Yasunori Matsuda

Revisit of Field Cancerization in Squamous Cell Carcinoma of Upper Aerodigestive Tract: Better Risk Assessment with Epigenetic Markers

We quantified field cancerization of squamous cell carcinoma in the upper aerodigestive tract with epigenetic markers and evaluated their performance for risk assessment. Methylation levels were analyzed by quantitative methylation-specific PCR of biopsied specimens from a training set of 255 patients and a validation set of 224 patients. We also measured traditional risk factors based on demographics, lifestyle, serology, genetic polymorphisms, and endoscopy. The methylation levels of four markers increased stepwise, with the lowest levels in normal esophageal mucosae from healthy subjects without carcinogen exposure, then normal mucosae from healthy subjects with carcinogen exposure, then normal mucosae from cancer patients, and the highest levels were in cancerous mucosae (P < 0.05). Cumulative exposure to alcohol increased methylation of homeobox A9 in normal mucosae (P < 0.01). Drinkers had higher methylation of ubiquitin carboxyl-terminal esterase L1 and metallothionein 1M (P < 0.05), and users of betel quid had higher methylation of homeobox A9 (P = 0.01). Smokers had increased methylation of all four markers (P < 0.05). Traditional risk factors allowed us to discriminate between patients with and without cancers with 74% sensitivity (95% CI: 67%–81%), 74% specificity (66%–82%), and 80% area under the curve (67%–91%); epigenetic markers in normal esophageal mucosa had values of 74% (69%–79%), 75% (67%–83%), and 83% (79%–87%); and both together had values of 82% (76%–88%), 81% (74%–88%), and 91% (88%–94%). Epigenetic markers done well in the validation set with 80% area under the curve (73%–85%). We concluded that epigenetics could improve the accuracies of risk assessment. Cancer Prev Res; 4(12); 1982–92. ©2011 AACR.

Estimation of the fraction of cancer cells in a tumor DNA sample using DNA methylation.

Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.

Hypomethylation of Alu repetitive elements in esophageal mucosa, and its potential contribution to the epigenetic field for cancerization

BackgroundAberrant hypermethylation of specific genes is present in esophageal squamous cell carcinomas (ESCCs). Such hypermethylation is also present in normal-appearing esophageal mucosae of ESCC patients and is considered to contribute to the formation of a field for cancerization. On the other hand, the presence of global hypomethylation in ESCCs or in their background esophageal mucosae is unknown.MethodWe collected 184 samples of esophageal mucosae (95 normal mucosae from healthy subjects, and 89 non-cancerous background mucosae from ESCC patients) and 93 samples of ESCCs. Methylation levels of repetitive elements (Alu, LINE1) and cancer/testis antigen genes (NY-ESO-1, MAGE-C1) were measured by bisulfite pyrosequencing and quantitative methylation-specific PCR, respectively.ResultsMethylation levels of Alu, LINE1, NY-ESO-1, and MAGE-C1 were significantly lower in ESCCs than in their background and normal mucosae. Also, in the background mucosae, a significant decrease of the Alu methylation level compared with the normal mucosae was present. In ESCCs, methylation levels of the two repetitive elements and the two cancer/testis antigen genes were correlated with each other.ConclusionThis is the first study to show the presence of global hypomethylation in ESCCs, and even in their non-cancerous background mucosae. Alu hypomethylation might reflect the severity of an epigenetic field for cancerization.

A two-year follow-up study of chronic fatigue syndrome comorbid with psychiatric disorders.

Aims:  Chronic fatigue syndrome patients often have comorbid psychiatric disorders such as major depressive disorders and anxiety disorders. However, the outcomes of chronic fatigue syndrome and the comorbid psychiatric disorders and the interactions between them are unknown. Therefore, a two‐year prospective follow‐up study was carried out on chronic fatigue syndrome patients with comorbid psychiatric disorders.

The Relationship Between Medial Temporal Lobe Atrophy and Cognitive Impairment in Patients With Dementia With Lewy Bodies

Background: The relationship between medial temporal lobe atrophy (MTA) and cognitive impairment in patients with dementia with Lewy bodies (DLB) remains unclear. We examined this relationship using voxel-based specific regional analysis system for Alzheimer disease (VSRAD) advance software, which allowed us to quantify the degree of MTA on images obtained from magnetic resonance imaging (MRI) scans. Methods: Thirty-seven patients diagnosed with DLB were recruited and scanned with a 1.5 Tesla MRI scanner. All MRI data were analyzed using VSRAD advance. The target volume of interest (VOI) included the entire region of the entorhinal cortex, hippocampus, and amygdala. The degree of MTA was obtained from the averaged positive z-score (Z score) on the target VOI, with higher scores indicating more severe MTA. Mini-Mental State Examination (MMSE) and the Revised Hasegawa Dementia Scale (HDS-R), which strengthened the measures of memory and language more than MMSE, were used to assess the presence of cognitive impairment. Results: A negative correlation was found between the Z score and MMSE total scores or the HDS-R total scores. A stepwise multiple regression analysis performed to adjust the covariate effects of sex, age, the onset age of the disease, duration of DLB, years of education, and donepezil treatment showed that the HDS-R total scores were independently associated with the Z score, whereas MMSE total scores were not. Conclusions: These results suggest that MTA is related to cognitive impairment in patients with DLB, particularly the regions of orientation, immediate and delayed recall, and word fluency.

