Naoki Yanagawa

Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor suppressor and tumor‐related genes. To determine the clinicopathological significance of gene promoter methylation in non‐small cell lung cancer (NSCLC), we examined the promoter methylation status of the APC, DAP‐kinase, E‐cadherin, GSTP1, hMLH1, p16, RASSF1A and RUNX3 genes in 75 NSCLCs and corresponding non‐neoplastic lung tissues by methylation‐specific PCR (MSP). The frequencies of methylation in NSCLCs and corresponding non‐neoplastic lung tissues were: 37% (28 of 75) and 48% (36 of 75) for APC, 28% (21 of 75) and 13% (10 of 75) for DAP‐kinase, 29% (22 of 75) and 15% (11 of 75) for E‐cadherin, 1% (1 of 75) and 0% (0 of 75) for GSTP1, 7% (5 of 75) and 0% (0 of 75) for hMLH1, 31% (23 of 75) and 0% (0 of 75) for p16, 43% (32 of 75) and 4% (3 of 75) for RASSF1A, and 20% (15 of 75) and 3% (2 of 75) for RUNX3, respectively. Methylation of p16 was more frequent in squamous cell carcinomas than in adenocarcinomas (P<0.05), and was associated with tobacco smoking (P<0.05). On the contrary, methylation of APC and RUNX3 was more frequent in adenocarcinomas than in squamous cell carcinomas (P<0.05). Thus, a different set of genes is thought to undergo promoter methylation, which leads to the development of different histologies. In addition, methylation of p16, RASSF1A and RUNX3 was mostly cancer‐specific (P<0.05), and may be utilized as a molecular diagnostic marker of NSCLCs.

Demethylation of the Synuclein γ Gene CpG Island in Primary Gastric Cancers and Gastric Cancer Cell Lines

Purpose: Whereas synuclein γ (SNCG) gene expression is usually highly tissue-specific and restricted to the nervous system, SNCG is expressed in advanced-stage breast and ovarian cancers. When overexpressed, SNCG stimulates cancer cell proliferation and metastasis. It is thought that the molecular mechanism of CpG island demethylation may underlie aberrant SNCG expression. To determine whether aberrant SNCG expression and demethylation play a role in gastric carcinogenesis, we examined the expression and methylation status of SNCG in primary gastric cancers, gastric cancer cell lines, and non-neoplastic gastric mucosal tissues. Experimental Design: Ten gastric cancer cell lines, 105 primary gastric cancers, and 10 non-neoplastic gastric mucosal tissues were examined. SNCG expression and methylation status were examined by reverse transcription-PCR and bisulfite-single-strand conformational polymorphism followed by direct sequencing, respectively. The relationship between SNCG methylation status and various clinicopathological factors of the primary gastric cancers was then analyzed. Results: SNCG mRNA expression was observed in 5 of 10 cell lines. Analysis of cell lines positive for SNCG expression revealed that most of the SNCG CpGs were demethylated. SNCG mRNA was not expressed in the 10 non-neoplastic gastric mucosal tissues, although several CpGs were demethylated. Of the 105 primary gastric cancers, 40 (38.1%) showed apparent SNCG demethylation, similar to the result obtained using cell lines. SNCG demethylation was more frequent in primary gastric cancers positive for lymph node metastasis (51%; 26 of 51) than in cancers without lymph node involvement (26%; 14 of 54; P < 0.05), and also more common in stage II-IV (48%; 27 of 56) than in stage I (27%; 13 of 49) cancers (P < 0.05). Conclusions: Aberrant SNCG gene expression can occur via CpG island demethylation, and tends to occur during the more progressive stages of gastric carcinogenesis.

Frequent epigenetic silencing of the p16 gene in non-small cell lung cancers of tobacco smokers.

