Junji Shibahara

Real‐time identification of liver cancers by using indocyanine green fluorescent imaging

We have often encountered difficulties in identifying small liver cancers during surgery. Fluorescent imaging using indocyanine green (ICG) has the potential to detect liver cancers through the visualization of the disordered biliary excretion of ICG in cancer tissues and noncancerous liver tissues compressed by the tumor.

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.

The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma

Expression profiling of hepatocellular carcinoma has demonstrated that glypican 3 (GPC3), a heparan sulfate proteoglycan anchored to the membrane, is expressed at a markedly elevated level in hepatocellular carcinoma. In this paper, two monoclonal antibodies against GPC3, GPC3-C02 and A1836A, were confirmed to specifically recognize GPC3 molecule in cells from hepatocellular carcinoma and hepatoblastoma cell lines by immunoblotting, and both were confirmed to recognize different epitopes of the GPC3 molecule by epitope mapping. Then, we evaluated the feasibility of GPC3-immunohistochemistry in the pathological diagnosis of benign and malignant hepatocellular lesions by applying these monoclonal antibodies to formalin-fixed and paraffin-embedded specimens. The immunoreactivity turned out to be identical in the two monoclonal antibodies and was thus confirmed to represent the actual expression of the GPC3 molecule. The expression was observed in the fetal liver, but not in normal adult liver, liver cirrhosis or hepatitis except for a tiny focus of a regenerative nodule of fulminant hepatitis. Diffusely positive staining of GPC3 was observed in malignant hepatocytes in hepatoblastomas and in hepatocellular carcinomas (47/56, 84%). GPC3 expression was independent of the differentiation and size of the hepatocellular carcinoma. On the other hand, there was only weak and focal staining in low-grade (2/8) and high-grade dysplastic nodules (6/8). GPC3 immunoreactivity was detected in only one of 23 metastatic lesions of colorectal carcinoma, and its expression was entirely absent in the liver cell adenoma (0/7), carcinoid tumor (0/1), and cholangiocellular carcinoma (0/16). When compared with immunohistochemistry of hepatocyte antigen and alpha-fetoprotein, GPC3-immunohistochemistry was siginificantly much more specific and sensitive for hepatocellular carcinomas. Thus, GPC3 was confirmed to be one of the oncofetal proteins now attracting attention for their promise both as markers of hepatocellular carcinoma in routine histological examination and as targets in monoclonal antibody-based hepatocellular carcinoma therapy.

Podoplanin is expressed in subsets of tumors of the central nervous system

Immunohistochemical analyses with the monoclonal antibody D2-40 were performed to ascertain the expression of podoplanin (a.k.a. T1-alpha, gp36, or aggrus) in tumors of the central nervous system (CNS) and to determine the diagnostic utility of the antibody. The analyses were performed on 325 tumors of various histologic types. The chief finding was almost constant immunoreactivity in ependymal tumors (37/40, 92.5%), choroid plexus papillomas (8/8, 100%), and meningiomas (100/100, 100%). The reactivity was considered “tissue-specific,” as the corresponding normal tissue of each tumor was also found to express podoplanin. In addition, expression, not committed to the lineages, was found in many other tumor types, including astrocytic tumors, medulloblastomas, and hemangioblastomas, with variable frequency and intensity. The way of expression was not fully understood, but the expression in astrocytic tumors seemed to be associated with pronounced fibrous properties or malignant phenotype, as was shown by high-frequent expression in pilocytic astrocytomas (12/12, 100%) and glioblastomas (29/35, 82.9%). The present study has shown that podoplanin is expressed in several types of CNS tumors with variable frequency and intensity. Given the widespread expression of podoplanin, the antibody D2-40 is of little use in diagnostic practice for CNS tumors.

SALL4 represents fetal gut differentiation of gastric cancer, and is diagnostically useful in distinguishing hepatoid gastric carcinoma from hepatocellular carcinoma.

The novel stem cell marker SALL4 has been identified as a diagnostic marker of germ cell tumors, especially yolk sac tumors, in gonadal organs. To clarify the significance of SALL4 as an oncofetal protein, we investigated SALL4 expression by immunohistochemistry in non-neoplastic stomach and gastric carcinoma with particular emphasis on á-fetoprotein (AFP)-producing gastric carcinoma, as AFP-producing gastric carcinoma shares expression of AFP and glypican 3 (GPC3) with yolk sac tumors and hepatic neoplasms. A total of 338 gastric carcinomas, 60 hepatocellular carcinomas, and 48 cholangiocellular carcinomas were studied by immunohistochemistry on tissue microarrays. In addition, more detailed whole tissue section immunohistochemistry was performed on non-neoplastic gastric tissue from 5 adult and 8 fetal specimens, 6 hepatoblastomas, and 31 cases of AFP-producing gastric carcinomas. SALL4 expression was observed in the neofetal stomach in gestational week 9 and disappeared thereafter. It was also identified by tissue microarray study in a fraction of gastric carcinomas (51 of 338, 15%), associated with older age (P=0.0001), male sex (P=0.0033), intestinal-type histology (P=0.0001), and synchronous liver metastasis (P=0.0047). AFP and GPC3 were closely associated with SALL4 expression in gastric carcinoma (both, P<0.0001), and a full-section study indicated that SALL4 was positive in all 31 cases of AFP-producing gastric carcinoma with diffuse staining in 24 cases (78%). Diffuse SALL4 expression was observed in the histologic patterns of hepatoid (89%), glandular (57%), and clear cell (39%) AFP-producing gastric carcinoma. In addition, SALL4 expression was completely negative in hepatoblastoma (n=6) and hepatocellular carcinoma (n=60). SALL4 is an oncofetal protein similar to AFP and GPC3, but it represents fetal gut differentiation in gastric carcinoma. SALL4 is a sensitive marker for AFP-producing gastric carcinoma and is especially useful to distinguish hepatoid gastric carcinoma from hepatocellular carcinoma.

