade 3 hand-foot syndrome., Conclusions: In mCRC patients, biweekly CAPIRI + bevacizumab appears effective and feasible as a second-line chemotherapy with relatively low toxicities, and has potential as a useful substitute for FOLFIRI + bevacizumab.

Background: CD3 +  and CD8 + T-cell infiltration were reported as positive predictive markers of survival in colorectal cancer (CRC) patients. Here, we demonstrate the prognostic significance of CD4 + and FOXP3 + T-cell densities in CRC., Methods: We quantified the intratumoural densities of CD3 + , CD8 + , CD4 +  and FOXP3 + T cells by immunohistochemistry and digital pathology in 342 CRC patients who underwent curative resection. Microsatellite instability was also assessed in 322 specimens. Patient demographics, clinicopathological features and survival rates were analysed., Results: High CD3 + , CD4 +  and FOXP3 + T-cell densities were associated with improved relapse-free survival (RFS); high CD8 + , CD4 +  and FOXP3 + T-cell densities were associated with improved disease-specific survival (DSS). Patients with low CD4 + and low FOXP3 + T-cell densities exhibited extremely poor prognoses. T stage, vascular/lymphatic invasion and CD4 + T-cell density were independent prognostic indicators for DSS. The distributions of CD4 + and FOXP3 + T-cell densities were not significantly different between the high microsatellite instability group and other groups, in contrast to those of CD3 + and CD8 + T-cell densities., Conclusions: Intratumoural CD4 + T-cell density and combined CD4 + and FOXP3 + T-cell densities were stronger prognostic indicators than other clinicopathological features. These results may facilitate the establishment of novel prognostic factors and therapeutic strategies for CRC.

Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI-H] CRC with CGI methylator phenotype, 28 MSI-H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The cancer stroma is important in cancer development, however, whether the aberrant expression of microRNAs (miRNAs) in the cancer stroma is associated with cancer progression remains to be fully elucidated. The aim of the present study was to identify the miRNAs associated with liver metastasis in the cancer stroma of human colorectal cancer (CRC). Using laser capture microdissection, cancer stroma was obtained from the primary lesion of six patients with CRC with liver metastasis (CRCwLM) and six patients with CRC without liver metastasis (CRCwoLM), and miRNA microarray analysis was performed. Candidate miRNA expression status in the stroma was validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis in 40 CRC cases (wLM, n=20; woLM, n=20), and the association between miRNA expression and clinicopathological factors was assessed in 101 advanced CRC samples. The localization of candidate miRNAs in CRCs was analyzed using in situ hybridization analysis (ISH). The microarray analysis identified six miRNAs with expression differing between the CRCwLM and CRCwoLM cancer stroma. Validation using RT‑qPCR analysis of the stroma showed that the expression levels of miR‑221 and miR‑222 in the cancer stroma were significantly higher in CRCwLM than in CRCwoLM. The RT‑qPCR analysis of 101 CRC samples showed that a high expression level of miR‑221 or miR‑222 in the cancer stroma was associated with liver metastasis, distant metastasis, and shorter overall survival rate of patients with CRC (P<0.05). Increased levels of miR‑221 and miR‑222 were observed in cancer cells and in fibroblasts in the stromal tissue in the ISH analysis. The results suggested that the overexpression of miR‑221 and miR‑222 in the cancer stroma is associated with the metastatic activity and malignant potential in patients with CRC.

We previously reported a phase I clinical trial of a peptide vaccine ring finger protein 43 (RNF43) and 34-kDa translocase of the outer mitochondrial membrane (TOMM34) combined with uracil-tegafur (UFT)/LV for patients with metastatic colorectal cancer (CRC), and demonstrated the safety and immunological responsiveness of this combination therapy. In this study, we evaluated vaccination-induced immune responses to clarify the survival benefit of the combination therapy as adjuvant treatment. We enrolled 44 patients initially in an HLA-masked fashion. After the disclosure of HLA, 28 patients were in the HLA-A*2402-matched and 16 were in the unmatched group. In the HLA-matched group, 14 patients had positive CTL responses specific for the RNF43 and/or TOMM34 peptides after 2 cycles of treatment and 9 had negative responses; in the HLA-unmatched group, 10 CTL responses were positive and 2 negative. In the HLA-matched group, 3-year relapse-free survival (RFS) was significantly better in the positive CTL subgroup than in the negative-response subgroup. Patients with negative vaccination-induced CTL responses showed a significant trend towards shorter RFS than those with positive responses. Moreover, in the HLA-unmatched group, the positive CTL response subgroup showed an equally good 3-year RFS as in the HLA-matched group. In conclusion, vaccination-induced CTL response to peptide vaccination could predict survival in the adjuvant setting for stage III CRC., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Cancer vaccines have been developed as a new therapeutic approach, however, their clinical benefit remains limited. We previously performed a phase II study for advanced colorectal cancer (CRC) using five human leukocyte antigen (HLA-A*24:02)-restricted peptides derived from kinase of the outer chloroplast membrane 1, translocase of outer mitochondrial membrane 34 (TOMM34), ring finger protein 43 (RNF43), vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2. In the present study the relationship between overall survival (OS) and several biomarkers, including cytotoxic T lymphocyte (CTL) and immunoglobulin G (IgG) responses to these five peptides, was investigated. In 89 advanced CRC patients treated with a combination therapy consisting of these five peptides and oxaliplatin-based chemotherapy, plasma was collected before and after 3 months of vaccine administration. IgGs reactive to each of the five peptides were assessed using the multiplex bead suspension Luminex system. Antigen-specific T-cell responses were estimated by enzyme-linked immunoSpot assay. Plasma levels of TOMM34 IgG (P<0.001), RNF43 IgG (P<0.001) and VEGFR2 IgG (P<0.001) were significantly increased after vaccination and stronger VEGFR2 IgG responses correlated significantly with OS in HLA-matched patients (P=0.034). CTL responses to VEGFR1 and VEGFR2 were also significantly increased in the HLA-matched group (P=0.049 and P<0.001, respectively). However, increased CTL response did not correlate with OS. Multivariate analysis indicated that IgG responses to VEGFR2 were the most significant predictor for OS in the HLA-A*24:02-matched group (P=0.04). Our findings indicated that VEGFR2 IgG responses may be an important immunological biomarker in the early course of treatment for CRC patients treated with therapeutic epitope peptides.

