Independent exercise for glottal incompetence to improve vocal problems and prevent aspiration pneumonia in the elderly: a randomized controlled trial.

OBJECTIVES: To evaluate the effect of a self-controlled vocal exercise in elderly people with glottal closure insufficiency. DESIGN: Parallel-arm, individual randomized controlled trial. METHODS: Patients who visited one of 10 medical centers under the National Hospital Organization group in Japan for the first time, aged 60 years or older, complaining of aspiration or hoarseness, and endoscopically confirmed to have glottal closure insufficiency owing to vocal cord atrophy, were enrolled in this study. They were randomly assigned to an intervention or a control group. The patients of the intervention group were given guidance and a DVD about a self-controlled vocal exercise. The maximum phonation time which is a measure of glottal closure was evaluated, and the number of patients who developed pneumonia during the six months was compared between the two groups. RESULTS: Of the 543 patients enrolled in this trial, 259 were allocated into the intervention group and 284 into the control; 60 of the intervention group and 75 of the control were not able to continue the trial. A total of 199 patients (age 73.9 ±7.25 years) in the intervention group and 209 (73.3 ±6.68 years) in the control completed the six-month trial. Intervention of the self-controlled vocal exercise extended the maximum phonation time significantly ( p < 0.001). There were two hospitalizations for pneumonia in the intervention group and 18 in the control group, representing a significant difference ( p < 0.001). CONCLUSION: The self-controlled vocal exercise allowed patients to achieve vocal cord adduction and improve glottal closure insufficiency, which reduced the rate of hospitalization for pneumonia significantly. CLINICAL TRIAL: gov Identifier-UMIN000015567.

Expression of Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma.

BACKGROUND: The side population (SP) of cancer cells is reportedly enriched with cancer stem cells (CSCs), however, the functional role and clinical relevance of CSC marker molecules upregulated in the SP of head and neck squamous carcinoma (HNSCC) cells are yet to be elucidated. Patients with clinical stage I/II (T1-2N0M0) tongue squamous cell carcinoma (TSCC) typically undergo partial glossectomy; however, development of delayed neck metastasis (DNM) tends to reduce their survival. In the present study, we aimed to determine the CSC markers in the SP of HNSCC cells along with their functions in cellular behaviors, and to clarify the association of these markers with DNM. METHODS: Flow cytometry was applied to isolate SP from main population (MP) in HNSCC cells. The expression of the CSC markers was examined by semi-quantitative RT-PCR and immunocytochemistry. In vitro proliferation, migration, and invasion assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Oct3/4 and Nanog were evaluated using surgical specimens from 50 patients with stage I/II TSCC. RESULTS: SPs were isolated in all three cell lines examined. Expression levels of Oct3/4 and Nanog were higher in SP cells than MP cells. Additionally, cell migration and invasion abilities were higher in SP cells than MP cells, whereas there was no difference in proliferation. Univariate analysis showed that expression of Oct3/4 and Nanog, vascular and muscular invasion, and mode of invasion were significantly correlated with DNM. Multivariate logistic regression revealed that Oct3/4 expression (risk ratio = 14.78, p = 0.002) and vascular invasion (risk ratio = 12.93, p = 0.017) were independently predictive of DNM. Regarding the diagnostic performance, Oct3/4 showed the highest accuracy, sensitivity, and NPV of 82.0 %, 61.5 %, and 86.8 %, respectively, while vascular invasion showed the highest specificity and PPV of 94.6 % and 71.4 %, respectively. CONCLUSION: These results suggest that Oct3/4 and Nanog represent probable CSC markers in HNSCC, which contribute to the development of DNM in part by enhancing cell motility and invasiveness. Moreover, along with vascular invasion, expression of Oct3/4 can be considered a potential predictor for selecting patients at high risk of developing DNM.

Prevalence of human papillomavirus in oropharyngeal cancer: a multicenter study in Japan.