Identification of ROBO1 as a Novel Hepatocellular Carcinoma Antigen and a Potential Therapeutic and Diagnostic Target

Purpose: Hepatocellular carcinoma is the most common primary malignancy of the liver and accounts for as many as one million deaths annually worldwide. The present study was done to identify new transmembrane molecules for antibody therapy in hepatocellular carcinoma. Experimental Design: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules. Results: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11). Conclusions: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.

Significance of IDH mutations varies with tumor histology, grade, and genetics in Japanese glioma patients.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found frequently in malignant gliomas and are likely involved in early gliomagenesis. To understand the prevalence of these mutations and their relationship to other genetic alterations and impact on prognosis for Japanese glioma patients, we analyzed 250 glioma cases. Mutations of IDH1 and IDH2 were found in 73 (29%) and 2 (1%) cases, respectively. All detected mutations were heterozygous, and most mutations were an Arg132His (G395A) substitution. IDH mutations were frequent in oligodendroglial tumors (37/52, 71%) and diffuse astrocytomas (17/29, 59%), and were less frequent in anaplastic astrocytomas (8/29, 28%) and glioblastomas (13/125, 10%). The pilocytic astrocytomas and gangliogliomas did not have either mutation. Notably, 28 of 30 oligodendroglial tumors harboring the 1p/19q co‐deletion also had an IDH mutation, and these alterations were significantly correlated (P < 0.001). The association between TP53 and IDH mutation was significant in diffuse astrocytomas (P = 0.0018). MGMT promoter methylation was significantly associated with IDH mutation in grade 2 (P < 0.001) and grade 3 (P = 0.02) gliomas. IDH mutation and 1p/19q co‐deletion were independent favorable prognostic factors for patients with grade 3 gliomas. For patients with grade 3 gliomas and without 1p/19q co‐deletion, IDH mutation was strongly associated with increased progression‐free survival (P < 0.0001) and overall survival (P < 0.0001), but no such marked correlation was observed with grade 2 gliomas or glioblastomas. Therefore, IDH mutation would be most useful when assessing prognosis of patients with grade 3 glioma with intact 1p/19q; anaplastic astrocytomas account for most of these grade 3 gliomas. (Cancer Sci 2012; 103: 587–592)

Pathology of the sympathetic nervous system corresponding to the decreased cardiac uptake in 123I-metaiodobenzylguanidine (MIBG) scintigraphy in a patient with Parkinson disease

Decreased cardiac uptake in (123)I-metaiodobenzylguanidine (MIBG) scintigraphy has been adopted as one of the most reliable diagnostic tests for Parkinson disease (PD) in Japan. To investigate the morphological basis for this finding, we performed a detailed neuropathological study of the cardiac sympathetic nervous system of a 71-year-old autopsy-proven PD patient, who presented with a marked decrease in cardiac uptake of MIBG, just 1 year prior to death. We carefully examined the intermediolateral column at several levels of the thoracic spinal cord, the sympathetic trunk and ganglia, and the nerve plexus of the anterior wall of the left ventricle and compared the findings with those of five age-matched controls. We found that the cardiac plexus was more heavily involved than the sympathetic ganglia in this patient with PD. Our study may provide further evidence that the markedly decreased cardiac uptake of MIBG observed in PD cases represents preferential involvement of the cardiac sympathetic nerve plexus in this disorder.

Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus

DNA hypermethylation may play a primary role in the genesis of Epstein‐Barr virus (EBV)‐associated gastric carcinoma (GC) (EB‐VaGC). Methylation‐specific PCR targeting CpG‐islands demonstrated markedly increased methylation of specific genes, such as p14, p15 and p16 genes, in EBVaGC in vivo. A high frequency of methylation was observed in an EBVaGC strain of severe combined immunodeficiency mice, and the expression of methylated genes in the strain was apparently lower than the expression of the unmethylated genes in EBV‐negative GC strains. Although over‐expression of DNA methyltransferases (DNMTs) is known to be associated with some human cancers, real‐time PCR demonstrated that DNMTs expression was suppressed in EBVaGC. The DNA methylation of specific genes, independently of DNMTs expression, may be important in the development of EBVaGC. (Cancer Sci 2003; 94: 76–80)

Papillary neuroepithelial tumor of the pineal region. A case report

We present here an unusual case of papillary neuroepithelial tumor of the pineal region. The patient was a 29-year-old female who presented with headaches. A computed tomography scan revealed a tumorous lesion at the pineal region and hydrocephalus. The resected tumor was composed of columnar and cuboidal cells showing characteristics of papillary growth. The tumor cells exhibited diffuse and intense immunoreactivity to cytokeratins and neural cell adhesion molecule. The tumor expressed abundant levels of transthyretin (prealbumin) and appeared ependymal in nature, with numerous microlumens delineated by punctate and ring-like patterns in epithelial membrane antigen staining. Reactivity to synaptophysin and glial fibrillary acidic protein was observed only in the infiltrated non-neoplastic pineal parenchyma. These histological characteristics matched the description of the recently reported papillary tumor of the pineal region thought to originate from the specialized ependyma of the subcommissural organ (SCO). Transthyretin expression of the present case further supports the likelihood of SCO origin, as transthyretin is one of the proteins presumed to be secreted by human SCO.