Shioto Suzuki

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.

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

Protein overexpression and gene amplification of HER-2 and EGFR in colorectal cancers: an immunohistochemical and fluorescent in situ hybridization study

Overexpression of HER-2 and the epidermal growth factor receptor (EGFR) has been observed in many cancers, sometimes accompanied by gene amplification. To assess whether novel chemotherapies targeting these overexpressed proteins may be effective for the treatment of colorectal cancers, we examined the exact frequency of HER-2 and EGFR overexpression, the relationship between gene amplification and protein expression, and the heterogeneity of gene amplification within and between primary and metastatic tumors. We evaluated 244 colorectal cancers immunohistochemically. All tumors found to overexpress HER-2 or EGFR were further analyzed for gene amplification by fluorescent in situ DNA hybridization. Overexpression of HER-2 and EGFR was found in 8 (3%) and 19 (8%) of the 244 colorectal carcinomas, respectively. Gene amplification was observed in 100 and 58% of the tumors exhibiting HER-2 and EGFR overexpression, respectively. HER-2 amplification in cancer cells was characterized by clusters of hybridization signals, suggesting amplicons in homogeneously staining regions that were predominant in most primary and metastatic tumors. EGFR amplification, observed as scattered signals reminiscent of amplicons in double minute chromosomes, or coamplification of EGFR with the centromeric regions was observed as a minor population within primary tumors, and found in variety of populations in metastatic tumors. Overexpression of HER-2 and EGFR were observed in only a small fraction of colorectal carcinomas, but were frequently accompanied by gene amplification.

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.

Relationship between genetic polymorphisms of drug-metabolizing enzymes (CYP1A1, CYP2E1, GSTM1, and NAT2), drinking habits, histological subtypes, and p53 gene point mutations in Japanese patients with gastric cancer

BackgroundGenetic polymorphisms of drug-metabolizing enzymes have recently been shown to affect susceptibility to chemical carcinogenesis. However, the molecular mechanisms of individual susceptibility to gastric cancer have not been fully understood. Therefore, we studied the relationship between the genetic polymorphisms of drug-metabolizing enzymes, drinking habits, histological subtypes, and p53 gene point mutations in Japanese patients with gastric cancer.MethodsThe genotypes of cytochromes P450 (CYP) 1A1 and 2E1, glutathione S-transferase (GST) M1, and N-acetyltransferase (NAT) were investigated by polymerase chain reaction (PCR), allele-specific PCR, or restriction fragment length polymorphism (RFLP) following PCR in 146 Japanese patients with gastric cancer (67 intestinal-type and 79 diffuse-type carcinomas) and 177 autopsied controls. In addition, p53 gene point mutations of exons 5 through 9 in gastric cancer tissues were determined.ResultsThe frequency of either being a habitual drinker or having a CYP2E1*1A/*1A genotype was significantly higher in patients with intestinal-type gastric cancer than in control subjects. The difference between the frequencies of habitual drinkers with the CYP2E1*1A/*1A genotype and non-drinkers with the CYP2E1*5B allele was much more significant in the intestinal-type cancer versus the control group. Among intestinal-type cancer patients with the CYP2E1*1A/*1A genotype, p53 point mutations were significantly more frequent in the group of habitual drinkers than in that of non-drinkers. On the other hand, the combination of GSTM1 null and CYP2E1*1A/*1A genotypes increased the risk for diffuse-type gastric cancer, but had no influence on the frequency of p53 gene mutations.ConclusionsThe present study suggests that individuals with both the CYP2E1*1A/*1A genotype and a history of habitual drinking have an increased risk of intestinal-type gastric cancer with a high frequency of p53 gene point mutations in the gastric mucosa.

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.

EGFR-dependent and independent activation of Akt/mTOR cascade in bone and soft tissue tumors.

To gain the insight into the involvement of signaling mediated by the mammalian target of rapamycin (mTOR) in the phenotype and biological profiles of tumors and tumor-like lesions of the bone and soft tissue, we analyzed the expression and phosphorylation (activation) of mTOR and its correlation with the status of upstream and downstream modulator proteins Akt, p70S6-kinase (S6K), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which we refer to collectively as mTOR cassette proteins. Immunohistochemical analysis of 140 cases showed activation of Akt in 55% (61% in malignant and 27% in benign), and mTOR expression in 61% (66% in malignant and 39% in benign). The preponderance of mTOR activation was found in tumors of peripheral nerve sheath (malignant peripheral nerve sheath tumor and schwannoma), skeletal muscle origin (rhabdomyosarcoma), and in those exhibiting epithelial nature (chordoma and synovial sarcoma). Together with the result of immunoblotting analysis, it was shown that many of those particular tumors with mTOR activation exhibited activation of Akt, S6K, and 4E-BP1, suggesting the constitutive activation of the Akt/mTOR pathway. In addition, although activation of the Akt/mTOR pathway was largely independent of activation of epidermal growth factor receptor (EGFR), mutation of EGFR was frequently accompanied by constitutive activation of Akt–mTOR–S6K/4E-BP1. By clinicopathological analysis, activation of Akt correlates with statistically higher probability of metastasis. We conclude that mTOR-mediated signaling proteins function not only in the proliferation of the tumor cells, but also in the differentiation and/or maintenance of morphological phenotypes in tumors of rhabdomyoblastic and nerve sheath cell origin. Furthermore, mTOR signaling may also modulate morphogenesis of tumors exhibiting epithelial nature. Additionally, activated Akt may have a function in metastasis. Overall, these results suggest that inhibitors of mTOR cassette may be useful as novel components of combined chemotherapy for a defined subset of bone and soft tissue sarcomas.

