Hisako Nozawa

Molecular differences between sporadic serrated and conventional colorectal adenomas.

Purpose: The purpose is to compare the molecular characteristics of serrated adenomas (SAs) with those of conventional adenomas (CADs) and hyperplastic polyps (HPs). Experimental Design: We evaluated the proliferative activity and molecular alterations in 47 SAs (25 pure-type and 22 mixed-type), 71 CADs, and 23 HPs. Results: The proliferative activity of SAs, as evaluated by Ki-67 expression, was intermediate between CADs and HPs. There was no significant difference in the incidence of KRAS or p53 mutations between the three histological groups. In the microsatellite instability (MSI) analysis, 21% of SAs (9 of 43) showed MSI at two or more loci (MSI-H); corresponding values were 5% of CADs (3 of 64) and 8% of HPs (1 of 13; SAs versus CADs, P = 0.0125). MSI-H was more likely to be found in pure-type SAs (36%; 8 of 22) than in mixed-type SAs (5%; 1 of 21; P = 0.0212). Loss of hMLH-1 expression was found in 8 of 9 SAs with MSI-H. The incidence of BRAF or KRAS mutations was 36 and 15% of SAs, respectively; the combined incidence of BRAF and KRAS mutations occurred in 49% of SAs. However, there was no significant difference in the incidence of BRAF or KRAS mutations between SAs with and without MSI-H. Conclusions: Genetic instability is more frequently implicated in the tumorigenesis of SAs, especially pure-type SAs, than in that of CADs. In contrast, activation of the Ras/Raf/MEK/MAP kinase cascade by BRAF or KRAS mutation, independently of the genetic instability, may be associated with the progression of about half of SAs.

Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C.

Infection with hepatitis C virus (HCV) is associated with lymphoproliferative disorders, represented by essential mixed cryoglobulinemia and B‐cell non‐Hodgkins lymphoma, but the pathogenic mechanism remains obscure. HCV may infect B cells or interact with their cell surface receptors, and induce lymphoproliferation. The influence of HCV infection of B cells on the development of lymphoproliferative disorders was evaluated in 75 patients with persistent HCV infection. HCV infection was more prevalent (63% vs. 16%, 14%, or 17% P < 0.05 for each), and HCV RNA levels were higher (3.35 ± 3.85 vs. 1.75 ± 2.52, 2.15 ± 2.94 or 2.10 ± 2.90 log copies/100 ng, P < 0.01 for each) in B cells than CD4+, CD8+ T cells or other cells. Negative‐strand HCV RNA, as a marker of viral replication, was detected in B cells from four of the 75 (5%) patients. Markers for lymphoproliferative disorders were more frequent in the 50 patients with chronic hepatitis C than the 32 with chronic hepatitis B, including cryoglobulinemia (26% vs. 0%, P < 0.001), low CH50 levels (48% vs. 3%, P = 0.012), and the clonality of B cells (12% vs. 0%, P < 0.01). By multivariate analysis, HCV RNA in B cells was an independent factor associated with the presence of at least one marker for lymphoproliferation (odds ratio: 1.98 [95% confidence interval: 1.36–7.24], P = 0.027). Based on the results obtained, the infection of B cells with HCV would play an important role in the development of lymphoproliferative disorders. J. Med. Virol. 81:619–627, 2009

Genetic polymorphism in cyclooxygenase‐2 promoter affects hepatic inflammation and fibrosis in patients with chronic hepatitis C

Summary.  Cyclooxygenase (COX)‐2 is involved in inflammation, anti‐apoptosis and carcinogenesis. The ‐1195GG genotype of single nucleotide polymorphism (SNP) in COX‐2 promoter was associated with low platelet counts in patients with chronic hepatitis C. Polymorphism of patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) gene (rs738409 C>G) have been reported to be associated with cirrhosis, and the major genotype of SNPs near interleukin (IL)28B are related to viral clearance. The present study was designed to assess the contribution of these SNPs to disease progression in patients with chronic hepatitis C. The study enrolled 220 Japanese patients with chronic hepatitis C. Three SNPs, ‐1195 COX‐2, PNPLA3 and IL28B (rs8099917), were genotyped in order to analyze their association with hepatic fibrosis and inflammation. The ‐1195GG genotype in COX‐2 was associated with advanced fibrosis and higher levels of inflammation in the liver tissues. The major genotype of IL28B was also associated with advanced fibrosis, but the polymorphism of PNPLA3 was neither associated with fibrosis nor inflammation. Multivariate analysis showed that ‐1195GG in COX‐2 is an independent factor associated with advanced fibrosis, while the major genotype of IL28B and HCV genotype 2 were other independent factors. In conclusion, the ‐1195GG genotype in COX‐2 is a genetic marker for liver disease progression, while the PNPLA3 genotypes are not associated with disease progression in Japanese patients with chronic hepatitis C.

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.

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.

