Akimasa Hayashi

Trans-ancestry mutational landscape of hepatocellular carcinoma genomes

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide–binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.

Distinct Clinicopathologic and Genetic Features of 2 Histologic Subtypes of Intrahepatic Cholangiocarcinoma

Previous studies have identified 2 clinically significant morphologic subtypes of intrahepatic cholangiocarcinoma (ICC) on the basis of anatomic location and/or histologic appearances. Recognizing that these classification schemes are not always applicable practically, this study aimed to establish a novel classification system based on mucin productivity and immunophenotype and to determine the rationale of this classification by examining the clinicopathologic and genetic characteristics of the 2 subtypes defined by this method. We retrospectively investigated 102 consecutive ICC cases and classified them on the basis of mucin productivity and immunophenotype (S100P, N-cadherin, and NCAM). We found that 42 and 56 cases were classified as type 1 and type 2 ICCs, respectively, and only 4 cases were of indeterminate type. Type 1 ICC, generally characterized by mucin production and diffuse immunoreactivity to S100P, arose less frequently in chronic liver diseases and showed higher levels of serum CEA and CA 19-9 than did type 2 ICC, which generally showed little mucin production and exhibited immunoreactivity to N-cadherin and/or NCAM. Type 1 ICC was characterized by several pathologic features, including higher frequencies of perineural invasion and lymph node metastasis. Although the log-rank test demonstrated that type 1 ICC had significantly worse survival, the multivariate Cox regression analysis showed no prognostic significance of this histologic subtype. Genetic analyses revealed that KRAS mutation was significantly more frequent in type 1 ICC, whereas IDH mutation and FGFR2 translocation were restricted to type 2 ICC. In conclusion, the present classification of ICC based on mucin productivity and immunophenotype identified 2 subtypes with clinicopathologic significance.

Concurrent Activation of Acetylation and Tri-Methylation of H3K27 in a Subset of Hepatocellular Carcinoma with Aggressive Behavior

Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0–300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0–3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both P<0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (P<0.01) and p53-positive staining (P<0.05), and poor prognosis (P<0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.

Intrahepatic cholangiocarcinoma frequently shows loss of BAP1 and PBRM1 expression, and demonstrates specific clinicopathologic and genetic characteristics with BAP1 loss

BAP1 and PBRM1 expression loss has been observed in multiple cancers, including intrahepatic cholangiocarcinoma (ICC). We investigated BAP1 and PBRM1 expression in ICC using immunohistochemistry, and analysed its association with clinicopathological and genetic features, including two histological subtypes.

Fatal amebic colitis after high-dose dexamethasone therapy for newly diagnosed multiple myeloma