Expression of epidermal growth factor receptor in gastric carcinomas.

BACKGROUND & AIMS Epidermal growth factor receptor belongs to the family of type I receptor tyrosine kinases. Overexpression of epidermal growth factor receptor has been observed in a variety of cancers with or without amplification of the gene. Novel chemotherapies targeting receptor tyrosine kinases might be effective for the treatment of cancers in which overexpression of this protein is a feature. The aim of this study was to assess the potential efficacy of epidermal growth factor receptor-targeted therapy in gastric cancer. This was achieved by determining the frequency of increased epidermal growth factor receptor expression in gastric cancers and investigating the relationship between protein overexpression and gene amplification. METHODS Immunohistochemical evaluation of 413 gastric cancers was carried out by using a monoclonal antibody to the epidermal growth factor receptor. The intensity of reactivity was scored by using a 4-tier system (negative, 1+, 2+, and 3+). All positive staining (>1+) tumors overexpressing the protein were then analyzed for gene amplification by fluorescence in situ hybridization by using a gene-specific probe. RESULTS High levels of overexpression (2+ or 3+ staining) were found in 9 of 413 (2.2%) patients, whereas low levels of overexpression (1+) were found in 34 (8.2%) of the study cohort. Fluorescence in situ hybridization analysis showed that more than 10 copies of the gene were recognized in all 5 cancers with 3+ staining and in 2 of the 4 tumors with 2+ staining. CONCLUSIONS Although a high level of overexpression of epidermal growth factor receptor is uncommon in gastric carcinomas, it almost exclusively occurs by gene amplification.

Critical and Diverse Involvement of Akt/Mammalian Target of Rapamycin Signaling in Human Lung Carcinomas

BACKGROUND: Aberrant signaling cascades emanating from epidermal growth factor receptor (EGFR) are involved in the complex network of oncogenic signaling in lung carcinomas. One representative cascade is the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. METHODS: The authors investigated the involvement of mTOR in the pathobiologic profiles of 150 specimens of lung carcinoma by immunohistochemistry and immunoblotting in correlation with the upstream and downstream proteins Akt and p70S6‐kinase (S6K), respectively. RESULTS: Immunohistochemistry revealed Akt activation in 44% of tumors and mTOR expression in 68.7% of tumors, and the preponderance of activation was observed in adenocarcinoma (AC) (100%). Phosphorylated mTOR (p‐mTOR) was observed in 53.3% of tumors and had the highest frequency in AC (89.7%). In AC, the frequency of p‐mTOR staining was higher in the well differentiated subtype, in particular, in the acinar structure. However, little correlation was observed between the activation of mTOR and Akt, except in the 5 AC specimens that harbored an EGFR gene mutation, which exhibited constitutive activation of both Akt and mTOR. Conversely, in squamous cell carcinomas, mTOR activation was associated with a significantly higher frequency of lymph node metastasis. CONCLUSIONS: The results of this study suggested the dual functions of mTOR. First, mTOR may function not only in the proliferation of tumor cells as an effector molecule downstream of EGFR but also possibly in the morphogenesis of AC. Second, the activation of mTOR may play a key role in metastasis in squamous cell carcinoma. Overall, the current results demonstrated the potential for the application of rapamycin, an mTOR inhibitor, as an additional novel component of chemotherapy for a defined subset of patients with lung carcinoma. Cancer 2009.

Aberration of epidermal growth factor receptor expression in bone and soft-tissue tumors: protein overexpression, gene amplification and activation of downstream molecules

In order to evaluate the involvement of epidermal growth factor receptor, and to analyze the correlation between gene aberration and protein expression in mesenchymal tumors, we examined protein expression by immunohistochemistry in 125 cases of bone and soft-tissue tumors. Furthermore, amplification of epidermal growth factor receptor gene was determined by fluorescence in situ hybridization. Positive immunostaining was found in 23 cases (18.4%). Among these 23 cases, one of malignant fibrous histiocytoma showed the highest degree (3+) of protein overexpression and gene amplification as clusters of hybridization signals, indicating homogeneously staining regions. The second case of malignant fibrous histiocytoma also showed a higher degree (2+) of overexpression and coamplification of the epidermal growth factor receptor gene with the centromeric regions, indicating polysomy of chromosome 7. The levels of expression observed in immunohistochemistry were confirmed by immunoblotting and found to be comparable. Moreover, although expression of phosphorylated epidermal growth factor receptor was detected in those two cases of malignant fibrous histiocytoma, constitutive activation of extracellular signal-related protein kinase 1/2 was not observed, suggesting that activation of epidermal growth factor receptor does not necessarily and constantly lead to signal transduction to the downstream molecules. In the remaining 123 cases, including 21 cases exhibiting weak (1+) immunoreactivity, no gene amplification nor polysomy was found. Collectively, expression of epidermal growth factor receptor was observed not infrequently in mesenchymal tumors, but ‘overexpression’ is rare and can be attributed to an increase in gene copy number, resulting from amplification or polysomy. Although cases that scored positive for protein expression and/or gene amplification could be qualified candidates for antiepidermal growth factor receptor therapies, further examination of the status of downstream molecules in the signal cascade, such as phosphorylated epidermal growth factor receptor and extracellular signal-related protein kinase 1/2, may be required as the process of therapeutic strategy.