Yutaka Tokumaru

Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients. Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control. Results: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-β2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-β2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-β2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation. Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Evaluation of Promoter Hypermethylation Detection in Body Fluids as a Screening/Diagnosis Tool for Head and Neck Squamous Cell Carcinoma

Purpose: To evaluate aberrant promoter hypermethylation of candidate tumor suppressor genes as a means to detect epigenetic alterations specific to solid tumors, including head and neck squamous cell carcinoma (HNSCC). Experimental Design: Using promoter regions identified via a candidate gene and discovery approach, we evaluated the ability of an expanded panel of CpG-rich promoters known to be differentially hypermethylated in HNSCC in detection of promoter hypermethylation in serum and salivary rinses associated with HNSCC. We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21 genes in a limited cohort of patients with HNSCC and normal controls. Using sensitivity and specificity for individual markers as criteria, we selected panels of eight and six genes, respectively, for use in salivary rinse and serum detection and tested these in an expanded cohort including up to 211 patients with HNSCC and 527 normal controls. Results: Marker panels in salivary rinses showed improved detection when compared with single markers, including a panel with 35% sensitivity and 90% specificity and a panel with 85% sensitivity and 30% specificity. A similar pattern was noted in serum panels, including a panel with 84.5% specificity with 50.0% sensitivity and a panel with sensitivity of 81.0% with specificity of 43.5%. We also noted that serum and salivary rinse compartments showed a differential pattern of methylation in normal subjects that influenced the utility of individual markers. Conclusions: Q-MSP detection of HNSCC in serum and salivary rinses using multiple targets offers improved performance when compared with single markers. Compartment-specific methylation in normal subjects affects the utility of Q-MSP detection strategies.

Inverse Correlation between Cyclin A1 Hypermethylation and p53 Mutation in Head and Neck Cancer Identified by Reversal of Epigenetic Silencing

Aberrant promoter hypermethylation of tumor suppressor genes is proposed to be a common feature of primary cancer cells. We recently developed a pharmacological unmasking microarray approach to screen unknown tumor suppressor gene candidates epigenetically silenced in human cancers. In this study, we applied this method to identify such genes in head and neck squamous cell carcinoma (HNSCC). We identified 12 novel methylated genes in HNSCC cell lines, including PGP9.5, cyclin A1, G0S2, bone-morphogenetic protein 2A, MT1G, and neuromedin U, which showed frequent promoter hypermethylation in primary HNSCC (60%, 45%, 35%, 25%, 25%, and 20%, respectively). Moreover, we discovered that cyclin A1 methylation was inversely related to p53 mutational status in primary tumors (P = 0.015), and forced expression of cyclin A1 resulted in robust induction of wild-type p53 in HNSCC cell lines. Pharmacological unmasking followed by microarray analysis is a powerful tool to identify key methylated tumor suppressor genes and relevant pathways.

PGP9.5 Promoter Methylation Is an Independent Prognostic Factor for Esophageal Squamous Cell Carcinoma

PGP9.5/UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family with a potential role in carcinogenesis. We previously identified PGP9.5 as a putative tumor-suppressor gene and methylation of the promoter as a cancer-specific event in primary cancer tissues. In this current study, we analyzed PGP9.5 methylation in 50 esophageal squamous cell carcinoma (ESCC) primary tumors with well characterized clinicopathologic variables including patient outcome. Two independent modalities for methylation analysis (TaqMan methylation-specific PCR and combined bisulfite restriction analysis) were used to analyze these samples. The two data sets were consistent with each other, as the 21 patients (42%) with highest methylation levels by TaqMan analysis all showed visible combined bisulfite restriction analysis bands on acrylamide gels. Using an optimized cutoff value by TaqMan quantitation, we found that patients with higher PGP9.5 methylation ratios in the primary tumor showed poorer 5-year survival rates than those without PGP9.5 methylation (P = 0.01). A significant correlation was also seen between PGP9.5 promoter methylation and the presence of regional lymph node metastases (P = 0.03). Multivariate analysis subsequently revealed that PGP9.5 methylation was an independent prognostic factor for ESCC survival (P = 0.03). These results suggest that PGP9.5 promoter methylation could be a clinically applicable marker for ESCC progression.