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air-liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer., Competing Interests: The authors declare no conflict of interest.

Background: Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer., Methods: We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues. Absolute copy numbers of F. nucleatum were measured by droplet digital PCR., Results: The detection rates of F. nucleatum were 63.9% (46/72) in normal-appearing mucosal tissues and 75.0% (75/100) in CRC tissue samples. The median copy number of F. nucleatum was 0.4/ng DNA in the normal-appearing colorectal mucosa in patients with colorectal cancer and 1.9/ng DNA in the colorectal cancer tissues (P = 0.0031). F. nucleatum copy numbers in stage IV colorectal cancer tissues were significantly higher than those in the normal-appearing mucosa in patients with colorectal cancer (P = 0.0016). The abundance of F. nucleatum in colorectal cancer tissues correlated with tumor size and KRAS mutation and was significantly associated with shorter overall survival times; this trend was notable in the patients with stage IV colorectal cancer. Focusing on normal-appearing mucosa in the patients with colorectal cancer, the F. nucleatum copy number was significantly higher in the patients with stage IV rather than stages I-III., Conclusion: These results suggest that determining F. nucleatum levels may help predict clinical outcomes in colorectal cancer patients. Further confirmatory studies using independent datasets are required to confirm our findings.

Background As TWIST1 methylation is specific to colorectal neoplasia, detection of TWIST1 methylation from faeces samples might be useful for colorectal neoplasia screening. However, because the content of human DNA in faeces is very small, it is very difficult to detect TWIST1 methylation by conventional bisulphite-based methylation assays. Therefore, we developed a new methylation assay without bisulphite treatment, the combined restriction digital PCR assay, and evaluated its sensitivity and specificity in combination with and without the faecal immunochemical test for haemoglobin for colorectal neoplasia detection from faeces samples. Methods For the combined restriction digital PCR assay, DNA was treated with three methylation-sensitive restriction enzymes and an exonuclease, followed by measurement of TWIST1 methylation level by droplet digital PCR. Faecal DNA testing and faecal immunochemical test for haemoglobin were performed on 109 patients with colorectal neoplasia and 10 control individuals. Results Basic performance testing showed that the combined restriction digital PCR assay enabled detection of 0.14% of the TWIST1 methylation level for the lymphocyte DNA. The combined restriction digital PCR assay from faeces samples had a sensitivity of 22.2% (95% confidence interval, 2.8-60.0%) for non-advanced adenoma, 47.1% (95% confidence interval, 23.0-72.2%) for advanced adenoma, and 33.7% (95% confidence interval, 23.7-45.0%) for colorectal cancer, and a specificity of 100.0%. Combination of faecal immunochemical test for haemoglobin and faecal combined restriction digital PCR assay increased sensitivity to 82.4% (95% confidence interval, 56.6-96.2%) for the detection of advanced adenoma. Conclusions We developed the combined restriction digital PCR assay, a possible highly sensitive methylation assay. Combination of faecal combined restriction digital PCR assay with faecal immunochemical test for haemoglobin may provide an alternative screening strategy for colorectal neoplasia, especially for potentially precancerous lesions.

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in a phase I study, in which peptide vaccines alone were given. Candidate miRNAs were selected by comparison of the better prognosis group with the poorer prognosis group. Next, we conducted microarray analysis of cancer tissues in a phase II study, in which peptide vaccines combined with chemotherapy were given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II cases, separating cancer tissues into cancer cells and stromal tissue using laser capture microdissection. Treatment effect in relation to overall survival (OS) and miRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that expression of miR-378a in stromal cells was the best among the three predictors (HR, 2.730; 95% CI, 1.027-7.585; P = 0.044). In conclusion, miR-125b-1 and miR-378a expression might be considered as novel biomarkers to predict the efficacy of vaccine treatment against colorectal cancer., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Cetuximab has activity against colorectal cancers. Recent studies demonstrated that cetuximab induces antibody-dependent cell-mediated cytotoxicity via immune cells, and a new immune-related mechanism of inducing immunogenic cell death. This study aimed to evaluate the immune responses induced by cetuximab in tumor microenvironments at liver metastasis sites of metastatic colorectal cancer patients. We assessed immune cell infiltration in the liver metastatic sites of 53 colorectal cancer patients. These patients were divided into three groups according to the treatment before operation: chemotherapy with cetuximab, chemotherapy without cetuximab, and no chemotherapy. The inflammatory cells in the liver metastatic sites were assessed by hematoxylin-eosin staining, focusing on the invasive margin. The overall inflammatory reaction and number of lymphoid cells were assessed with a four-point scoring system. We then assessed immune cell infiltration (CD3, CD8 and CD56) in 15 liver metastatic sites. Hematoxylin-eosin staining demonstrated more inflammatory cells in the chemotherapy with cetuximab group than in the other groups (P < 0.001). Of note, inflammatory cells were found in intratumoral areas, and the destruction of cancer cell foci was observed in the chemotherapy with cetuximab group. Moreover, a higher infiltration of CD3+ (P = 0.003), CD8+ (P = 0.003) and CD56+ (P = 0.001) cells was observed in the chemotherapy with cetuximab group than in the other groups. These results suggest that cetuximab might have an immune-enhancing effect. As such, the immune-related mechanism of action of cetuximab may enhance the efficacy of combination therapy, such as chemotherapy and immunotherapy using therapeutic peptides., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population. Methods Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR. Results The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group. Conclusions This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.