BACKGROUND: The incidence rates of oropharyngeal squamous cell carcinoma (OPSCC) have risen steadily in the USA and in northern Europe. These increases are thought to be a consequence of persistent infection with high-risk human papillomavirus (HPV) in OPSCC patients. HPV is an emerging etiologic factor in OPSCC. In Japan, the incidence of OPSCC has significantly increased over the last three decades. However, the population of HPV-positive OPSCC patients is currently unknown. We examined the nationwide trends with regard to HPV incidence in OPSCC patients at 21 specific sites, and examined the relationship between the presence of HPV and survival in OPSCC patients in Japan. METHODS: Tumor samples were obtained from patients with OPSCC prior to treatment, and HPV infection was investigated by polymerase chain reaction (PCR). Hybrid Capture 2 (HC2) was also adopted for swab examination on the surface of fresh tumors. RESULTS: HPV was detected by PCR in 79 (50.3%) out of 157 OPSCC patients. The clinical features of HPV-positive OPSCC were low differentiation, a tendency to involve the lateral wall, and high nodal staging. The sensitivity and specificity of HC2 were 93.7 and 96.2%, respectively, indicating its utility as a screening test. HPV-positive patients had significantly better overall survival and disease-free survival than HPV-negative patients.

Large cell neuroendocrine carcinoma of the submandibular gland: case report and literature review.

Large cell neuroendocrine carcinoma (LCNEC) of the salivary gland is extremely rare. We report on a case of LCNEC in the submandibular gland. A 58-year-old male had a four-month history of an enlarging mass in his left submandibular region. He underwent lymph node resection and metastasis of LCNEC was suspected. Magnetic resonance imaging of the neck showed a solid submandibular gland tumor with marginal blurring. Positron-emission tomography and upper gastrointestinal endoscopy showed no evidence of malignancy other than in the left submandibular gland. He underwent left submandibular gland resection and left upper neck dissection. The final diagnosis was LCNEC of the submandibular gland; surgical margin was negative. Fourteen months later he is free of tumors. This is the first report of LCNEC of the submandibular gland. LCNEC of the salivary gland shows high-grade malignancy like that of the lung. According to past reports, two of four patients died despite multidisciplinary treatments. There are no standard treatments for LCNEC of the salivary glands. More studies are needed to define prognostic factors and establish therapeutic methods.

Present and Future of EGFR Inhibitors for Head and Neck Squamous Cell Cancer.

Although EGFR is expressed at high levels in head and neck squamous cell carcinomas (HNSCCs) and mutations are extremely rare, monotherapy with EGFR inhibitors has shown limited success. The PI3kinase/Akt pathway is responsible for cellular survival, and inhibition of phosphatidylinositol (PI) synthesis has antiproliferative, anti-invasive, and antiangiogenesis effects on HNSCC. Molecular crosstalk has been observed between EGFR and IGF1R signaling through the PI3kinase/Akt pathway in HNSCC, as has molecular crosstalk between the NFκB and STAT3 signaling pathways. Therefore, the combination of an EGFR antagonist with an agent that inhibits the activation of both Akt and NFκB may overcome resistance to EGFR antagonists in HNSCC.

[Hyponatremia with cisplatin administration in head and neck cancer patients].

Hyponatremia is one of the most common electrolyte disorders encountered in clinical practice of medical anticancer treatment. Cisplatin (CDDP) is a well-known chemotherapeutic agent that associates with hyponatremia. We retrospectively studied clinical features of hyponatremia CDDP administration. The incidence of hyponatremia at the first administration was 64. 1%. The significant risk factors of hyponatremia are body weight less than 60 kg, creatinin clearance less than 60mL/min, and sodium depletion and intake loss due to treatment-induced anorexia, nausea, vomiting and diarrhea. The mechanism of hyponatremia induced by CDDP is thought to be mainly renal salt wasting, and sometimes the syndrome of inappropriate secretion of antidiuretic hormone(SIADH).

Identification of stem-like cells in head and neck cancer cell lines.

This study investigated the existence of stem-like cells in established head and neck squamous cell carcinoma (HNSCC) lines, HSC3 and HSC4. Flow cytometric analysis confirmed the presence of side population (SP) cells excluding Hoechst 33342 in HSC4 cells (0.37+/-0.06%) but not HSC3 cells in a reserpine-sensitive manner. After sorting, the SP cells generated both SP and main population (MP) cells in culture while MP cells generated MP cells only. Higher expression of stem cell markers was detected in SP than in MP cells. These results suggest that cancer stem-like cells exist in head and neck squamous cell carcinoma.

