Asuka Murata

PIK3CA Mutation Is Associated with a Favorable Prognosis among Patients with Curatively Resected Esophageal Squamous Cell Carcinoma

Purpose: PIK3CA encodes the catalytic subunit of PI3K, p110α. Mutant PIK3CA stimulates the AKT pathway and promotes cancer cell proliferation. PIK3CA mutations have been associated with poor prognosis in patients with colorectal or lung cancer. In contrast, the relationship between PIK3CA mutations and favorable prognoses has been shown in breast cancer. However, the influence of PIK3CA mutations on the prognosis of patients with esophageal squamous cell carcinoma (ESCC) remains unclear. Experimental Design: Using a nonbiased database of 219 curatively resected ESCCs and eight esophageal cancer cell lines, we evaluated PIK3CA mutational status by pyrosequencing. The expression of p53 and phosphorylated AKT (i.e., AKT activation) was evaluated by immunohistochemistry. Results: PIK3CA mutations in exon 9 and/or 20 were detected in 46 cases (21%). No ESCC cell line harbored PIK3CA mutations. PIK3CA mutations were significantly associated with phosphorylated AKT expression, but not with p53 expression, sex, age at surgery, tobacco use, alcohol use, or histologic grade. Compared with wild-type PIK3CA cases, patients with PIK3CA mutations in exons 9 and/or 20 experienced significantly better disease-free survival [log-rank P = 0.0089; univariate HR: 0.37, 95% confidence interval (CI): 0.15–0.75, P = 0.0042; multivariate HR: 0.34, 95% CI: 0.10–0.86, P = 0.021] and overall survival (log-rank P = 0.012; univariate HR: 0.38, 95% CI: 0.16–0.78, P = 0.0060; multivariate HR: 0.35, 95% CI: 0.10–0.90, P = 0.028). Conclusion: PIK3CA mutations in ESCC are associated with longer survival, suggesting its role as a prognostic biomarker. Future studies are needed to confirm this association and to elucidate the exact mechanisms by which PIK3CA mutations affect tumor behavior. Clin Cancer Res; 19(9); 2451–9. ©2013 AACR.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer

Background:LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs.Methods:Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined.Results:The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1–90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI.Conclusion:Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

LINE-1 Hypomethylation, DNA Copy Number Alterations, and CDK6 Amplification in Esophageal Squamous Cell Carcinoma

Purpose: Global DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis. DNA methylation of the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level, and is attracting interest as a useful marker for predicting cancer prognosis. Our previous study using more than 200 esophageal squamous cell carcinoma (ESCC) specimens demonstrated the significant relationship between LINE-1 hypomethylation and poor prognosis. However, the mechanism by which LINE-1 hypomethylation affects aggressive tumor behavior has yet to be revealed. Experimental Design: To examine the relationship between LINE-1 hypomethylation and DNA copy number variations, we investigated LINE-1–hypomethylated and LINE-1–hypermethylated ESCC tumors by comparative genomic hybridization array. Results: LINE-1–hypomethylated tumors showed highly frequent genomic gains at various loci containing candidate oncogenes such as CDK6. LINE-1 methylation levels were significantly associated with CDK6 mRNA and CDK6 protein expression levels in ESCC specimens. In our cohort of 129 patients with ESCC, cases with CDK6-positive expression experienced worse clinical outcome compared with those with CDK6-negative expression, supporting the oncogenic role of CDK6 in ESCC. In addition, we found that the prognostic impact of LINE-1 hypomethylation might be attenuated by CDK6 expression. Conclusion: LINE-1 hypomethylation (i.e., global DNA hypomethylation) in ESCC might contribute to the acquisition of aggressive tumor behavior through genomic gains of oncogenes such as CDK6. Clin Cancer Res; 20(5); 1114–24. ©2014 AACR.

Primary neuroendocrine tumor in the liver treated by hepatectomy: report of a case.

A 49-year-old woman was admitted to our hospital under suspicion of an enlarging hepatic tumor, which had been previously diagnosed to be a cavernous hemangioma. Computed tomography revealed three enhanced tumors, one measuring 15 cm in diameter within the right lobe of the liver and two intrahepatic metastases in Couinaud’s hepatic segments 3 and 5. We diagnosed the patient to have primary liver cancer, and suspected a combined liver tumor preoperatively. We performed a right trisectionectomy with radiofrequency ablation of the intrahepatic metastasis in S3. According to the immunohistochemical findings of the resected specimen and the findings of postoperative imaging studies, the tumor was diagnosed to be a primary neuroendocrine tumor in the liver. The patient is presently alive without recurrence at 33 months after the operation.