Various vaccine treatments against metastatic colorectal cancer have been developed and applied. However, to improve the efficacy of immunotherapy, biomarkers that can predict the effects are needed. It has been reported that various microRNAs (miRNAs) in peripheral blood may be useful as non-invasive biomarkers. In this study, miRNAs influencing the efficacy of vaccine treatment were screened for in a microarray analysis of 13 plasma samples that were obtained from patients prior to vaccine treatment. To validate the screening results, real-time RT-PCR was performed using 93 plasma samples obtained from patients prior to vaccine treatment. Four candidate miRNAs were selected according to the results of the comprehensive analysis of miRNA expression, which were ranked using the Fisher criterion and the absolute value of the log2 ratio in the screening analysis. The validation analysis showed that in the HLA-A*2402‑matched patient group (vaccine-treated group), patients with a high expression of plasma miR-6826 had a poorer prognosis than those with a low expression (P=0.048). In contrast, in the HLA-A*2402-unmatched patient group (control group), there was no difference between the patients with high or low plasma miR-6826 expression (P=0.168). Similar results were obtained in the analysis of miR-6875 (P=0.029 and P=0.754, respectively). Moreover, multivariate analysis of the Cox regression model indicated that the expression of miR-6826 was the most significant predictor for overall survival (P=0.003, hazard ratio, 3.670). In conclusion, plasma miR-6826 and miR-6875 may be predictive biomarkers for a poor response to vaccine treatment. Although further clarification is needed regarding the functions of miR-6826 and miR-6875 and their relationship to immune‑related molecules, plasma miR-6826 and miR-6875 may be useful negative biomarkers for predicting the efficacy of vaccine treatment.

