Keiichi Iwaya

Nuclear p53 immunoreaction associated with poor prognosis of breast cancer

p53 protein has been frequently detected at high levels in the nuclei of human breast cancer cells. We analyzed inununohistochemically the association between nuclear localization of p53 protein and clinical and histological parameters of breast cancer patients. Surgically resected tissues of 73 primary breast cancers were processed by acetone fixation and paraffin embedding and examined using an anti‐p53 monoclonal antibody, Pabl801. p53 immunoreactivity was detected in the nuclei of cancer cells in 17 cases (23%). The nuclear p53 immunoreaction was closely associated with overexpression of c‐erbB‐2 protein (P<0.05), high histologic grade (P<0.01), advanced clinical stage (P<0.05), and negative estrogen receptor status (P< 0.01). When 31 cases which had been followed up for more than 50 months were examined, a positive nuclear p53 immunoreaction was found to he significantly associated with shorter overall survival of patients (P<0.01). These results suggest that inununohistochemical examination of nuclear p53 protein is clinically useful as an indicator of breast cancer aggressiveness.

Cytoplasmic and/or nuclear staining of beta-catenin is associated with lung metastasis

Beta-catenin is involved in cell motility in the extracellular matrix, and is expressed in normal and neoplastic mesenchymal cells. In order to clarify whether beta-catenin expression in the cytoplasm and/or nucleus is associated with a propensity for pulmonary metastasis in osteosarcoma, the LM8 murine osteosarcoma cell line with a high metastatic potential to the lung was compared with original Dunn cells in terms of the beta-catenin expression level. Both osteosarcoma cell lines lost membrane localization of beta-catenin. However, beta-catenin gene had no mutation in exon 3 by direct sequence analysis. A large number of LM8 cells showed diffuse cytoplasmic and/or nuclear staining of beta-catenin (30.8 per high power field (HPF)), while a much smaller number of Dunn cells showed expression of beta-catenin (7.7 per HPF). Cells with positive staining of beta-catenin were frequently seen at the invasive front and in intravenous tumor deposits within the metastatic lesions to the lung. Thus, LM8 cells express a larger amount of the beta-catenin protein than Dunn cells, as judged by immunoblot analysis. In five resected cases of pulmonary metastasis, translocation of beta-catenin to the cytoplasm and/or nucleus of osteosarcoma cells was detected, although seven primary osteosarcomas cells that did not metastasize for more than five years did not show beta-catenin expression. These data indicate that the cytoplasmic and/or nuclear staining of beta-catenin is a biological marker of metastatic potential of osteosarcoma to the lung.

Nuclear accumulation of beta-catenin in intestinal-type gastric carcinoma: correlation with early tumor invasion

Abstract. Mutation of the adenomatous polyposis coli gene, which is known to be an early event in the carcinogenesis of intestinal-type gastric carcinoma, leads to accumulation of beta-catenin. In addition, beta-catenin has been found to activate down stream signaling molecules in the wingless/Wnt pathway. In this study, the clinical significance of nuclear accumulation of beta-catenin was evaluated in gastric carcinoma. Immunohistochemical staining showed nuclear localization in 16 (12%) of 139 (94 intestinal-type and 45 diffuse-type) gastric carcinomas, and all 16 lesions with nuclear staining were intestinal-type adenocarcinomas. Of the 16 cases, 15 were in the early clinical stage. In the remaining case, the lesion had invaded the subserosal layer and showed strong nuclear staining at the invasive front. In 14 of the 16 cases with nuclear localization, there were no abnormal mobility shifts detected using polymerase chain reaction-single strand conformational polymorphism analysis. This was confirmed using direct sequencing analysis, which revealed the wild-type sequence in the 12 cases tested. Nuclear accumulation of beta-catenin did not correlate with lymph node metastasis or 5-year survival. These findings suggest that high intranuclear levels of beta-catenin protein play an important role in early tumor growth and may function in initiation of invasive processes in intestinal-type gastric carcinoma.