Promoted cell proliferation by connexin 30 gene transfection to head-and-neck cancer cell line.

BACKGROUND: There have been inconsistent results regarding the contribution of the connexin family of genes to tumor cell proliferation. MATERIALS AND METHODS: We aimed to clarify the role of connexin 30 (Cx30), by transfecting three kinds of vectors that express either full length Cx30 (Cx30-Full), Cx30 devoid of C-terminal region (Cx30-DelC) or Cx30 C-terminal region (Cx30-CT), in HSC-4, a head-and-neck cancer cell line. RESULTS: Transfected Cx30-Full was localized on the plasma membrane, while Cx30-DelC and Cx30-CT was expressed in the cytoplasm or circumnuclear sites. We studied the effect on the growth rate followed by immunostaining with anti-Ki-67 (MIB-1). The MIB indices of HSC-4 cells transfected with Cx30-Full and Cx30-DelC, but not Cx30-CT were shown to be significantly higher than that of the controls. CONCLUSION: Our results demonstrated that Cx30 enhanced the proliferation of HSC-4 cells and the proliferating activity was considered to be achieved without the transport of the protein onto the plasma membrane.

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 (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta 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. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha 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.

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.

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.

Appropriate diameter for screws to fix the maxilla following Le Fort I osteotomy: an investigation utilizing finite element analysis.

BACKGROUND: After Le Fort I osteotomy, there is sometimes a secondary deformity (relapse), with the lower segment deviating from the intraoperatively fixed position. It is hyopothesized that the structural stability of the reconstructed maxilla is affected by the diameter of the fixation screws. The present study aims to elucidate the relationship between the diameters of the screws and the structural stability of the maxilla after Le Fort I osteotomy. METHODS: 3D models were produced on a workstation from 20 dry skulls and a Le Fort I operation was simulated on them. The upper and lower segments of the divided maxilla in each of the 20 models were connected using four plates and 16 screws. Five different diameters of the fixation screws were tested. Thus altogether 100 models were produced. A 180N load was applied to the molar region for each model. Using finite element analysis, the resultant stresses and deviations of the lower segments were calculated. Finally, referring to these values, the relationships between screw diameters and stability of the lower segment were evaluated. RESULT: The stability of the lower segment was greatest when the diameter of the fixation screws was equal to the thickness of the bone at each fixation site. CONCLUSION: In performing Le Fort I osteotomy, it is recommended that bone thickness is measured at each fixation site in advance, and the diameter of the fixation screws matched accordingly; thereby optimum stability of the reconstructed maxilla can be anticipated.

Decreased expression of connexin-30 and aberrant expression of connexin-26 in human head and neck cancer.

BACKGROUND: The relation of connexins and carcinogenesis has been studied in various organs and cell lines, with connexins currently being considered tumor suppressors. MATERIALS AND METHODS: The expression of connexin-26 (Cx26) and connexin-30 (Cx30) in human head and neck carcinomas, as well as in adjacent normal mucosa, was evaluated by immunohistochemistry. Furthermore, the expression of these connexins with regard to apoptosis and cell differentiation was investigated. RESULTS: In the cancer tissues, Cx30 expression was drastically reduced compared to apparently normal mucosa while Cx26 expression was almost the same. The intracellular distribution patterns of Cx26 and Cx30 in cancer cells differed between cancer locations. In the cancer tissues, expression of Cx26 and Cx30 was not related to apoptosis assessed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) reaction or to cell differentiation assessed by immunoreaction to involucrin. CONCLUSION: The present results suggest that connexins may play a role in the carcinogenesis of head and neck cancer.

PGP9.5 methylation in diffuse-type gastric cancer.