We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Despite the comparable clinical benefit of XELOX (capecitabine with oxaliplatin) and FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin), the value of XELOX treatment in combination with cetuximab for metastatic colorectal cancer (mCRC) remains largely unknown., Patients and Methods: In this clinical trial we evaluated the efficacy and safety of weekly/biweekly cetuximab administration combined with biweekly XELOX in patients with previously untreated v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) wild type mCRC. The primary end point was response rate (RR) with confirmation, and the secondary end points included progression-free survival (PFS), overall survival (OS), disease control rate (DCR), dose intensity, and the safety of the protocol treatment., Results: Forty patients who fulfilled the inclusion criteria participated in this study. The median treatment cycle number was 8 and the median dose intensities were 218 mg/m 2 /wk for cetuximab, 34 mg/m 2 /wk for oxaliplatin, and 821 mg/m 2 /d for capecitabine. One patient showed complete response and partial response was observed in 19 patients, giving an overall RR of 50% (95% confidence interval [CI], 33.8%-66.2%). Stable disease was obtained in 13 patients, resulting in a DCR of 82.5% (95% CI, 67.2%-92.7%). The PFS was 6.5 months (95% CI, 3.5-9.6 months), and the OS was 24.3 months (95% CI, 14.9-33.7 months). The safety profile revealed the common Grade 3/4 adverse events to be acneiform eruption (12.5%), peripheral neuropathy (7.5%), and elevated alanine transaminase levels (7.5%). Grade 3/4 thrombocytopenia and neutropenia occurred only in 5.0% and 2.5% of the patients, respectively. Grade 1 hand-foot syndrome (HFS) was not uncommon (20%), whereas Grade 2/3 HFS occurred in only 3 patients (7.5%). No deaths were reported within 30 days of the last dose., Conclusion: Cetuximab with XELOX showed a confirmed overall RR of 50%, which was within the previously reported range of RR. The safety profile showed an acceptable rate and severity of adverse events. In light of the several advantages of XELOX, including convenience and the reported cost-saving aspects, further study of this combination therapy is warranted., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Results from several randomised trials suggest that the sequential use of cytotoxic agents in patients with metastatic colorectal cancer (mCRC) has the potential to improve overall survival compared with combination chemotherapy. This study is designed to investigate whether sequential treatment with bevacizumab-based first-line treatment with oxaliplatin is superior to combination treatment of mCRC., Methods and Analysis: The C-cubed (C(3)) study is a two-arm, multicentre, open-label, randomised phase III trial in Japan comparing the efficacy and safety of sequential capecitabine or 5-fluorouracil plus bevacizumab (Cape/5-FU-Bmab) with escalation to capecitabine or 5-fluorouracil plus oxaliplatin plus bevacizumab (CapeOX/mFOLFOX6-Bmab) versus combination CapeOX/mFOLFOX6-Bmab as the first-line treatment of mCRC. In the sequential arm (Arm A: oxaliplatin 'wait-and-go'), treatment escalation from Cape/5-FU-Bmab to CapeOX/mFOLFOX6-Bmab is recommended in the case of progressive disease. In the combination arm (Arm B: oxaliplatin 'stop-and-go'), de-escalation from CapeOX/mFOLFOX6-Bmab to Cape/5-FU-Bmab is possible after 12 weeks of treatment. Re-escalation to CapeOX/mFOLFOX6-Bmab after progressive disease is considered only for patients who received de-escalation of oxaliplatin after 12 weeks of treatment not caused by oxaliplatin-associated toxicity. A target sample size of 304 evaluable patients is considered sufficient to validate an expected HR for time to failure of strategy of the sequential approach 'wait-and-go' compared to the combination approach 'stop-and go' with 80% power and 2-sided 5% α in case of a true HR<0.69., Ethics and Dissemination: This study is conducted according to the standards of Good Clinical Practice and in compliance with the Declaration of Helsinki 2013 and local regulations, and has been submitted and approved by the Ethical Committee of the Non-Profit Organization MINS Institutional Review Board. The protocol and the trial results, even inconclusive, will be presented at international oncology congresses and published in peer-reviewed journals., Trial Registration Number: UMIN000015405, Pre-results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: The clinical benefit of cetuximab combined with oxaliplatin-based chemotherapy remains under debate. The aim of the present multicenter open-label Phase II study was to explore the efficacy and safety of biweekly administration of cetuximab and mFOLFOX-6 or XELOX as first-line chemotherapy in patients with metastatic colorectal cancer., Methods: Sixty-two patients with previously untreated KRAS/BRAF wild-type metastatic colorectal cancer were recruited to the study between April 2010 and May 2011. Patients received one of two treatment regimens, either cetuximab plus mFOLFOX-6 (FOLFOX + Cmab) or cetuximab plus biweekly XELOX (XELOX + Cmab), according to their own preference. Treatment was continued until disease progression or the appearance of intolerable toxicities. The primary endpoint was response rate; secondary endpoints were progression-free survival, overall survival, disease control rate, dose intensity, conversion rate to surgical resection, and safety., Results: The response rates in the FOLFOX + Cmab (n = 37) and XELOX + Cmab (n = 25) groups were 64.9 % (24/37) and 72.0 % (18/25), respectively. The median PFS in the FOLFOX + Cmab and XELOX + Cmab groups was 13.1 months (95 % confidence interval [CI] 12.1-17.5) and 13.4 months (95 % CI 10.1-17.9), respectively. Neutropenia was the most frequent grade 3/4 adverse event in both groups (33.9 %), followed by anorexia, acneiform eruption, skin fissure and paronychia. A waterfall plot of tumor diameter showed prominent shrinkage of the tumors in 88.7 % of patients., Conclusions: The results of the present study indicate that biweekly cetuximab plus mFOLFOX-6/XELOX is an effective and tolerable treatment regimen. Biweekly administration of cetuximab requires only one hospital visit every 2 weeks, and may become a convenient treatment option for patients with KRAS/BRAF wild-type metastatic colorectal cancer., Trial Registration: This study is registered with University Hospital Medical Information Network (UMIN 000003253 ). Registration date is 02/24/2010.

Background and Objectives: Cetuximab has shown efficacy in patients with metastatic colorectal cancer (mCRC). Recent studies have demonstrated that cetuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC), mediated via the fragment c gamma receptors(FcgR)in mCRC. Since the establishment of KRAS mutations as a major negative predictor of efficacy, additional biomarkers have been found to be useful for the improvement of selection of patients likely to be responsive to cetuximab. We investigated the relationship between polymorphisms and the outcome of mCRC patients treated with cetuximab., Methods: In this study, 57 patients were evaluated. The relationships of FcgR polymorphisms with response rate (RR), progression-free survival (PFS), and overall survival (OS) were analyzed., Results: The FcgR polymorphisms were not significantly related to RR, PFS, and OS. Compared with the other haplotypes, the haplotype containing the 131H and 158V alleles was related to a lower RR (p=0.018). The diplotypes containing 131H and 158V alleles had significantly lower RR than the other diplotypes (p=0.038)., Conclusion: Our data suggest that FcgR polymorphisms may be associated with the outcome of mCRC patients treated with cetuximab and FOLFIRI. However, these results are currently controversial, and detailed investigations are needed to confirm the relationship between FcgRs polymorphisms and cetuximab efficacy.

A therapeutic vaccine against minimal residual cancer cells is needed for the treatment of patients with colorectal cancer. Several gene therapy studies have revealed that the combination of a suicide gene and cytokine gene might induce effective antitumor immunity. In this study, we constructed an interleukin (IL)-18 and herpes simplex virus-thymidine kinase (HSV-TK) expression vector driven by the human telomerase reverse transcriptase (hTERT) promoter to study the efficacy of combination gene therapy with IL-18 and the HSV-TK suicide gene. Low immunogenic colon 26 cells were used for transfection and inoculation into syngeneic BALB/c mice. Large established tumors of colon 26 transfectants expressing IL-18 and HSV-TK driven by the hTERT promoter were completely eradicated after GCV administration in syngeneic BALB/c mice. Immunohistochemical analysis at the tumor rejection sites revealed enormous infiltrations of CD8+ T lymphocytes as well as CD4+ T lymphocytes and CD11b+ monocytes. Moreover, established distant tumors were completely eradicated by vaccination with the IL-18 and HSV-TK transfectants in combination with GCV. These data suggest that the IL-18 and suicide gene therapy can elicit antitumor specific immunity. In conclusion, gene therapy with IL-18 and HSV-TK plasmid vector driven by the hTERT promoter may be useful for cancer vaccination.