Abstract 1357: Clinical, pathological and prognostic value of LINE-1 methylation in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objectives:Cancer cells exhibit 2 types of deoxyribonucleic acid (DNA) methylation alterations: genome-wide DNA hypomethylation and site-specific CpG island promoter hypermethylation. Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. As long interspersed nucleotide element-1 (LINE-1) retrotransposon constitutes a substantial portion (approximately 17%) of the humangenome, the level of LINE-1 methylation is regarded to be a surrogate marker of genome-wide DNA methylation. Importantly, LINE-1 hypomethylation is associated with poor prognosis in several type of cancers, suggesting the potencial role of the LINE-1 methylation level as a useful marker for predicting cancer prognosis. However, clinical, pathological and prognostic significance of LINE-1 hypomethylaiton in hepatocellular carcinoma (HCC) remains unclear. Methods: Using 160 HCC resected specimens, we quantified the LINE-1 methylation by utilizing the bisulfite pyrosequencing technology. This assay amplifies a region of LINE-1 element (position 305-331 in accession no. [X58075][1]), which includes 4 CpG cites. The average of the relative amounts of C in the 4 CpG sites was used as the overall LINE-1 methylation level.Results: We first examined the LINE-1 methylation level in 85 HCC tissues and matched normal hepatic parenchyma samples. The cancer tissues exhibited significantly lower levels of LINE-1 methylation (median, 64.5 ; range, 99.1-21.5) than the matched normal hepatic parenchyma (median, 81.0 ; range, 97.2-35.8) (P 0.09). In the Kaplan-Meier analysis, LINE-1 hypomethylators (methylation level < 74.4) experienced significantly shorter disease-free survival than those with hypermethylators (log-rank P=0.029). Over-all survival is not associated with LINE-1 methylation ( log-rank P=0.16). Conclusions: Genome-wide DNA hypomethylation, as measured in LINE-1, is associated with disease-free survival among patients with HCC, suggesting that LINE-1 hypomethylation may be a biomarker that can be used to identify patients who will experience an inferior outcome. Citation Format: kazuto harada, Yoshirumi Baba, Akira Chikamoto, Takatsugu Ishimoto, Keisuke Kosumi, Asuka Murata, Naoya Yoshida, Toru Beppu, Hideo Baba. Clinical, pathological and prognostic value of LINE-1 methylation in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2014-1357 [1]: /lookup/external-ref?link_type=GEN&access_num=X58075&atom=%2Fcanres%2F74%2F19_Supplement%2F1357.atom

Abstract 420: Tet family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma

Objective: DNA methylation is a fundamental epigenetic process that modulates gene expression. Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Although the base 5-methylcytosine (5mc) and the DNA methyltransferase (DNMT) family of enzymes that catalyze DNA methylation are well-studied, the mechanisms of DNA demethylation are poorly understood. The Ten-eleven translocation (Tet) family of enzymes (Tet1, Tet2, and Tet3) has been implicated in DNA demethylation. These enzymes possess the dioxygenase activity and convert 5-mC to 5-hydroxymethylcytosine (5-hmC). Frequent Tet mutational inactivation or reduction has been reported to associate with decreased 5-hmC levels in various types of myeloid leukemia and melanoma. However, the status of 5hmC and Tet expression in esophageal squamous cell carcinoma (ESCC) remains still unknown. Method: We analyzed the 5-hmC status in DNA from ESCC frozen tissues utilizing the ELISA test, and 5-hmC expression was evaluated by immunohistochemistry. We next examined the expression levels of Tet family mRNA in both normal and tumor tissues by RT-qPCR. Results: 5-hmC level was significantly lower in ESCC than in paired normal tissue in ELISA test (n=33, P Conclusion: The 5hmC expression was decreased in ESCC tissues, and was associated with Tet2 expression level, suggesting that 5hmC and Tet2 might play a crucial role in the development of ESCCs. Citation Format: Asuka Murata, Yoshifumi Baba, Ryuichi Karashima, Satoshi Ida, Yu Imamura, Takatsugu Ishimoto, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Tet family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2014-420

Granulocyte-colony-stimulating factor producing esophageal squamous cell carcinoma: a report of 3 cases

Although different types of granulocyte-colony-stimulating factor (G-CSF)-producing carcinomas have been reported, G-CSF-producing esophageal squamous cell carcinoma (SCC) is extremely rare. The prognosis of G-CSF-producing esophageal cancer has generally been reported to be very poor. Here, we report three cases of G-CSF-producing SCC who were successfully treated by use of multimodal therapy.