Optimal use of a panel of methylation markers with GSTP1 hypermethylation in the diagnosis of prostate adenocarcinoma

Purpose: In this study, we tested the ability of a panel of hypermethylation markers to improve the sensitivity of histologic prostate cancer detection in sextant needle biopsies. Experimental Design: We obtained fresh-frozen sextant biopsies from 72 excised prostates and directly compared blinded histologic review and quantitative real-time methylation-specific PCR for hypermethylation of four genes, Tazarotene-induced gene 1 (TIG1), adenomatous polyposis coli (APC), retinoic acid receptor β2 (RARβ2), and glutathione S-transferase π (GSTP1) to detect the presence of prostate cancer. Results were compared with the final surgical pathological review of the resected prostates as the gold standard. Results: Histologic review alone detected carcinoma with a sensitivity of 64% (39 of 61 cases) and 100% specificity. Quantitative real-time methylation-specific PCR for TIG1, APC, RARβ2, and GSTP1 detected carcinoma with a sensitivity of 70%, 79%, 89%, and 75%, respectively, with 100% specificity for all of the genes. Using this panel of methylation markers in combination with histology resulted in the detection of 59 of 61 (97%) cases of prostate with 100% specificity, a 33% improvement over histology alone. Conclusion: The use of a panel of methylation markers as an adjunct to histologic review may substantially augment prostate cancer diagnosis from needle biopsies.

Biallelic inactivation of the RIZ1 gene in human gastric cancer

The distal short arm of chromosome 1 is commonly deleted in a variety of human neoplasms including gastrointestinal cancer. Genetic alterations of the retinoblastoma protein-interacting zing-finger gene (RIZ)1 including loss of heterozygosity (LOH) at 1p36, frameshift mutations, and promoter hypermethylation were reported previously in several cancers. In this study, we evaluated RIZ1 in 30 primary gastric cancers and found frameshift mutations in two cases (6.7%). Moreover, using real-time quantitative methylation-specific PCR, methylation of the RIZ1 promoter was detected in 11 (37%) cases. In all 11 cases with methylation, inactivation of the second allele occurred through frameshift mutation, LOH or promoter methylation. Our results suggest that RIZ1 is a specific target of inactivation in human gastric cancer.

The role of PGP9.5 as a tumor suppressor gene in human cancer

PGP9.5 is a controversial molecule from an oncologic point of view. We recently identified frequent methylation of PGP9.5 gene exclusively in primary head and neck squamous cell carcinoma (HNSCC), suggesting that it could be a tumor suppressor gene. On the other hand, PGP9.5 was reported to be overexpressed in a subset of human cancers presumably due to intrinsic oncogenic properties or as a result of transformation. To demonstrate that PGP9.5 possesses tumor suppressive activity, we examined forced expression by stable transfection of PGP9.5 in 4 HNSCC cell lines. Although all 4 cell lines demonstrated reduced log growth rates in culture after transfection, only 2 cell lines with wild type p53 (011, 022) demonstrated decreased growth in soft agar. In 2 cell lines with mutant p53 (013, 019), we observed no altered growth in soft agar and increased sensitivity to UV irradiation. We then tested for and found a high frequency of promoter methylation in a larger panel of primary tumors including HNSCC, esophageal SCC, gastric, lung, prostate and hepatocellular carcinoma. Our data support the notion that PGP9.5 is a tumor suppressor gene that is inactivated by promoter methylation or gene deletion in several types of human cancers.

HOP/OB1/NECC1 Promoter DNA Is Frequently Hypermethylated and Involved in Tumorigenic Ability in Esophageal Squamous Cell Carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2′-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPα and HOPβ, respectively), and HOPα and HOPβ were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPβ silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPα silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer. (Mol Cancer Res 2008;6(1):31–41)

A Promoter Methylation Pattern in the N-Methyl-d-Aspartate Receptor 2B Gene Predicts Poor Prognosis in Esophageal Squamous Cell Carcinoma

Purpose: To investigate whether the promoter methylation pattern in N-methyl-d-aspartate receptor 2B (NMDAR2B) is correlated with clinical features of human esophageal squamous cell carcinoma (ESCC), the methylation status of the gene was examined at three different sites (P1, P2, and P3) where two CpG islands reside within 1 kb upstream of the transcription start site. Experimental Design: Three independent modalities for methylation analysis (bisulfite sequencing, combined bisulfite restriction analysis, and TaqMan methylation-specific PCR) were done to analyze total 67 ESCC tissues that included 43 primary tumors with well-characterized clinicopathologic variables including patient outcome. Results: Using an optimized cutoff value based on quantitative methylation-specific PCR, we found that patients with higher NMDAR2B methylation ratio in the proximal region (P1) showed a worse 5-year disease-specific survival rate than those without NMDAR2B methylation (P < 0.006). A significant correlation was also seen between NMDAR2B promoter methylation and the presence of vascular permeation (P = 0.03). Conclusion: NMDAR2B promoter methylation could be a clinically applicable marker in ESCC.