To predict precisely severe toxicity of irinotecan, we evaluated the association of UGT1A variants, haplotypes and the combination of UGT1A genotypes to severe toxicity of irinotecan. UGT1A1*6 (211G>A), UGT1A1*28 (TA6>TA7), UGT1A1*60 (-3279T>G), UGT1A7 (387T>G), UGT1A7 (622T>C), and UGT1A9*1b (-118T9>T10, also named *22) were genotyped in 123 patients with metastatic colorectal cancer who had received irinotecan-based chemotherapy. Among the 123 patients, 73 were enrolled in either of two phase II studies of the FOLFIRI (leucovorin, 5-fluorouracil and irinotecan) regimen; these patients constituted the training population, which was used to construct the predicting system. The other 50 patients constituted the validation population; these 50 patients either had participated in a phase II study of irinotecan/5'-deoxy-5-fluorouridine or were among consecutive patients who received FOLFIRI therapy. This prediction system used sequential forward floating selection based on statistical pattern recognition using UGT1A genotypes, gender and age. Several UGT1A genotypes [UGT1A1*6, UGT1A7 (387T>G), UGT1A7 (622T>C) and UGT1A9*1b] were associated with the irinotecan toxicity. Among the haplotypes, haplotype-I (UGT1A1: -3279T, TA6, 211G; UGT1A7: 387T, 622T; UGT1A9: T10) and haplotype-II (UGT1A1: -3279T, TA6, 211A; UGT1A7: 387G, 622C; UGT1A9: T9) were also associated with irinotecan toxicity. Furthermore, our new system for predicting the risk of irinotecan toxicity was 83.9% accurate with the training population and 72.1% accurate with the validation population. Our novel prediction system using statistical pattern recognition depend on genotypes in UGT1A, age and gender; moreover, it showed high predictive performance even though the treatment regimens differed among the training and validation patients.

Background and Objectives: Cetuximab shows activity in KRAS (Kirsten rat sarcoma viral oncogene homolog) wild-type metastatic colorectal cancer (mCRC). Recent studies have demonstrated that cetuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in mCRC. We investigated the associations of FcγR (fragment C γ receptor) and EGFR (epidermal growth factor receptor) polymorphisms with the outcome of mCRC patients treated with cetuximab and FOLFIRI (folic acid/5-fluorouracil/irinotecan) as second-line therapy in the FLIER (Cetuximab Plus Folinic Acid/5-Fluorouracil/Irinotecan in KRAS Wild-Type Metastatic Colorectal Cancer as a Second-Line Treatment) study., Methods: A total of 57 patients were evaluated in this study. The association of each polymorphism with the response rate, progression-free survival, and overall survival was analyzed., Results: A tendency for longer overall survival was observed in patients with the EGFR CA repeat ≥36 genotype than in those with the ≤35 genotype (600 versus 483 days, P = 0.051). The haplotype containing the 131H and 158V alleles was associated with a lower response rate than the other haplotypes (P = 0.018). These results are contrary to previously published results., Conclusion: Our data suggest that FcγR and EGFR CA repeat polymorphisms may be associated with the outcome of mCRC patients treated with cetuximab and FOLFIRI, although further investigations will be needed to confirm the association of FcγR and EGFR polymorphisms with the efficacy of cetuximab.

Background: We previously reported a phase I study of a cancer vaccine using five novel HLA-A*2402-restricted peptides, and demonstrated the safety and the promising potential of our five-peptide cocktail for advanced colorectal cancer. The objective of this analysis was to investigate predictive biomarkers for the prior selection of patients who are likely to have clinical benefit from such therapy., Patients and Methods: Seventeen patients with colorectal cancer who were treated with the five peptides underwent a complete blood count, serum chemistry tests and enzyme-linked ImmunoSpot assay before the treatment as predictive markers of high reactivity to the peptides., Results: Interleukin-6 level was a significant predictor for overall survival of patients treated with the peptide cocktail (p=0.017). A high neutrophil/lymphocyte ratio was likely to have some association with the poor induction of peptide-specific immune reaction., Conclusion: Interleukin-6 level might be a good predictive biomarker for clinical benefit of patients treated with this peptide vaccine., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: We previously conducted a phase I trial for advanced colorectal cancer (CRC) using five HLA-A*2402-restricted peptides, three derived from oncoantigens and two from vascular endothelial growth factor (VEGF) receptors, and confirmed safety and immunological responses. To evaluate clinical benefits of cancer vaccination treatment, we conducted a phase II trial using the same peptides in combination with oxaliplatin-based chemotherapy as a first-line therapy., Methods: The primary objective of the study was the response rates (RR). Progression free survival (PFS), overall survival (OS), and immunological parameters were evaluated as secondary objective. The planned sample size was more than 40 patients for both HLA2402-matched and -unmatched groups. All patients received a cocktail of five peptides (3 mg each) mixed with 1.5 ml of IFA which was subcutaneously administered weekly for the first 12 weeks followed by biweekly administration. Presence or absence of the HLA-A*2402 genotype were used for classification of patients into two groups., Results: Between February 2009 and November 2012, ninety-six chemotherapy naïve CRC patients were enrolled under the masking of their HLA-A status. Ninety-three patients received mFOLFOX6 and three received XELOX. Bevacizumab was added in five patients. RR was 62.0% and 60.9% in the HLA-A*2402-matched and -unmatched groups, respectively (p=0.910). The median OS was 20.7 months in the HLA-A*2402-matched group and 24.0 months in the unmatched group (log-rank, p=0.489). In subgroup with a neutrophil/lymphocyte ratio (NLR) of <3.0, patients in the HLA-matched group did not survive significantly longer than those in the unmatched group (log-rank, p=0.289) but showed a delayed response., Conclusions: Although no significance was observed for planned statistical efficacy endpoints, a delayed response was observed in subgroup with a NLR of <3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination., Trial Registration: Trial registration: UMIN000001791.

