Baseline soluble MICA levels act as a predictive biomarker for the efficacy of regorafenib treatment in colorectal cancer.

BACKGROUND: To evaluate the effect of regorafenib on soluble MHC class I polypeptide-related sequence A (MICA) (sMICA) level in vitro. In addition, we clinically examined whether its plasma levels were associated with regorafenib activity in terms of progression-free survival (PFS) in patients with CRC. METHODS: Human CRC cell line HCT116 and HT29 cells were treated with regorafenib and its pharmacologically active metabolites, M2 or M5 at the same concentrations as those in sera of patients. We also examined the sMICA levels and the area under the plasma concentration-time curve of regorafenib, M2 and M5. RESULTS: Regorafenib, M2, and M5 significantly suppressed shedding of MICA in human CRC cells without toxicity. This resulted in the reduced production of sMICA. In the clinical examination, patients with CRC who showed long median PFS (3.7 months) had significantly lower sMICA levels than those with shorter median PFS (1.2 months) (p = 0.045). CONCLUSIONS: MICA is an attractive agent for manipulating the immunological control of CRC and baseline sMICA levels could be a predictive biomarker for the efficacy of regorafenib treatment.

Retinoids Decrease Soluble MICA Concentration by Inhibiting the Enzymatic Activity of ADAM9 and ADAM10.

BACKGROUND/AIM: The association between MHC class I polypeptide-related sequence A (MICA) and hepatocellular carcinoma (HCC) development was identified in our previous genome-wide association study. Decreasing soluble MICA (sMICA) through MICA sheddases suppression facilitates natural killer (NK) cell-mediated cytotoxicity. The expression of ADAM9 in HCC has been correlated with poor prognosis, and our recent study showed that its suppression contributes to cancer elimination by decreasing sMICA. MATERIALS AND METHODS: Human HCC cell line PLC/PRF/5 and HepG2 cells were used. sMICA levels were measured by ELISA. Expression of retinoid X receptors (RXRs) and retinoic acid receptors (RARs) was knocked down by siRNA. RESULTS: In our screening of FDA-approved drugs in vitro, retinoids were found to be efficient ADAM9 and ADAM10 inhibitors. Treatment with retinoids reduced sMICA levels in human HCC cells. Interestingly, the effects were abrogated by depletion of the retinoid receptor RXRα. CONCLUSION: Retinoids can be potential novel agents for HCC treatment.

Leukotriene receptor antagonists enhance HCC treatment efficacy by inhibiting ADAMs and suppressing MICA shedding.

In our previous genome-wide association study, we demonstrated the association between MHC class I-related chain A (MICA) and hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C. Increasing membrane-bound MICA (mMICA) in cancer cells by reducing MICA sheddases facilitates natural killer (NK) cell-mediated cytotoxicity. Our recent study clarified that A disintegrin and metalloproteases (ADAM), including ADAM9, are MICA sheddases in HCC, and that the suppression of ADAMs increases mMICA, demonstrating the rationality of mMICA-NK targeted therapy. Furthermore, we showed that regorafenib suppresses ADAM9 transcriptionally and translationally. A library of FDA-approved drugs was screened for more efficient inhibitors of ADAM9. Flow cytometry evaluation of the expression of mMICA after treatment with various candidate drugs identified leukotriene receptor antagonists as potential ADAM9 inhibitors. Furthermore, leukotriene receptor antagonists alone or in combination with regorafenib upregulated mMICA, which was in turn downregulated by leukotriene C4 and D4 via ADAM9 function. Our study demonstrates that leukotriene receptor antagonists could be developed as novel drugs for immunological control and suppression of ADAM9 in HCC. Further, leukotriene receptor antagonists should be explored as combination therapy partners with conventional multi-kinase inhibitors for developing therapeutic strategies with enhanced efficacies for HCC management and treatment.

Enhanced B-cell differentiation driven by advanced cirrhosis resulting in hyperglobulinemia.