Epidemiological studies have demonstrated a causal link between tobacco smoking and lung cancer. We investigated the association between inactivation of the p16 gene and tobacco smoking in 51 non‐small cell lung cancers (NSCLCs). Aberrations of the p16 gene were studied by PCR single‐strand conformation polymorphism analysis, followed by direct sequencing, microsatellite analysis, methylation‐specific PCR, and immunohistochemistry. Mutations were detected in 3.9% (2/51) of the tumors; the tumors carrying mutations were from smokers. The incidences of loss of heterozygosity, homozygous deletion, and promoter methylation in 37 smokers vs. 14 non‐smokers were; 45.9% vs. 28.6%, 16.2% vs. 7.1%, and 35.1% vs. 7.1%, respectively. Among these, only the association between promoter methylation and tobacco smoking was statistically significant (P<0.05). Therefore, epigenetic aberration is considered to be a major causative event in p16 silencing by tobacco smoking. Loss of p16 protein expression was apparent in 49% (25/51) of the tumors, and was associated with tobacco smoking (P<0.05) and with histological type (P<0.05). These findings suggest that tobacco smoking leads to inactivation of the p16 gene mainly through the epigenetic mechanism, ultimately increasing the risk of NSCLC, especially the squamous cell histological type.

Detection of HPV-DNA, p53 alterations, and methylation in penile squamous cell carcinoma in Japanese men

Penile carcinoma is a rare disease, accordingly there are few studies on molecular changes, and these results also vary greatly. A total of 26 penile squamous cell carcinomas in Japanese men were studied with respect to HPV, p53 alterations, and methylation of gene promoter region. HPV‐DNA was detected in three of 26 patients (11.5%). Overexpression of p53 was observed in 13 of 26 patients (50%), and p53 gene mutations were detected in four of 26 patients (15.4%). The frequency of methylation was as follows: DAPK, 26.9% (7/26); FHIT, 88.4% (23/26); MGMT, 19.2% (5/26); p14, 3.8% (1/26); p16, 23.1% (6/26); RAR‐β, 23.1% (6/26); RASSF1A, 11.5% (3/26); and RUNX3, 42.3% (11/26). As for correlation between HPV and p53 alterations, and methylation status, mutations of the p53 gene were detected only in HPV‐negative patients, and methylation was more frequently found in HPV‐negative than in HPV‐positive patients. The present results suggest that the majority of penile squamous cell carcinomas in Japanese men are unrelated to HPV, and gene alterations accumulate more frequently in HPV‐unrelated penile carcinomas.

Frequent epigenetic silencing of the FHIT gene in penile squamous cell carcinomas

Methylation of normally unmethylated CpG-rich islands in or near the promoter region has been associated with transcriptional inactivation of tumor-suppressor and tumor-related genes in human cancers. However, so far, only a few studies have searched for DNA methylation in penile carcinoma (PC). On the other hand, human papillomavirus (HPV) has been reported to be associated with the pathogenesis of PC. To elucidate the methylation status of PC and HPV infection, the methylation status of eight genes (DAPK, FHIT, MGMT, p14, p16, RAR-β, RASSF1A, and RUNX3), the incidences of the HPV status, and the expression of Fhit protein were examined in 25 PCs. The frequencies of methylation were: 28% for DAPK, 92% for FHIT, 20% for MGMT, 4% for p14, 24% for p16, 24% for RAR-β, 12% for RASSF1A, and 44% for RUNX3. Negative expression of Fhit protein was observed in 22 of the 25 cancers (88%). Among those 22, 20 showed methylation of the FHIT gene. HPV-DNA was detected in three of the 25 cancers (12%). Methylation of FHIT gene was frequently found than HPV infection, therefore methylation of the FHIT gene is suggested to play an important role in the pathogenesis of penile squamous cell carcinoma.

Peripheral T‐cell lymphoma arising from an intraglandular lymph node in the parotid gland: A case report and literature review

T/NK‐cell lymphoma of the salivary gland is rare. A 58‐year‐old man complained of a tumor mass in the left parotid gland region and he was diagnosed to have a left parotid tumor. The tumor was subsequently resected, revealing a diffuse growth pattern of medium to large sized atypical cells. The tumor was surrounded by fibrous connective tissue in the form of a capsule, and those were positive for CD3, CD4, CD5 and CD30, but negative for Bcl2, CD8, CD10, CD15, CD20, CD25, CD56, CD79a, CD246, EMA, granzyme B, TdT and TIA‐1. There was no molecular evidence of Epstein‐Barr virus (EBV) infection. It was diagnosed as peripheral T‐cell lymphoma (PTCL) arising from an intraglandular lymph node in the parotid gland. In conclusion, Only 17 cases of primary T/NK‐cell lymphoma of the salivary glands have been recorded until now, and the characteristics of these are not clear yet. Additional study is needed.