Involvement of Arp2/3 complex in the process of colorectal carcinogenesis

Increased motility is one of the characteristics of cancer cells, and actin polymerization and disassembly are essential for cellular motility. Since actin-related protein (Arp) 2/3 complex acts as a nucleus for actin polymerization, in this study, we immunohistochemically investigated the expression of Arp2 and Arp3 in 175 colorectal tumors in various stages of neoplastic progression. Arp2 and Arp3 showed identical expression patterns, and both were expressed in the stromal cells around neoplastic tubules or glands and in the tumor cells themselves. The frequency of expression of Arp2 and Arp3 (Arp2&3) by the stromal cells increased with the atypia of the colorectal neoplasms, from 5.5% (3/55) in adenoma with mild or moderate atypia, to 11.8% (2/17) in adenoma with severe atypia, 53.3% (16/30) in intramucosal carcinoma, and 91.8% (67/73) in invasive carcinoma (P<0.0001). The frequency of expression of Arp2&3 in the tumor cells was similar and was 1.8% (1/55) in adenoma with mild or moderate atypia, 23.5% (4/17) in adenoma with severe atypia, 23.5% (7/30) in intramucosal carcinoma, and 32.9% (24/73) in invasive carcinoma. Expression of Arp2&3 by the stromal cells was significantly correlated with nuclear accumulation of p53 in the tumor cells and stromal expression of CD10. These results suggest that formation of Arp2/3 complex by both neoplastic and stromal cells contributes to the increased motility of both cell types and thus provides suitable conditions for invasion.

Correlation between liver metastasis of the colocalization of actin‐related protein 2 and 3 complex and WAVE2 in colorectal carcinoma

Directed movement of normal cells occurs when actin‐related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma. (Cancer Sci 2007; 98: 992–999)

Coexpression of Actin-Related Protein 2 and Wiskott-Aldrich Syndrome Family Verproline-Homologous Protein 2 in Adenocarcinoma of the Lung

Purpose: Highly invasive and metastatic cancer cells, such as adenocarcinoma of the lung cells, form irregular protrusions by assembling a branched network of actin filaments. In mammalian cells, the actin-related protein 2 and 3 (Arp2/3) complex initiates actin assembly to form lamellipodial protrusions by binding to Wiskott-Aldrich syndrome (WASP)/WASP family verproline-homologous protein 2 (WAVE2). In this study, colocalization of Arp2 and WAVE2 in adenocarcinoma of the lung was investigated to elucidate its prognostic value. Experimental Design: Immunohistochemical staining of Arp2 and WAVE2 was done on mirror sections of 115 adenocarcinomas of the lung from pathologic stage IA to IIIA classes. Kaplan-Meier disease-free survival and overall survival curves were analyzed to determine the prognostic significance of the coexpression of Arp2 and WAVE2. Results: Immunoreactivity for both Arp2 and WAVE2 was detected in the same cancer cells in 78 (67.8%) of the 115 lung cancer specimens. The proportion of cancer cells expressing both Arp2 and WAVE2 was significantly higher in cases with lymph-node metastasis (P = 0.0046), and significantly lower in bronchioloalveolar carcinomas (P < 0.0001). The patients whose cancer cells coexpressed them had a shorter disease-free survival time (P < 0.0001) and overall survival time (P < 0.0001). Multivariate Cox regression analysis revealed that coexpression of Arp2 and WAVE2 is an independent risk factor for tumor recurrence. Conclusions: Coexpression of Arp2 and WAVE2 is correlated with poorer patient outcome, and may be involved in the mechanism of cancer metastasis.

Hybrid Carcinomas of the Salivary Glands: Report of Nine Cases with a Clinicopathologic, Immunohistochemical, and p53 Gene Alteration Analysis

Hybrid carcinomas of the salivary gland are a recently defined and rare tumor entity, consisting of two histologically distinct types of carcinoma within the same topographic area. In this study, we examined nine such cases, which mainly arose in the parotid gland (seven cases), with an additional one each from submandibular and lacrimal glands, and analyzed their clinicopathologic profiles, including immunohistochemical features and p53 gene alterations. The prevalence of hybrid carcinomas was 0.4% among the 1863 cases of parotid gland tumors in our series. The nine patients comprised five men and four women, ranging in age from 40 to 81 years (mean, 62 y). Tumor size ranged from 2 to 10 cm (mean, 4.2 cm). Of the seven patients who were followed up, two were alive with disease and five were alive with no evidence of disease, although the follow-up period was short. Three cases had cervical lymph nodal metastases. The combinations of carcinoma components in our hybrid carcinomas were as follows: epithelial–myoepithelial carcinoma and basal cell adenocarcinoma in two cases, epithelial–myoepithelial carcinoma and squamous cell carcinoma in one case, salivary duct carcinoma and adenoid cystic carcinoma in two cases, myoepithelial carcinoma and salivary duct carcinoma in one, acinic cell carcinoma and salivary duct carcinoma in one, and squamous cell carcinoma and salivary duct carcinoma in two. Although the proportion of each carcinoma component in a tumor mass varied from case to case, the minor component always represented ≥ 10% of the area. Differences in cellular composition were studied by immunohistochemistry and electron microscopy. The Ki-67–labeling index apparently differed between the two carcinoma elements in five cases. Diffusely positive p53 immunoreactivity was observed in four cases, restricted to the more aggressive component in each pair. Furthermore, p53 gene alteration analysis of these p53-positive cases revealed that all and three cases demonstrated loss of heterozygosity at p53 microsatellite loci and p53 gene point mutations, respectively, which were detected only in the p53-immunoreactive carcinoma component. Therefore, there is the possibility that such molecular-genetic events take an integral part for inducing the transformation from histologically lower to higher grade tumor during the hybrid carcinoma genesis of the salivary glands.