BACKGROUND AND AIM: The mechanism underlying hyperglobulinemia in cirrhosis, a long appreciated phenomenon, has never been clearly understood. The aim of this study is to investigate the basis for changes in humoral immunity observed in cirrhosis. METHODS: We retrospectively reviewed our medical record to analyze serum immunoglobulin (Ig) levels in patients with liver disease. We also prospectively analyzed peripheral blood mononuclear cells and sera from liver disease patients. Peripheral blood mononuclear cell surface marker expressions were measured by flow cytometry and serum B-cell-activating factor was measured by enzyme-linked immunosorbent assay. Expression of specific gene expression in magnetically separated B cells was also analyzed by real-time polymerase chain reaction. RESULTS: In retrospective analysis, we found that advancing cirrhosis, irrespective of underlying etiology or hepatocellular carcinoma, resulted in progressively increasing levels of serum IgG and IgA. In prospective analysis using clinical samples, we demonstrated that advancing cirrhosis stage was associated with increased toll-like-receptor (TLR)9 expression in CD27+ B cell and serum B-cell-activating factor levels but decreased CD27+ memory B-cell frequency. The remaining CD27+ B cells in peripheral blood exhibited increased activation-induced cytidine deaminase mRNA expression. Finally, we also demonstrated isolated B cells from advanced cirrhosis were more reactive to TLR9 stimulation that drove antibody secreting cells differentiation leading to hyperimmunoglobulinemia in vitro. CONCLUSIONS: Enhanced TLR9-induced differentiation into antibody secreting cell may explain peripheral reductions of circulating CD27+ memory B cells as well as increased serum Ig levels in cirrhosis.

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer.

Distinct molecular features of different macroscopic subtypes of colorectal neoplasms.

BACKGROUND: Colorectal adenoma develops into cancer with the accumulation of genetic and epigenetic changes. We studied the underlying molecular and clinicopathological features to better understand the heterogeneity of colorectal neoplasms (CRNs). METHODS: We evaluated both genetic (mutations of KRAS, BRAF, TP53, and PIK3CA, and microsatellite instability [MSI]) and epigenetic (methylation status of nine genes or sequences, including the CpG island methylator phenotype [CIMP] markers) alterations in 158 CRNs including 56 polypoid neoplasms (PNs), 25 granular type laterally spreading tumors (LST-Gs), 48 non-granular type LSTs (LST-NGs), 19 depressed neoplasms (DNs) and 10 small flat-elevated neoplasms (S-FNs) on the basis of macroscopic appearance. RESULTS: S-FNs showed few molecular changes except SFRP1 methylation. Significant differences in the frequency of KRAS mutations were observed among subtypes (68% for LST-Gs, 36% for PNs, 16% for DNs and 6% for LST-NGs) (P<0.001). By contrast, the frequency of TP53 mutation was higher in DNs than PNs or LST-Gs (32% vs. 5% or 0%, respectively) (P<0.007). We also observed significant differences in the frequency of CIMP between LST-Gs and LST-NGs or PNs (32% vs. 6% or 5%, respectively) (P<0.005). Moreover, the methylation level of LINE-1 was significantly lower in DNs or LST-Gs than in PNs (58.3% or 60.5% vs. 63.2%, P<0.05). PIK3CA mutations were detected only in LSTs. Finally, multivariate analyses showed that macroscopic morphologies were significantly associated with an increased risk of molecular changes (PN or LST-G for KRAS mutation, odds ratio [OR] 9.11; LST-NG or DN for TP53 mutation, OR 5.30; LST-G for PIK3CA mutation, OR 26.53; LST-G or DN for LINE-1 hypomethylation, OR 3.41). CONCLUSION: We demonstrated that CRNs could be classified into five macroscopic subtypes according to clinicopathological and molecular differences, suggesting that different mechanisms are involved in the pathogenesis of colorectal tumorigenesis.

Molecular features of colorectal polyps presenting Kudo's type II mucosal crypt pattern: are they based on the same mechanism of tumorigenesis?