Parathyroid carcinoma with anaplastic feature: Association of a p53 gene mutation with anaplastic transformation

Parathyroid carcinoma is a rare neoplasm that accounts for only 1–3% of cases of primary hyperparathyroidism. Parathyroid carcinoma is a well‐differentiated tumor that is sometimes difficult to differentiate histopathologically from its benign counterpart, parathyroid adenoma. The molecular mechanism of parathyroid carcinogenesis remains unknown, and investigators have reported that abnormalities of the p53 gene do not play a significant role in parathyroid carcinogenesis, unlike in other human malignancies. The present report describes parathyroid carcinoma with anaplastic transformation of differentiated parathyroid carcinoma in a patient with primary hyperparathyroidism. Nuclear accumulation of p53 protein was found in anaplastic carcinoma cells but not in differentiated carcinoma cells. Polymerase chain reaction–single‐strand conformation polymorphism followed by direct sequencing showed that anaplastic carcinoma cells carried a missense mutation at codon 248 (CGG to CAG) of the p53 gene, while the remaining differentiated carcinoma cells had the wild‐type p53 gene. These findings suggest that the p53 gene mutation is associated with anaplastic transformation of parathyroid carcinoma.

Synchronous Double Malignant Tumors Consisting of Stomach and Hodgkin's Lymphoma with Collision between Gastric Adenocarcinoma and Hodgkin's Lymphoma in the Stomach

We report the rare case of a 72-year-old man with double cancers (gastric adenocarcinoma and Hodgkin’s lymphoma) with collision between gastric adenocarcinoma and Hodgkin’s lymphoma. Abdominal computed tomography showed increased wall thickness in the fundus region of the stomach and multiple lymph node swellings in the lesser curvature, periceliac and left cardial regions. Upper gastrointestinal endoscopy showed an ulcer approximately 5 cm in diameter with a malignant appearance in the fundus region of the stomach. On histopathologic examination, two completely different tumors were recognized in the stomach. One tumor was a poorly differentiated adenocarcinoma characterized by poorly developed tubular structures associated with prominent lymphoid infiltration of the stroma. The other tumor was found to have proliferated in the wall of the stomach, with diffuse granulomatous lesions and bordering the adenocarcinoma. Large atypical lymphoid cells with prominent nucleoli and enlarged mononuclei or multinuclei were seen in the latter tumor. Hodgkin’s lymphoma was also found in the swollen lesser curvature lymph nodes. As a result, gastric adenocarcinoma and metastasis of Hodgkin’s lymphoma were collided in the stomach. In conclusion, this case might be helpful in exploring the occurrence mechanism of tumor collision between lymphoma and carcinoma.

Epstein-Barr virus clonality and plasmacytosis in a patient with atypical angioimmunoblastic T cell lymphoma