Dear Editor, High-dose dexamethasone therapy is widely used as induction therapy for multiple myeloma; infection is one of the major complications associated with this therapy. Amebiasis, which is caused by the protozoan Entamoeba histolytica, is a common infection with a worldwide morbidity rate of 1% [1]. Amebic colitis, the most wellknown manifestation of amebiasis, can deteriorate into the fulminant form in immunocompromised patients with a mortality rate of >40% [2, 3]. Here, we describe a case of fatal amebic colitis with concurrent cytomegalovirus (CMV) infection that developed after a single course of high-dose dexamethasone therapy for newly diagnosed multiple myeloma. A 60-year-old Japanese man was referred to our hospital because of hyperproteinemia. He had no other symptoms such as abdominal pain or diarrhea. Serum electrophoresis detected monoclonal IgA lambda protein, and bone marrow examination revealed extensive infiltration of plasma cells. We therefore diagnosed multiple myeloma, Durie–Salmon stage IIIA, and International Staging System stage II. He received high-dose dexamethasone therapy (40 mg/day, days 1–4, 9–12, and 17– 20). Three weeks later, he was hospitalized for severe bloody diarrhea, dehydration, and high fever. Stool and blood cultures yielded negative results for pathogenic bacteria. Colonoscopy revealed multiple erosions, and biopsy examination of these lesions showed vegetative forms of E. histolytica. Some CMV-positive cells were also detected in the colonic mucosa (Fig. 1). Repeated stool examinations revealed the presence of vegetative forms of E. histolytica, and he was diagnosed with amebic colitis. He reported a history of sexual activity with prostitutes when he had traveled to amebiasis-endemic areas 6 months before, but denied having sex with men. After initial improvement with oral metronidazole therapy, his diarrhea deteriorated. A CMV antigenemia assay revealed elevated levels, and colonoscopy revealed exacerbated hemorrhagic erosive colitis with multiple ulcers; these findings were consistent with CMV colitis. Despite treatment with ganciclovir, the gastrointestinal hemorrhage was highly progressive, and he died 40 days after the diagnosis of amebic colitis. An autopsy revealed hemorrhagic enterocolitis with mucosal exfoliation throughout the colon. On immunohistochemical analysis, neither vegetative forms of E. histolytica nor immunoreactive CMV cells were observed. Multiple myeloma is often complicated by infectious diseases, but fulminant amebic colitis has not been reported as a complication of therapy for myeloma. Amebic colitis is a common disease, and approximately 90% of patients are asymptomatic [4]. Several studies have reported that steroid C. I. Kobayashi :G. Yamamoto :Y. Imai :M. Kurokawa (*) Department of Hematology & Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Clinicopathologic Characteristics of Hepatocellular Carcinoma With Reactive Ductule-like Components, a Subset of Liver Cancer Currently Classified as Combined Hepatocellular-Cholangiocarcinoma With Stem-Cell Features, Typical Subtype.

The aim of this study was to elucidate the clinicopathologic characteristics of hepatocellular carcinoma with reactive ductule-like components (HCC-RD), corresponding to combined hepatocellular-cholangiocarcinoma (CHC) with stem cell features, typical subtype. Retrospective clinicopathologic analysis was performed on HCCs surgically treated at the University of Tokyo Hospital between 1995 and 2013. RD components were defined as neoplastic ductular structures composed of small “stem/progenitor-like” cells. There were 46 HCC-RDs, comprising about 3% of all HCCs. Thirty-eight cases of CHC, classical type (classical CHC), were identified during the study period. When compared with conventional HCC, HCC-RD was characterized by younger patient age (P=0.016), higher frequency of female patients (P<0.001), and higher serum &agr;-fetoprotein levels (P=0.005). Serum carbohydrate antigen 19-9 elevation was also more frequently observed in HCC-RD than in conventional HCC (P=0.002). Histologically, clear cell constituents and interstitial fibrosis were more frequent in HCC-RD than in conventional HCC (P=0.003 and <0.001, respectively). When compared with HCC-RD and conventional HCC, classical CHC was characterized by a poorly differentiated HCC component, frequent vascular invasion, and lymph node metastasis (P<0.05). There was little prognostic difference between HCC-RD and conventional HCC, whereas overall and disease-free survival in classical CHC was significantly worse than in conventional HCC. In conclusion, although HCC-RDs do have some unique clinicopathologic characteristics, they have no prognostic significance, and it is not reasonable to include these tumors in the CHC category.

CPT2 downregulation adapts HCC to lipid-rich environment and promotes carcinogenesis via acylcarnitine accumulation in obesity

Objective Metabolic reprogramming of tumour cells that allows for adaptation to their local environment is a hallmark of cancer. Interestingly, obesity-driven and non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) mouse models commonly exhibit strong steatosis in tumour cells as seen in human steatohepatitic HCC (SH-HCC), which may reflect a characteristic metabolic alteration. Design Non-tumour and HCC tissues obtained from diethylnitrosamine-injected mice fed either a normal or a high-fat diet (HFD) were subjected to comprehensive metabolome analysis, and the significance of obesity-mediated metabolic alteration in hepatocarcinogenesis was evaluated. Results The extensive accumulation of acylcarnitine species was seen in HCC tissues and in the serum of HFD-fed mice. A similar increase was found in the serum of patients with NASH-HCC. The accumulation of acylcarnitine could be attributed to the downregulation of carnitine palmitoyltransferase 2 (CPT2), which was also seen in human SH-HCC. CPT2 downregulation induced the suppression of fatty acid β-oxidation, which would account for the steatotic changes in HCC. CPT2 knockdown in HCC cells resulted in their resistance to lipotoxicity by inhibiting the Src-mediated JNK activation. Additionally, oleoylcarnitine enhanced sphere formation by HCC cells via STAT3 activation, suggesting that acylcarnitine accumulation was a surrogate marker of CPT2 downregulation and directly contributed to hepatocarcinogenesis. HFD feeding and carnitine supplementation synergistically enhanced HCC development accompanied by acylcarnitine accumulation in vivo. Conclusion In obesity-driven and NASH-driven HCC, metabolic reprogramming mediated by the downregulation of CPT2 enables HCC cells to escape lipotoxicity and promotes hepatocarcinogenesis.