Background: This study was the first multicenter phase II study of cetuximab plus folinic acid/5-fluorouracil/irinotecan (FOLFIRI) in KRAS wild-type mCRC as a second-line treatment in Japan including BRAF and PIK3CA genotyping., Patients and Methods: Tumors of 112 pre-registered patients were genotyped for KRAS, BRAF, and PIK3CA. The primary study end-point was response rate, and secondary end-points were progression-free survival (PFS), overall survival (OS), and safety., Results: Sixty-seven patients (59.8%) were EGFR-positive and KRAS wild-type. The mean age of the enrolled patients (n=60) was 62.6 years (range=37-82 years). The response rate was 31.7% and stable disease was observed in 53.3%. No objective response was observed in patients with BRAF or PIK3CA mutations. The median PFS and OS were 7.4 and 18.2 months, respectively. Grade-3/4 adverse events were leucopenia (26.7%), neutropenia (43.3%), paronychia (10.0%), fissure (10.0%) and acne-like rash (5.0%)., Conclusion: Second-line cetuximab plus FOLFIRI was effective and well-tolerated.

Background: To evaluate the safety of combination vaccine treatment of multiple peptides, phase I clinical trial was conducted for patients with advanced colorectal cancer using five novel HLA-A*2402-restricted peptides, three peptides derived from oncoantigens, ring finger protein 43 (RNF43), 34 kDa-translocase of the outer mitochondrial membrane (TOMM34), and insulin-like growth factor-II mRNA binding protein 3 (KOC1), and the remaining two from angiogenesis factors, vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2., Methods: Eighteen HLA- A*2402-positive colorectal cancer patients who had failed to standard therapy were enrolled in this study. 0.5 mg, 1.0 mg or 3.0 mg each of the peptides was mixed with incomplete Freund's adjuvant and then subcutaneously injected at five separated sites once a week. We also examined possible effect of a single site injection of "the cocktail of 5 peptides" on the immunological responses. ELISPOT assay was performed before and after vaccinations in the schedule of every 4 weeks., Results: The vaccine treatment using multiple peptides was well tolerated without any severe treatment-associated systemic adverse events. Dose-dependent induction of peptide-specific cytotoxic T lymphocytes was observed. The single injection of "peptides cocktail" did not diminish the immunological responses. Regarding the clinical outcome, one patient achieved complete response and 6 patients revealed stable disease for 4 to 7 months. The median overall survival time (MST) was 13.5 months. Patients, in which we detected induction of cytotoxic T lymphocytes specific to 3 or more peptides, revealed significantly better prognosis (MST; 27.8 months) than those with poorer immune responses (MST; 3.7 months) (p = 0.032)., Conclusion: Our cancer vaccine treatment using multiple peptides is a promising approach for advanced colorectal cancer with the minimum risk of systemic adverse reactions., Clinical Trial Registration: UMIN-CTR number UMIN000004948.

Aim: This is a multicenter phase II study to assess the efficacy and toxicity of FOLFIRI treatment agents in full and the influence of UGT1A1*28 polymorphism in Japanese patients with advanced/metastatic colorectal cancer (mCRC)., Patients and Methods: Fifty patients with mCRC participated in this study. Treatment consisted of FOLFIRI (irinotecan; 150 mg/m(2)) as second-line chemotherapy; 34 patients consented to the evaluation of UGT1A1 genotype., Results: The overall response rate was 12% for all 50 evaluable patients; 31 patients (62.0%) had stable disease, and only in 12 (24.0%) did disease progress. The median progression-free survival was 5.8 months. The tolerance treatment was acceptable, with only 15 out of 50 patients (30%) experiencing grade 3/4 neutropenia, and grade 4 thrombocytopenia was observed in only one case. Grade 3 non-hematological adverse reactions included stomatitis in three, diarrhea in one, and a clinically insignificant increase in serum alkaline phosphatases in one patient, respectively. There was no definite relation between the UGT1A1*28 polymorphism and toxicity., Conclusion: Standard FOLFIRI regimen can be administered to Japanese patients. The results showed good tolerability and efficacy for second-line FOLFIRI, provided that evaluation of UGT1A1 polymorphism is properly implemented before the start of the chemotherapy.