Attention to anomalies of the right pulmonary vein in subcarinal lymph node dissection in radical esophagectomy for cancer

Here, we report on an anomaly of the right pulmonary vein in the subcarinal area and emphasis the importance of its focus in subcarinal lymph node dissection. A 51-year-old Japanese man underwent thoracoscopic radical esophagectomy with regional lymph node dissection for esophageal carcinoma T1bN1M0, stage IIB. While dissecting the subcarinal lymph node, we encountered a thick vein crossing posterior to the intermediate right bronchus. We recognized the anomalous right pulmonary vein (anomalous V2) that drained into the left atrium from the right posterior segment (S2). We cautiously dissected the subcarinal lymph node and were able to preserve the anomalous vein. This anomalous pulmonary vein can cause serious complications. If the anomalous V2 had been injured or ligated, congestion of the right S2 or cardiac tamponade might have develop. Since anomalous pulmonary veins are easily identified by enhanced computed tomography, careful observation is essential for avoiding their unnecessary injury.

Cytomegalovirus-associated ulceration of gastric conduit after chemoradiotherapy following esophagectomy for cancer

A 64-year-old man underwent radical esophagectomy for cancer and simultaneous reconstruction using the gastric conduit through the posterior mediastinum. Two courses of adjuvant chemotherapy were performed. Twenty-eight months postoperatively, recurrence of the cancer was detected in the mediastinal lymph nodes, and he underwent concurrent chemoradiotherapy and boost chemotherapy. Endoscopy was then performed to investigate the cause of epigastralgia, and multiple ulcerations were found in the lesser curvature of the gastric conduit. Although a proton-pump inhibitor was orally administered, the ulceration was intractable. Re-examination of the original biopsy specimens and serological testing revealed positivity for cytomegalovirus. The ulcers began to heal after administration of foscarnet sodium. After the treatment, no signs of exacerbation associated with reinstitution of chemotherapy were observed.

Abstract 5375: Androgen excess induces aberrant DNA methylation in the prostate.

Aberrant DNA methylation is deeply involved in prostate carcinogenesis. However, inducers of aberrant DNA methylation in the prostate are almost unknown. Here, we investigated whether androgen excess can induce aberrant DNA methylation or not, using a rat prostate cancer model. First, by analysis of reported data of expression microarray after treatment of a prostate cancer cell line with a demethylating agent, we isolated two marker genes (Mmp23, Nkx3.1) whose promoter CpG islands were aberrantly methylated in testosterone and 3,2′-dimethyl-4-aminobiphenyl-induced invasive adenocarcinomas. These two genes and three genes previously isolated (Aebp1, Amn1, Tgfbr2) [Yamashita et al. Cancer Res. 2008] were used as markers. Then, we analyzed temporal profiles of DNA methylation levels of the five genes in the dorsolateral lobe of rats treated with excessive androgen. Testosterone was administrated every five weeks for 15, 30, 40 and 50 weeks from six weeks old by subcutaneous implantation of a Silastic tube containing 40 mg testosterone propionate. Aberrant DNA methylation of two genes (Amn1, Mmp23) was gradually induced during the testosterone treatment while that of the other three genes was not. The former two genes had lower mRNA expression levels in the normal prostate than the latter three genes, which was in accordance with the known fact that genes with low transcription are susceptible to DNA methylation. Third, we investigated molecular mechanisms for aberrant DNA methylation induction by androgen excess. Androgen excess did not induce mRNA expression of DNA methyltransferases; Dnmt1, Dnmt3a or Dnmt3b in the rat prostrate. As for histone modifications, it did not induce significant changes in either H3K4me3 or H3K27me3 levels of the five genes methylated in rat prostate cancer. In contrast, it induced infiltration of lymphocytes and neutrophils in the prostate. Temporal profiles of expression levels of specific inflammation-related genes (Cxcl2, Il1b, Nos2 and Tnf) paralleled the methylation levels of Amn1 and Mmp23. Androgen excess did not induce bacterial infection as determined by copy number of bacterial DNA, but induced increase in Ki67-positive cells in regions with and without infiltration of inflammatory cells. The inflammatory response, known as a major mechanism for aberrant DNA methylation induction, along with cell proliferation was likely to be the cause of aberrant DNA methylation induction in the rat prostate with androgen excess. These results demonstrated that androgen excess can induce aberrant DNA methylation in the prostate, and suggested that inflammatory response underlies aberrant DNA methylation induction by androgen excess. Citation Format: Satoshi Yamashita, Satoru Takahashi, Yasunori Matsuda, Ken Gyobu, Toshikazu Ushijima. Androgen excess induces aberrant DNA methylation in the prostate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2013-5375