Sarcomatoid Variant of Salivary Duct Carcinoma Clinicopathologic and Immunohistochemical Study of Eight Cases With Review of the Literature

Salivary duct carcinoma (SDC) is an uncommon, high-grade tumor. We present 8 cases of sarcomatoid SDC, which has been defined recently as a rare variant of SDC. The 8 patients (5 men, 3 women) had a mean age of 63.6 years. Histologically, all tumors were characterized by a biphasic neoplasm composed of both SDC and sarcomatoid elements. In 3 cases, sarcomatoid components showed osteosarcomatous heterologous differentiation. A residual pleomorphic adenoma was detected in 5 tumors. The sarcomatoid component showed focal immunoreactivity for cytokeratin in 4 cases and epithelial membrane antigen in all 8 cases. Diffuse p53 immunostaining was detected in 3 cases, and it was coexpressed in both components. Our observations support the histogenetic theory of a common origin of the carcinomatous and sarcomatoid populations. Of the 13 patients, including our 8, reported to have sarcomatoid SDC arising in a major salivary gland and for whom long-term follow-up data were available, 7 have died of disease (mean survival, 15.6 months). These results indicate that sarcomatoid SDC is a highly aggressive tumor, similar to conventional SDC.

Genome-wide association study of clinically defined gout identifies multiple risk loci and its association with clinical subtypes

Objective Gout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only. Methods A GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls. Results Five gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10−8), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10−12; OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10−23; OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10−9; OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case–control ORs for two distinct types of gout (r=0.96 [p=4.8×10−4] for urate clearance and r=0.96 [p=5.0×10−4] for urinary urate excretion). Conclusions Our findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma

Breast carcinoma with a high histologic grade is highly invasive and metastatic. One reason for its irregular morphology is the formation of excessive protrusions due to assemblages of branched actin filament networks. In mammalian cells, the actin-related protein 2 and 3 (Arp2/3) complex initiates actin assembly to form lamellipodial protrusions by binding to the Wiskott–Aldrich syndrome (WASP)/WASP family verproline-homologous protein2 (WAVE2), a member of the WASP protein family. In this study, the localization Arp2 and WAVE2 in breast carcinoma was investigated to clarify whether coexpression of the two proteins is associated with histologic grade or patient outcome. Immunohistochemical staining of Arp2 and WAVE2 was performed on mirror specimens of 197 breast carcinomas, and the association between coexpression of the two proteins and clinicopathologic factors was examined. Kaplan–Meier disease-free survival and overall survival curves were analyzed to determine the prognostic significance of Arp2 and WAVE2 coexpression in breast carcinoma. Coexpression of Arp2 and WAVE2 was detected in 64 (36%) of 179 invasive ductal carcinomas and in 2 (11%) of 18 ductal carcinomas in situ, but was not detected in any adjacent non-cancerous tissue. The proportion of cancer cells expressing both Arp2 and WAVE2 was significantly higher in cases with high histologic grade (P<0.0001), and cases with lymph node metastasis (P=0.0150). The patients whose cancer cells showed such coexpression had shorter disease-free (P=0.0002) and overall survival (P=0.0122) than patients whose cancer cells expressed only one or none of Arp2 and WAVE2. Multivariate Cox regression analysis revealed that coexpression of Arp2 and WAVE2 is an independent factor for both tumor recurrence (P=0.0308) and death (P=0.0455). These results indicate that coexpression of Arp2 and WAVE2 is a significant prognostic factor that is closely associated with aggressive morphology of invasive ductal carcinoma of the breast.