Gastric Cancer With Primitive Enterocyte Phenotype: An Aggressive Subgroup of Intestinal-type Adenocarcinoma.

A primitive cell-like gene expression signature is associated with aggressive phenotypes of various cancers. We assessed the expression of phenotypic markers characterizing primitive cells and its correlation with clinicopathologic and molecular characteristics in gastric cancer. Immunohistochemical analysis of a panel of primitive phenotypic markers, including embryonic stem cell markers (OCT4, NANOG, SALL4, CLDN6, and LIN28) and known oncofetal proteins (AFP and GPC3), was performed using tissue microarray on 386 gastric cancers. On the basis of the expression profiles, the 386 tumors were clustered into 3 groups: group 1 (primitive phenotype, n=93): AFP, CLDN6, GPC3, or diffuse SALL4 positive; group 2 (SALL4-focal, n=56): only focal SALL4 positive; and group 3 (negative, n=237): all markers negative. Groups 1 and 2 predominantly consisted of intestinal-type adenocarcinoma, including 13 fetal gut-like adenocarcinomas exclusively in group 1. Group 1 was significantly associated with higher T-stage, presence of vascular invasion and nodal metastasis when compared with groups 2 and 3. Group 1 was associated with patients’ poor prognosis and was an independent risk factor for disease-free survival. Group 1 showed frequent TP53 overexpression and little association with Epstein-Barr virus or mismatch repair deficiency. Further analysis of the Cancer Genome Atlas data set validated our observations and revealed that tumors with primitive phenotypes were mostly classified as “chromosomal instability” in the Cancer Genome Atlas’ molecular classification. We identified gastric cancer with primitive enterocyte phenotypes as an aggressive subgroup of intestinal-type/chromosomal instability gastric cancer. Therapeutic strategies targeting primitive markers, such as GPC3, CLDN6, and SALL4, are highly promising.

Small colorectal cancers resembling submucosal tumor with massive submucosal invasion and lymph node metastasis: A report of two cases and review of the literature

Colorectal cancer resembling submucosal tumor (SMT) is very rare. We herein report two cases of small colon carcinoma resembling SMT (80-year-old female and 67-year-old male), which massively invaded into the submucosal layer and accompanied marked lymphatic invasion and lymph node metastasis. We also reviewed the reported cases of colorectal carcinoma resembling SMT (SMT-like group, n=70) and analyzed the clinicopathological characteristics of this group compared with typical colorectal carcinoma cases operated at our institution (control group, n=1723). Tumors in the SMT-like group were significantly smaller in size compared with the control group; the median diameter measured 22mm vs. 37mm (P<0.01), respectively. Histologically, although the tumors in the SMT-like group were small in diameter, they almost all invaded into the submucosal (T1) or deeper layer (T2-4), and the rate of poorly differentiated adenocarcinoma or mucinous adenocarcinoma was significantly higher than that in the control group (48.6% vs. 7.7%; P<0.01). In the subgroup analysis of T1 tumors, the rate of lymphatic invasion in the SMT-like group was also significantly higher than that in the control group (43.8% vs. 15.4%; P<0.01). Carcinoma resembling SMT appears to be invasive and has a high risk of lymphatic invasion even if small in size. Therefore, surgical treatment with dissection of the regional lymph nodes might be necessary in cases with any signs of massive submucosal invasion.