Lymphotropic hepatitis C virus has an interferon-resistant phenotype

Summary.  Hepatitis C virus (HCV) infects and associates with B cells, leading to abnormal B‐cell activation and development of lymphoproliferative and autoimmune disorders. This immune perturbation may in turn be associated with the resistance of HCV against the host immune system. The objective of this study was to analyse the effects of HCV infection of B cells on the efficacy of interferon (IFN)‐based therapy. The study enrolled 102 patients with chronic hepatitis C who were treated with pegylated IFN plus ribavirin. HCV RNA titres in B cells were compared in patients with rapid viral responder (RVR) vs non‐RVR, sustained viral responder (SVR) vs non‐SVR and null viral responder (NVR) vs VR. The levels of HCV RNA in B cells were significantly higher in non‐RVR, non‐SVR and NVR groups. Association between the therapy outcome and the positive B‐cell HCV RNA was also investigated in relation to other known viral and host factors. Multivariable analyses showed that the positive B‐cell HCV RNA and the minor single‐nucleotide polymorphism near the IL28B gene (rs8099917) were independent factors associated with NVR in patients infected with HCV genotype 1. When these two factors were combined, the sensitivity, specificity, positive and negative predictive values for NVR were 92.3%, 98.2%, 92.3% and 98.2%, respectively. Genotype 1 and the presence of one or no mutations in the IFN‐sensitivity determining region were associated with higher levels of B‐cell HCV RNA. B‐cell‐tropic HCV appears to have an IFN‐resistant phenotype. B‐cell HCV RNA positivity is a predictive factor for resistance to IFN‐based therapy.

Magnitude of CD8+ T‐cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)‐specific CD8+ T‐cell responses and the clinical course of acute HCV infection.

Reactivation of epstein–barr virus in B cells of patients with chronic hepatitis C†

Hepatitis C virus (HCV) infection is associated with lymphoproliferative disorders. HCV infection of B cells is a predictive factor for lymphoproliferative disorders in patients with chronic hepatitis C, although its molecular mechanisms remain unknown. Epstein–Barr virus (EBV) is a B cell‐tropic virus with the potential to cause lymphoproliferative disorders, and its reactivation is induced by several viruses and cytokines. The possibility that HCV infection triggers reactivation of EBV and induces lymphoproliferative disorders were investigated. Expression of EBV mRNAs was analyzed by RT‐PCR in patients infected with HCV and control subjects, and correlations between reactivation of EBV and markers for lymphoproliferative disorders were investigated. BZLF1 mRNA, a starter molecule of reactivation, was detected in peripheral blood mononuclear cells from 12 of 52 (23%), patients infected with HCV and the frequency was higher than in healthy subjects [3 of 43 (9%), P = 0.032]. But the presence of the BZLF1 mRNA was not associated with an abnormality of markers for lymphoproliferative disorders. This study on BZLF1 mRNA expression among lymphoid cell subsets showed that reactivation of EBV was observed specifically in B cells. The BZLF1 mRNA disappeared following anti‐viral therapy and remained negative after eradication of HCV in patients with a sustained viral response, while the EBER1 RNA, a marker for persistence of EBV, was detected throughout the therapy. Infection with HCV induces reactivation of EBV in B cells, but this reactivation was not associated directly with lymphoproliferative disorders triggered by HCV. J. Med. Virol. 82:2064–2072, 2010.

Abstract 5278: Mutation in protein tyrosine phosphatase receptor type K (PTPRK) enhances progression of colon cancer through WNT signaling

Recent comprehensive genome analyses revealed the accumulation of genetic alterations in colorectal cancers (CRCs). However, it is still unknown which genetic alterations are closely associated with certain phenotype of CRCs, such as tumors with highly invasive character regardless of its small size. Here we examined the small invasive CRCs using high-throughput sequencing in order to clarify the common molecular features in those CRCs. Seven small invasive CRCs (types 0-IIa+IIc or 0-Is+IIc, less than 20mm in diameter and with submucosal massive invasion) were examined by exon sequencing. Seven thousands six variants (SNVs and indels) were detected. Among them, PTPRK was frequently mutated (3 cases, 43%; 1 nonsense and 2 missense mutations). We further examined public database (TCGA and COSMIC) and found that 50 of 617 (8.17%) CRCs contain PTPRK mutation. Those cases are frequent in proximal colon, extensively methylated (CIMP-H), microsatellite instability-high (MSI-H) and pathologically mucinous type. Consistent with clinical behavior, invasion assay in CRC cell lines revealed that CRCs with PTPRK domain D1 mutation showed high invasiveness via WNT signaling activity. These data indicate that PTPRK domain D1 mutation contributes to the invasiveness and progression of proximal CRCs, especially CIMP-H and MSI-H cases. Citation Format: Masayuki Tojo, Kazuo Konishi, Keiko Shinjo, Fumiharu Ohka, Keisuke Katushima, Akira Hatanaka, Norihisa Ichimura, Hisako Nozawa, Tomoe Shimazaki, Hitoshi Yoshida, Yutaka Kondo. Mutation in protein tyrosine phosphatase receptor type K (PTPRK) enhances progression of colon cancer through WNT signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2015-5278