BACKGROUND AND STUDY AIMS: The molecular features of serrated polyps (SPs) with hyperplastic crypt pattern, also called Kudo's type II observed by chromoendoscopy, were evaluated. METHODS: The clinicopathological and molecular features of 114 SPs with a hyperplastic pit pattern detected under chromoendoscopy (five dysplastic SPs, 63 sessile serrated adenoma/polyps (SSA/Ps), 36 microvesicular hyperplastic polyps (MVHPs), and 10 goblet cell-rich hyperplastic polyps (GCHPs)) were examined. The frequency of KRAS and BRAF mutations and CpG island methylator phenotype (CIMP) were investigated. RESULTS: Dysplastic SPs and SSA/Ps were frequently located in the proximal colon compared to others (SSA/Ps vs. MVHPs or GCHPs, P < 0.0001). No significant difference was found in the frequency of BRAF mutation among SPs apart from GCHP (60 % for dysplastic SPs, 44 % for SSA/Ps, 47 % for MVHPs, and 0 % for GCHPs). The frequency of CIMP was higher in dysplastic SPs or SSA/Ps than in MVHPs or GCHPs (60 % for dysplastic SPs, 56 % for SSA/Ps, 32 % for MVHPs, and 10 % for GCHPs) (SSA/Ps vs. GCHP, P = 0.0068). When serrated neoplasias (SNs) and MVHPs were classified into proximal and distal lesions, the frequency of CIMP was significantly higher in the proximal compared to the distal SNs (64 % vs. 11 %, P = 0.0032). Finally, multivariate analysis showed that proximal location and BRAF mutation were significantly associated with an increased risk of CIMP. CONCLUSIONS: Distinct molecular features were observed between proximal and distal SPs with hyperplastic crypt pattern. Proximal MVHPs may develop more frequently through SSA/Ps to CIMP cancers than distal MVHPs.

Clinicopathological and molecular features of colorectal serrated neoplasias with different mucosal crypt patterns.

OBJECTIVES: Endoscopic examination shows that serrated neoplasias (SNs), such as serrated adenomas and sessile serrated adenomas, exhibit different mucosal crypt patterns. However, it remains unclear whether advanced serrated polyps with different mucosal crypt patterns have different clinicopathological or molecular features. METHODS: We classified the mucosal crypt patterns of 86 SNs into three types (hyperplastic, adenomatous, and mixed pattern) and evaluated their clinicopathological and molecular features. RESULTS: We found significant differences in the proliferative activity status between SNs with mixed/adenomatous patterns and those with the hyperplastic patterns. SNs with the hyperplastic pattern were frequently located in the proximal colon and had a macroscopically superficial appearance, whereas SNs with the adenomatous pattern were often located in the distal colon and had a protruding appearance. Furthermore, a significant difference was observed in the frequency of the CpG island methylator phenotype (CIMP), involving the methylation of two or more CIMP-related genes (MINT1, MINT2, MINT31, p16, and MLH1), between SNs with the hyperplastic pattern and those with the mixed/adenomatous patterns (18/32 (56%) vs. 8/28 (29%) or 7/26 (27%); P=0.0309 or P=0.0249, respectively). Moreover, the prevalence of KRAS mutations was significantly higher in SNs with the adenomatous pattern than in those with the hyperplastic pattern (7/26 (27%) vs. 1/32 (3%); P=0.0173). In comparison with other patterns, the mixed pattern was detected more frequently in mixed serrated polyps (MSPs), which contain separate histological components. Some MSPs exhibited concordant molecular alterations among the different histological components. CONCLUSIONS: The clinicopathological and molecular features of SNs correlated strongly with their mucosal crypt patterns, which were observed using chromoendoscopy.

Primary micropapillary carcinoma of the colon: a case report and literature review.

We report a case of micropapillary carcinoma (MPC) of the transverse colon. A 56-year-old woman was admitted to our hospital with hematochezia. A lower gastrointestinal examination revealed an irregular ulcerative tumor of approximately 60 mm diameter with marginal elevation in the transverse colon. Abdominal computed tomography showed multiple swollen lymph nodes. A histological examination of the resected specimen revealed that cancer cells had invaded the subserosa. Microscopically, small papillary cells proliferated with lacuna spaces and the cribriform glandular configuration was observed. Immunohistochemically, the basal surface of the neoplastic cell clusters was diffusely positive for MUC1. No primary tumor was observed except for the colon. Therefore, this tumor was diagnosed as a primary MPC of the colon. Since a colorectal MPC was first reported in 2005, seven case reports and three pathological reviews have been presented in the English literature. MPC has an aggressive behavior with a high incidence of lymphovascular invasion and nodal metastases. We should take intensive chemotherapy for colorectal MPC into account, even if surgical resection is curative.