Unlabelled: Retrospective studies have suggested that UDP-glucuronosyltransferase (UGT)1A1, UGT1A7, and UGT1A9 predict severe toxicity and efficacy of irinotecan-containing regimens. We prospectively evaluated the impact of UGT1A genotypes and haplotypes on severe toxicity and efficacy in patients treated with fluorouracil, leucovorin, and irinotecan combination chemotherapy (FOLFIRI) for metastatic colorectal cancer (mCRC) from the two prospective multicenter phase II studies in Japan. The FLIGHT1 study was a first-line FOLFIRI trial, and FLIGHT2 was a FOLFOX-refractory, second-line FOLFIRI trial. A total of 73 patients agreed to additional analysis, and were genotyped for UGT1A polymorphisms, UGT1A1*28 (TA6>TA7), UGT1A1*6 (211G>A), UGT1A1*27 (686C>A), UGT1A1*60 (-3279T>G), UGT1A1*93 (-3156G>A), UGT1A7 (-57T>G), UGT1A7*3 (387T>G, 622T>C), and UGT1A9*22 (T9>T10). Of 73 patients, 34 developed G3/4 severe hematological toxicities. The toxicities were significantly more frequent in patients with UGT1A1*6 (211A), UGT1A7 (387G), and UGT1A9*22 reference alleles (T9). Haplotype I, which consists of all favorable alleles, was associated with a significant reduction in hematologic toxicity (P = 0.031). In contrast, haplotype II, which contains four high-risk alleles, showed significantly higher hematologic toxicity than the other haplotypes (P = 0.010). Six out of seven patients who were homozygous for UGT1A1*28 or *6 experienced severe hematological toxicity despite the fact that their response rate was not impaired (42.9%). We concluded that UGT1A polymorphisms, especially UGT1A1*6, are important for the prediction of severe toxicity of FOLFIRI in northeast Asian populations. In this regard, haplotype analyses should substantially impact the prediction of severe hematological toxicities of FOLFIRI. (, Clinical Trial Registration: UMIN000002388 and UMIN000002476)., (© 2013 Japanese Cancer Association.)

Aim: We performed a phase II study of irinotecan with 5'-deoxy-5-fluorouridine (5'-DFUR) for metastatic colorectal cancer based on UDP-glucuronosyltransferase (UGT) 1A1 polymorphism., Patients and Methods: A total of 28 patients were enrolled. The dose of irinotecan was 150 mg/m(2) for patients with the *1/*1 wild-type genotype, and 70 mg/m(2) for those with the *1/*28 mutated genotype. The primary end-point was the response rate (RR); secondary end-points were safety, time to treatment failure (TTF), and overall survival (OS)., Results: In 28 patients total, genotype was wild-type in 22 and mutated in six. The RR was *1/*1 (22.7%; wild-type) vs. *1/*28 (16.7%; mutated); the median TTF was 5 months vs. 4.5 months, and the median OS was 13 months vs. 17.5 months, respectively. None of these differences were significant. Toxicities of grade 3 or higher were neutropenia (9.0% vs. 0%, respectively) and diarrhea (13.6% vs. 0%, respectively)., Conclusion: This genotype-oriented therapy was effective and safe, and thus appears useful for patients who have complications or advanced age.

Our previous report revealed that the expression of Frizzled-7 (FZD7) in colorectal cancer (CRC) and its possible role in CRC progression. In this study we measured the expression levels of candidate FZD7 ligands, Wnt3 and Wnt11 in colon cancer cell lines (n = 7) and primary CRC tissues (n = 133) by quantitative RT-PCR. We also examined the functional effects of Wnt11 with the use of Wnt11 transfectants of colon cancer HCT-116 cells. Wnt11 transfectants showed the increased proliferation and migration/invasion activities compared to mock cells. Western blot analysis of transfectants revealed that phosphorylation of JNK and c-jun was increased after Wnt11 transfection. Wnt11 mRNA expression was significantly higher in the stage I, II, III, or IV tumor tissues than in non-tumor tissues (overall P < 0.003), while there was no significant difference in Wnt3 mRNA expression between tumor and non-tumor tissues. In addition, Wnt11 mRNA expression was significantly higher in patients with recurrence or death after surgery than in those with no recurrence (disease free) after surgery (P = 0.018). We also compared the expression levels of Wnt11 mRNA with those of FZD7 mRNA in the same CRC samples. Wnt11 mRNA expression was significantly higher in patients with higher FZD7 mRNA levels than in those with lower FZD7 mRNA levels (P = 0.0005). The expression levels of Wnt11 mRNA were correlated with those of FZD7 mRNA (P < 0.0001). These data suggest that Wnt11 may play an important role in CRC progression., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Colorectal cancer (CRC) is the second most common malignancy in Japan. Treatment with inhibitors of the vascular endothelial growth factor (VEGF) signalling pathway has proven benefit in metastatic CRC. Cediranib is an oral highly potent VEGF signalling inhibitor that inhibits all three VEGF receptors., Patients and Methods: In this phase II, double-blind, placebo-controlled study, 172 patients with metastatic CRC were randomised to receive once-daily cediranib (20 or 30 mg) or placebo, each combined with modified FOLFOX6 (mFOLFOX6). The primary objective was comparison of progression-free survival (PFS)., Results: The comparison of cediranib 20 mg versus placebo met the primary objective of PFS prolongation [hazard ratio = 0.70 (95% confidence interval 0.44-1.11), P = 0.167], which met the protocol-defined criterion of P < 0.2. Median PFS was 10.2 versus 8.3 months, respectively. The PFS comparison for cediranib 30 mg versus placebo did not meet the criterion. The most common adverse events (AEs) in the cediranib-containing groups were diarrhoea and hypertension., Conclusions: Cediranib 20 mg plus mFOLFOX6 met the predefined criteria in terms of improved PFS compared with placebo plus mFOLFOX6. Cediranib 20 mg was generally well tolerated and the AE profile was consistent with previous studies.