Diffuse-type gastric cancer (DGC) is the most deadly form of gastric cancer and is frequently accompanied by peritoneal dissemination and metastasis. The specific molecular events involved in DGC pathogenesis remain elusive. Accumulating evidence of epigenetic inactivation in tumor suppressor genes led us to conduct a comprehensive screen to identify novel methylated genes in human cancers using pharmacologic unmasking and subsequent microarray analysis. We compared differential RNA expression profiles of DGC and intestinal-type gastric cancer (IGC) cell lines treated with 5-aza-2'-deoxycytidine using microarrays containing 22,284 genes. We identified 16 methylated genes, including many novel genes, in DGC cell lines and studied PGP9.5 with particular interest. In primary gastric cancers, PGP9.5 was found to be more frequently methylated in DGCs (78%) than in IGCs (36%; DGC versus IGC, P < 0.05). Furthermore, real-time methylation-specific PCR analysis of PGP9.5 showed relatively higher methylation levels in DGC than in IGC. Our data thus implicate a molecular event common in the DGC phenotype compared with IGC.

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.

[Aberrant promoter hypermethylation of tazarotine-induced gene 1 (TIG1) in head and neck cancer].

We tested the methylation status of tazarotine induced gene 1 (TI(G1) in head and neck cancer cell lines and primary tumors by the methylation-specific polymerase chain reaction (MSP). MSP showed that the TIG1 promoter was methylated in all cell lines. We then used MSP to check the methylation status of TIG1 in primary head and neck cancer (n = 50). MSP showed TIG1 methylation in 31 (62%) head and neck cancers and no methylation in any normal samples. To confirm MSP results, we directly sequenced dense CpG regions. We found that promoter regions contained methylated cytosines. We thus observed a cancer-specific pattern of TIG1 methylation in primary head and neck cancer. Our results support the notion that promoter methylation is an important mechanism of TIG1 gene inactivation and occurs frequently in head and neck cancer. TIG1 methylation represents a new molecular marker for targeting diagnostic and therapeutic approaches in these cancers.

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.

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 beta2 (RARbeta2), and glutathione S-transferase pi (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, RARbeta2, 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.

Quantitative GSTP1 methylation levels correlate with Gleason grade and tumor volume in prostate needle biopsies.

PURPOSE: Methylation of the GSTP1 gene promoter region is the most common epigenetic change in prostate cancer. The quantitative GSTP1 methylation assay, a recently developed methylation specific and polymerase chain reaction based technique, allows the accurate discrimination of benign prostate tissue and prostate cancer even in small, formalin fixed prostate needle biopsies. In this study we investigated whether GSTP1 methylation correlated with Gleason grade and tumor volume in prostate biopsies. We also compared quantitative GSTP1 methylation with the histological diagnosis in each biopsy. MATERIALS AND METHODS: Formalin fixed, paraffin embedded prostate needle cores (25 mu) were used for the GSTP1 methylation study. After excluding 98 prostate needle cores (47%) with insufficiently extracted DNA 109 biopsies from 40 patients with prostate cancer of various Gleason grades and tumor volume, and benign prostate tissue were tested for GSTP1 methylation using quantitative fluorogenic real-time methylation specific polymerase chain reaction. RESULTS: A total of 13 benign and 96 cancer biopsy parts were included in this study. GSTP1 methylation was positive (GSTP1-to-ACTB ratio 5 or greater) in 83 of 96 cancer parts (86.5%). All 13 benign parts (100%) were negative for GSTP1 methylation with a methylation level of 0 in 12 and 1 in 1. The sensitivity and specificity of quantitative GSTP1 methylation assay were 86.5% and 100%, respectively. Quantitative GSTP1 methylation correlated with Gleason grade, demonstrating higher GSTP1 methylation in higher Gleason grade tumors (Spearman correlation coefficient 0.377, p < 0.0001). Likewise, GSTP1 methylation also correlated with the cancer percent per biopsy (Spearman correlation coefficient 0.567, p < 0.0001). A multiple linear regression model predicted the greater contribution of cancer percent than Gleason grade to quantitative GSTP1 methylation levels. CONCLUSIONS: We confirmed that the quantitative GSTP1 methylation assay reliably discriminates benign and malignant prostate tissues, thus, augmenting the routine histological evaluation of prostate needle biopsies. In addition, quantitative GSTP1 methylation also correlated with prostate cancer Gleason grade and cancer volume, suggesting that quantitative GSTP1 methylation may be of prognostic significance.