Dear Editor, Marked plasmacytosis with angioimmunoblastic T cell lymphoma (AITL) is rare [1–5], and its pathology is unclear. AITL sometimes involves polyor monoclonal proliferation of B cells that are positive or negative for the Epstein-Barr virus (EBV) genome [6]. However, the relationship between EBV-infection and plasmacytosis in AITL has not been comprehensively investigated. An 87-year-old Japanese man complaining of malaise and anorexia was referred to our hospital. His physical examination identified systemic lymphadenopathy and splenomegaly. Laboratory findings were as follows: WBCs, 6400/μL (28% immature to mature plasma cells) (Fig. 1a, b); IgG, 3790 mg/ dL; IgM, 843 mg/dL; IgA, 153 mg/dL; IgE, 379 mg/dL; and a κ/λ ratio of serum-free light chain, 0.5 (normal 0.26–1.65). Immature to mature plasma cells were diverse and positive for CD19, CD30, and CD138, but negative for CD20 or light chain restriction, suggesting their polyclonal proliferation (Fig. 1c). A bone marrow specimen revealed 18% plasma cell-like cells and 10% lymphocytes, which phenotypes were compatible with those of the peripheral blood cells. The histologic features of an inguinal lymph node (LN) showed diffuse and polymorphic proliferation of small-to large-sized abnormal lymphoid cells, eosinophils, numerous plasma cells, and endothelial proliferation (Fig. 1d, e). The smallto medium-sized cells were positive for CD2, CD3, and PD-1, but negative for CD10 and CXCL13 (Fig. 1f). The positive ratio of CD4 to CD8 was 2:1. The larger cells were positive for CD20 and CD79α (Fig. 1g, h). More than half of CD79α-positive cells were positive for CD138 (Fig.1i). Numerous immunoglobulin light chain-positive cells were detected without κ/λ restriction. CD21 staining revealed focally expanded follicular dendritic cell meshworks with some encircling of endothelial venules (Fig. 1j). The large Hodgkin and Reed-Sternberg (HRS)-like cells were mostly positive for CD30, CD20, and PAX5 and negative for CD15 (Fig. 1k). Few EBV-encoded small RNA (EBER)-positive cells were detected in the LN (Fig. 1l). CD68-positive cells were not prominent. Flow cytometry analyses detected two cellular populations in the LN, peripheral blood (PB), and bone marrow. These two populations comprised CD3or CD19-positive populations. Polymerase chain reactions for T cell receptor-β (TCR-β) chain gene rearrangement and for immunoglobulin heavy (IgH) chain gene rearrangement studies detected a clonal TCR-β gene rearrangement in both the LN and PB, but a clonal IgH chain gene rearrangement was only detected in the LN. The TCR-β gene rearrangement band size of the LNwas identical to that of the PB (data not shown). The EBV-DNA concentration was 1.9 × 10 copies/10 PB cells. Terminal repeat analysis for the clonality of the EBV genome revealed a single band in the PB, but no ladders in the LN (Fig. 1m). The EBV-associated antibodies positive for EBV viral capsid antigen IgG and negative for EBV early antigen suggested the latent infection. The present patient developed pneumonia without chemotherapy and passed away shortly after the definite diagnosis of AITL was made. The diagnosis of AITL remains a matter of debate [6]. The presence of monoclonal B cell proliferation including HRSlike cells has induced a pathogenetic or diagnostic confusion with AITL [6–8]. In our patient, large B cells predominantly negative for EBER, a finding supported by the EBV-clonality Okuyama S and Tajima K equally contributed to this study.

Comparison of HER2 immunohistochemical results using a monoclonal antibody (SV2-61γ) and a polyclonal antibody (for Dako HercepTest) in advanced gastric cancer.

We compared a monoclonal antibody (SV2‐61γ) and a polyclonal antibody (Dako HercepTest) in immunohistochemical assessments of human epidermal growth factor receptor 2 (HER2) expression in 73 samples of advanced gastric cancer. Results were scored as 0 to 3+, and equivocal or discordant (SV2‐61γ/Dako HercepTest = 0/2+, 0/3+, 1+/3+ or 2+/3+) cases were subjected to fluorescence in situ hybridization (FISH) analysis. The frequencies of HER2 scores of 2+ or 3+ were 15.1% (11/73) using SV2‐61γ and 38.4% (28/73) using Dako HercepTest. All of the equivocal or discordant cases with a HER2 score of 3+ using Dako HercepTest exhibited amplification of the HER2 gene regardless of the HER2 score determined with SV2‐61γ. The results of the HER2 tests differed according to the antibodies used for immunohistochemistry that preceded FISH analysis, being 15.1% (11/73) using SV2‐61γ and 23.3% (17/73) using Dako HercepTest. Thus, therapeutic decisions might be markedly influenced by the selection of antibody used in the HER2 test.