Background: Regulatory T-cells (Tregs) actively engage in the maintenance of immunological self-tolerance and immune homeostasis. The purpose of the present study was to determine how oxaliplatin plus infusional 5-fluorouracil and leucovorin (FOLFOX) and irinotecan plus infusional 5-fluorouracil and leucovorin (FOLFIRI) affect Tregs and other immune effectors., Patients and Methods: A total of 27 patients with metastatic colorectal cancer received the FOLFOX (n=17) or FOLFIRI (n=10) chemotherapeutic regimen. Blood samples were collected from patients before and 7 days after chemotherapy. The prevalence of Tregs co-expressing CD4(+)FoxP3(+) was analyzed with flow cytometry., Results: The percentage and the number of CD4(+)FoxP3(+) Tregs were significantly reduced after FOLFOX and FOLFIRI in the patients who had high levels of Tregs before chemotherapy. On the other hand, the total number of lymphocytes and the population of CD4(+) T lymphocytes were unchanged., Conclusion: FOLFOX and FOLFIRI may enhance antitumor immunity via suppression of Tregs.

Objectives: This is the first phase II study to evaluate the efficacy and tolerability of the first-line FOLFIRI, as well as the influence of uridine diphosphate glucuronosyl transferase 1, family polypeptide A1 gene (UGT1A1) 28/6 polymorphism, in Japanese metastatic colorectal cancer patients., Methods: Fifty-two patients were enrolled in this study and were administrated FOLFIRI (irinotecan; 150 mg/m(2)) as first-line chemotherapy. Thirty-nine patients accepted the evaluation of UGT1A1 genotypes. In patients with UGT1A1 28 homozygosity, the starting dose was reduced (100 mg/m(2)) according to the Food and Drug Administration recommendation and our previous phase I study., Results: After a median follow-up period of 22 months, complete response was achieved in 1.9%, partial response in 38.5 %, stable disease in 51.9% and progressive disease in 3.9%. The overall response rate was 40.4%, the disease control rate was 92.3% and the median overall survival time was 22.3 months. The major toxicity was grade 3-4 neutropenia in 44.2%. There was no definite relation between UGT1A1 28, 6 polymorphisms and toxicity. However, homozygosity for UGT1A1 28 or UGT1A1 6 and double heterozygosity for both UGT1A1 28 and UGT1A1 6 were significantly associated with severe neutropenia in metastatic colorectal cancer patients (P< 0.001)., Conclusions: FOLFIRI is effective and tolerable for Japanese metastatic colorectal cancer patients. Homozygosity for UGT1A1 28 or 6 and heterozygosity for both UGT1A1 28 and 6 are associated with severe neutropenia.

Objective: We evaluated DNA amplificability to achieve a 100% success rate in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissue samples obtained from a recent clinical trial., Methods: We evaluated the effects of deparaffinization, formalin fixation or storage time, and amplicon size on the amplificability of DNAs extracted from 19 formalin-fixed and paraffin-embedded colorectal cancer tissue samples. We subjected to KRAS mutation analysis 112 samples from metastatic colorectal cancer patients in 31 hospitals enrolled in a Phase II trial of a second-line FOLFIRI (5-fluorouracil+ leucovorin + irinotecan) + cetuximab regimen., Results: Deparaffinization, formalin fixation and storage times did not appear to affect the recovery and amplificability of DNAs. However, amplicon size had a remarkable effect on the amplificability of DNAs. The smaller fragments with a size of ≤278 bp (96-278 bp) were successfully amplified with polymerase chain reaction in all samples tested, whereas the larger fragments with a size of ≥298 bp (298-565 bp) were not amplified. All samples from our clinical trial were successfully analyzed using three sets of primers with the amplicon sizes of 201, 221 and 240 bp, and KRAS mutations in exons 2 and 3 were detected in 49 of the 112 cases (43.8%)., Conclusions: These data suggest that the evaluation of DNA amplificability and amplicon size is important for the success of mutation detection tests such as the KRAS test with dideoxy sequencing using formalin-fixed and paraffin-embedded tissue samples in the clinical setting.

It is very important for immunotherapy to release Th2-dominated and Treg-dominated immunological conditions in patients with malignant diseases. In the present study, we assessed the population of CD4+IL-10+T-cells and CD4+Foxp3+T-cells in peripheral blood in patients with colorectal cancer using a flow cytometric analysis, and we investigated whether Th2-dominated and Treg-dominated condition could be modulated by PSK. Peripheral blood samples were collected preoperatively from 40 patients with colorectal cancer before and after oral administration of PSK (3 g/day × 1 week). After PSK treatment, CD4+IL-10+T-cell percentages decreased in 63% of patients and CD4+Foxp3+T-cells percentages decreased in 63% of patients. However, no correlation was found between the ratio of CD4+IL-10+T-cell percentages and that of CD4+Foxp3+T-cell percentages after/before PSK treatment. These results suggest that PSK could release Th2-dominated and Treg-dominated immunological condition in patients with colorectal cancer. Further examinations are needed to investigate the after/before percentage ratio of CD4+Foxp3+T-cells can be useful predicting parameters for the selection of responders of PSK.