Akishi Ooi

EGFR protein overexpression and gene amplification in squamous cell carcinomas of the esophagus

Overexpression of epidermal growth factor receptor (EGFR) is observed in many cancers, sometimes accompanied by gene amplification. Recently, several clinical therapies targeting EGFR were developed, but the eligibility criteria for these therapies is not fully established. To develop such eligibility criteria for esophageal squamous cell carcinoma (ESCC), we sought to clarify: (i) the exact frequency of EGFR overexpression, (ii) the relationship between protein overexpression and gene amplification, (iii) the relationship between gene amplification and specific gene mutations and (iv) the correlation between the status of EGFR and clinical or pathological features. Immunohistochemistry revealed that EGFR protein is overexpressed in 53 (50%) of the 106 ESCC examined. Fluorescence in situ hybridization (FISH) indicated clear EGFR gene amplification in 15 of the 53 tumors, somewhat higher EGFR copy in 32 cases, and no increase in 6 cases. Gene amplification was significantly associated with high level overexpression. Direct sequencing of exons 19 and 21 of EGFR revealed no mutations in 15 tumors exhibiting gene amplification, and no mutations in 25 tumors not exhibiting gene amplification. Overexpression of EGFR was significantly correlated with depth of invasion of the tumor.In conclusion, anti‐EGFR therapies may be appropriate for patients with ESCC. We assume that combined analyses by immunohistochemistry/FISH would clarify aberrations in protein and gene function, and could help to identify those patients who may benefit from anti‐EGFR therapy.

Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay.

c‐erb‐2 amplification and overexpression are currently attracting a great deal of attention because a new adjuvant therapy using an antibody against the c‐erbB‐2 gene product, trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), has proved effective in treating breast cancer with amplification and/or overexpression of c‐erbB‐2. Aberrations of c‐erbB‐2 have also been detected in ovarian, endometrial and gastric carcinomas at varied frequencies. Amplification of the c‐erbB‐2 locus (17q12‐q21.32), overexpression of c‐erbB‐2 protein (p185) and serum levels of soluble c‐erbB‐2 protein fragments (p105) were examined in gastric cancer patients using fluorescence in situ hybridization (FISH), immunohistochemistry and enzyme‐linked immunosorbent assay (ELISA), respectively. Overexpression of c‐erbB‐2 protein was found in 29 (8.2%) of the 352 gastric carcinomas analyzed. In FISH analysis, all tumors with 3+ immunostaining and 1 of 5 tumors with 2+ staining showed high‐level amplification of c‐erbB‐2. Pre‐operative serum p105 was quantified in serum specimens from 129 patients with gastric cancer and 28 patients with benign diseases. There were no significant differences in the serum p105 levels among 11 patients with c‐erbB‐2‐overexpressing carcinomas, 118 patients with c‐erbB‐2 non‐overexpressing carcinomas and 28 controls, although a single case of gastric carcinoma overexpressing c‐erbB‐2 with extensive liver metastasis had a higher level than the cut‐off value. The mechanisms of overexpression of p185 and high‐level amplification of c‐erbB‐2 in gastric adenocarcinomas seem similar to those well‐established in breast cancers. Patients having gastric adenocarcinoma with c‐erbB‐2 amplification are potential candidates for a new adjuvant therapy using humanized monoclonal antibody.

Amplification and overexpression of c-erbB-2, epidermal growth factor receptor, and c-met in biliary tract cancers

Carcinomas of the biliary tract have a poor prognosis. It is important to understand the molecular genetic characteristics of these tumours in order to employ newer effective treatments and to improve patient prognosis. There is increasing evidence that overexpression of tyrosine kinase growth factor receptors such as ErbB‐2, epidermal growth factor receptor (EGFR), and Met may play important roles in the development of biliary tract carcinomas. The aim of this study was to assess the potential for novel chemotherapies targeting these receptors. Overexpression of the tyrosine kinase receptor proteins was examined by immunohistochemistry in 221 biliary tract carcinomas, of which 28 were from the intrahepatic bile duct, 78 from the extrahepatic bile duct, 89 from the gall bladder, and 26 from the ampulla of Vater. Positively stained tumours were further examined for gene amplification by fluorescence in situ hybridization. Overexpression of ErbB‐2 was found in 15.7%, 11.5%, and 5.1% of carcinomas of the gall bladder, ampulla of Vater, and extrahepatic bile duct, respectively, and gene amplification was present in 79% of these. Overexpression of EGFR was found in 8.1% of tumours with no predominant location and was also associated with gene amplification with high frequency (77%). Met overexpression, most frequent in intrahepatic bile duct carcinomas (21.4%), was not associated with gene amplification. It is proposed that the new adjuvant chemotherapies could be directed to carcinomas of the biliary tract in which ErbB‐2 and EGFR are overexpressed. Copyright

Two genetic variants of CD38 in subjects with autism spectrum disorder and controls.

The neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play a role in the etiology of ASD. Here, we first examined the immunohistochemical expression of CD38 in the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalized with OT in secretory neurons. In studies of the association between CD38 and autism, we analyzed 10 single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly from a case-control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource Exchange (AGRE). The SNPs of CD38, rs6449197 (p<0.040) and rs3796863 (p<0.005) showed significant associations with a subset of ASD (IQ>70; designated as high-functioning autism (HFA)) in the U.S. 104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan to replace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6-4.6% of the Japanese population and was associated with ASD in the smaller case-control study. The SNP was clustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.

Inhibition of GSK‐3β activity attenuates proliferation of human colon cancer cells in rodents

The authors’ recent discovery that glycogen synthase kinase‐3β (GSK‐3β) participates in colon cancer cells’ survival and proliferation prompted us to investigate whether GSK‐3β inhibition alters proliferation of colon cancer cells in vivo. Groups of four or five athymic mice (Balb/c, nu/nu) with subcutaneous xenografts of SW480 human colon cancer cells were treated with dimethyl sulfoxide (DMSO) or different doses (1, 2 and 5 mg/kg body weight) of either small‐molecule GSK‐3β inhibitor (SB‐216763 and AR‐A014418) by intraperitoneal injection three times per week for 5 weeks. Compared with DMSO (a diluent of the GSK‐3β inhibitors) as a control, either GSK‐3β inhibitor significantly inhibited proliferation of cancer cell xenografts in the rodents in a dose‐dependent manner. Histochemical and immunohistochemical analysis of tumor xenografts demonstrated a significant, dose‐dependent decrease in fractions of proliferating cells and an increase in the incidence of apoptosis of cancer cells in mice treated with either GSK‐3β inhibitor. No adverse events or effects were observed in the rodents during the course of treatment, except for rare lethal accidents due to intraperitoneal injection. Morphological examination showed no apparent pathologic changes in major organs including the lungs, liver, pancreas, kidneys, spleen and large bowel of rodents treated with DMSO and the GSK‐3β inhibitors. The results indicate that the GSK‐3β inhibitors would be a novel class of therapeutic agent for colon cancer. (Cancer Sci 2007; 98: 1388–1393)

Expression of glial fibrillary acidic protein gfap in peripheral nerve sheath tumors a comparative study of immunoreactivity of gfap vimentin s 100 protein and neurofilament in 38 schwannomas and 18 neurofibromas

Immunoreactivity of glial fibrillary acidic protein (GFAP) in 38 schwannomas and 18 neurofibromas was evaluated and compared with the reactivity of vimentin, S-100 protein, and neurofilament protein. All cases were positive for vimentin and S-100 protein. GFAP was positively stained in the neoplastic cells of 15 of 38 schwannomas (38%) and in two of 18 neurofibromas (11%). The extensively stained GFAP-positive tumors tended to be deeply situated in the body. The GFAP-positive cells were usually spindle-shaped and appeared preferentially in the perivascular region of hyalinized, thick blood vessels.

Trastuzumab-Mediated Antibody-Dependent Cellular Cytotoxicity against Esophageal Squamous Cell Carcinoma

Purpose: In the present study, we investigated the degree of protein expression and gene amplification of HER-2 in esophageal squamous cell carcinoma (SCC) cell lines and freshly isolated tumors, and trastuzumab-mediated biological activity, in particular antibody-dependent cellular cytotoxicity (ADCC) against HER-2–expressing esophageal SCC cell lines. Experimental Design: Ten different SCC cell lines with various levels of HER-2 status evaluated by flow cytometry, immunocytochemistry (HercepTest), and fluorescence in situ hybridization were evaluated for ADCC, growth inhibitory, or apoptosis-inducing activities mediated by trastuzumab. Results: Trastuzumab induced ADCC against HER-2–expressing esophageal SCC and the activities reflected the degree of HER-2 expression analyzed by flow cytometric analysis, but not by HercepTest nor fluorescence in situ hybridization analysis. Furthermore, trastuzumab-mediated ADCC against transforming growth factor-β–producing SCC was enhanced by the treatment with SB-431542, which is a selective inhibitor of the phosphorylation induced by transforming growth factor-β. There were very marginal effects of anti-proliferative or apoptosis-inducing activities mediated by trastuzumab for HER-2–expressing esophageal SCC. Conclusion: HER-2–expressing esophageal SCC cells could be killed by trastuzumab-mediated ADCC and the activity reflected the degree of HER-2 expression detected by flow cytometry.

Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice.

Blocking monoclonal antibodies (mAbs) specific to mouse interleukin‐1 receptor antagonist (IL‐1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL‐1ra. A sensitive and specific ELISA against mouse IL‐1ra was also established. In Propionibacterium αcnes‐induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor‐α levels but not those of IL‐1ra, IL‐1, and IL‐6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL‐1ra level was more than 20 times as high as serum IL‐1 levels. Immunohistochemical analysis demonstrated that IL‐1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL‐1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL‐1ra in this liver injury model. J. Leukoc. Biol. 58: 90–98; 1995.

Clinicopathological significance of platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor-A expression, PDGF receptor-β phosphorylation, and microvessel density in gastric cancer

BackgroundAngiogenesis is important in the growth and metastasis of various kinds of solid tumors, including gastric cancers. The angiogenic process is triggered by several key growth factors, including vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B, that are secreted by tumors. Our aim was to define: i) the expression pattern of VEGF-A and PDGF-B in tumor cells and the activation of PDGF receptor (PDGFR)-β tyrosine kinase in stromal cells of human gastric adenocarcinomas; and ii) the relationship between VEGF-A and PDGF-B expression and microvessel density (MVD), to determine if there is a rationale for a new therapeutic strategy.MethodsA series of 109 gastric adenocarcinoma cases that had undergone surgical resection was examined immunohistochemically using antibodies against VEGF-A, PDGF-B, and CD34, followed by further examination of PDGFR-β phosphorylation by immunoblotting analysis.ResultsMVD was higher in diffuse-type than intestinal-type cancers (p < 0.001). VEGF-A overexpression correlated to PDGF-B overexpression in both the intestinal-type (p < 0.005) and diffuse-type (p < 0.0001) groups, indicating that VEGF-A and PDGF-B are secreted simultaneously in the same tumor, and may thus play important roles together in angiogenesis. However, several differences between intestinal-type and diffuse-type cancers were observed. In the diffuse-type cancer group, higher MVD was related to the PDGF-B proportion (p < 0.05) and VEGF-A overexpression (p < 0.05), but not to PDGF-B overexpression or the VEGF-A proportion. On the other hand, in the intestinal-type cancer group, higher MVD was correlated to overexpression (p < 0.005), intensity (p < 0.05), and proportion (p < 0.05) of PDGF-B, but not of VEGF-A. In addition, phosphorylation of PDGFR-β was correlated with depth of cancer invasion at statistically significant level.ConclusionsOur results indicate that PDGF-B, which is involved in the maintenance of microvessels, plays a more important role in angiogenesis in intestinal-type gastric carcinomas than VEGF-A, which plays a key role mainly in the initiation of new blood vessel formation. In contrast, VEGF-A has a critical role for angiogenesis more in diffuse-type cancers, but less in those of intestinal type. Thus, a therapy targeting the PDGF-B signaling pathway could be effective for intestinal-type gastric carcinoma, whereas targeting VEGF-A or both VEGF-A and PDGF-B signaling pathways could be effective for diffuse-type gastric carcinomas.

Medullary carcinoma with lymphocytic infiltration of the stomach. Clinicopathologic study of 27 cases and immunohistochemical analysis of the subpopulations of infiltrating lymphocytes in the tumor

The current study attempts to clarify the possible immune response that occurs in medullary carcinoma with lymphocytic infiltration of the stomach by an immunohistochemical analysis of the subpopulations of tumor‐infiltrating lymphocytes. This carcinoma was histologically characterized by the sparse population of small nests consisting of poorly differentiated carcinoma cells, widely separated by intervening nondesmoplastic stroma infiltrated uniformly with abundant lymphocytes frequently accompanied by lymph follicles. An immunohistochemical analysis revealed that T‐cells were evenly distributed throughout the tumor with intimate contact with individual carcinoma cells, except the lymph follicles consisted mainly of B‐cells. Because of the similarities of morphologic features and subpopulations of tumor‐infiltrating lymphocytes of this carcinoma to the normal lymphoid tissue, an organized immune response combined with cell‐mediated and humoral immunities against the invading carcinoma cells seemed to occur in this type of gastric carcinoma, resulting in a excellent prognosis compared with that in ordinary gastric carcinoma.