Abstract 2965: Relationship between LINE-1 methylation level, allelic imbalance and PIK3CA mutation in esophageal squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Epigenetic changes such as DNA methylation, histone modification, and loss of genome imprinting play a crucial role in esophageal squamous cell carcinogenesis, along with genomic and genetic alterations. DNA methylation is a fundamental epigenetic process that modulates gene expression. Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Genome-wide DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis. Since LINE-1 or the L1 retrotransposon constitutes a substantial portion (17%) of the human genome, the level of LINE-1 methylation is regarded to be a surrogate marker of global DNA methylation. Our group has reported that esophageal squamous cell carcinomas (ESCCs) patients with LINE-1 hypomethylated tumor experience poorer prognosis compared with those with LINE-1 hypermethylated tumors (Ann Surg, 2012). Method: To examine the potential mechanism by which LINE-1 hypomethylation affects tumor aggressive behavior, we performed an array-based comparative genomic hybridization (array-CGH; Agilent 400K) analysis utilizing LINE-1 hypomethylated ESCC tissues (n=3) and LINE-1 hypermethylated ESCC tissues (n=3). In addition, we assessed the relationship between LINE-1 methylation level and mutations of KRAS, BRAF, and PIK3CA using a non-biased cohort of 202 ESCCs and Pyrosequencing technology. Results: Array-CGH analysis revealed that genomic gains and losses frequently occurred only in LINE-1 hypomethylated ESCCs; genomic gains were found at 1q42, 1q44, 2p16, 3q13, 3q29, 6q22, 7p21-22, 7q11, 7q31, 8q11, 8q21, 9q21-22, 10q11, 10q26, 11p11, 11q22, 17q11-12, 17q21, and 20p12, while losses were found at 2q35, 3p21, 3p14, 4q25, 4q28, 5p31 and 6p21. On the other hand, LINE-1 hypermethylated tumors showed no significant genomic gain and bare genomic loss (10q11). KRAS mutation was extremely rare (1/202; 0.5%), and BRAF mutation was absent (0/202; 0%). PIK3CA in exon 9 and/or 20 mutation was detected in 45 of 202 cases (22%). Interestingly, PIK3CA wild-type tumors showed significantly lower LINE-1 methylation level compared with PIK3CA mutated tumors (p=0.027). Conclusion: Our data suggest a relationship between LINE-1 methylation level (global DNA methylation level), allelic imbalances (copy number alterations) and oncogene mutation (PIK3CA). This relationship may confer a poor prognosis in ESCCs. Further studies are necessary to elucidate mechanism(s) by which LINE-1 hypomethylation affects tumor behavior. Citation Format: Yoshifumi Baba, Masayuki Watanabe, Asuka Murata, Hironobu Shigaki, Shiro Iwagami, Masaaki Iwatsuki, Takatsugu Ishimoto, Hideo Baba. Relationship between LINE-1 methylation level, allelic imbalance and PIK3CA mutation in esophageal squamous cell carcinoma. [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 2965. doi:10.1158/1538-7445.AM2013-2965

Abstract 648: Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer.

Background: Cancer cells exhibit two types of DNA methylation changes: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. As LINE-1 or L1 retrotransposon constitutes a substantial portion of the human genome, the level of L1 methylation is regarded to be a surrogate marker of global DNA methylation. In Colorectal cancer (CRC), L1 methylation in primary tumor has been shown to be highly variable, and L1 hypomethylation is strongly associated with poor prognosis. Nonetheless, no study has examined L1 methylation level in metastatic lesions [liver, lung, and lymph node (LN)] from CRCs, and assessed the heterogeneity of L1 methylation level within CRC tumor. Methods: A total of 65 patients with liver and/or lung metastases from CRC who were undergoing hepatic and/or lung resection at Kumamoto University Hospital were enrolled in this study. The bisulfite-pyrosequencing technology was utilized to quantify the L1 methylation level in 60 liver metastases, matched 26 primary tumors and matched 6 LN metastases, and 6 paired lung metastatic and primary tumors. The heterogeneity within primary tumor (superficial area vs. invasive area) was also evaluated (n=6). Results: The distribution of the L1 methylation level in liver metastases (n=60) was as follows: mean, 68.9; median, 71.0; SD, 10.8; range, 37.1-90.1 (all in 0-100 scale). L1 methylation level of liver metastases was not associated with sex, age at hepatic resection, tumor stage of primary lesion, or tumor size. L1 methylation level of liver metastases was significantly related with that of matched primary tumors (n=26; Pearson correlation coefficient r=0.71, p=0.0001). LN metastases showed similar L1 methylation level of matched primary tumors and liver metastases (n=6); the distribution of the L1 methylation level in matched 6 LN metastases was as follows: mean, 71.0; SD, 9.37; range, 59.3-81.9. In addition, lung metastases and matched primary tumors exhibited similar L1 methylation level; the distributions of the L1 methylation level in lung metastases and matched primary tumors (n=6) were as follows: mean, 65.6 and 71.3; range, 55.7-82.2 and 66.1-79.6, respectively. Interestingly, the heterogeneity of L1 methylation level was not observed; the difference between superficial area and invasive area was within 7 in all 6 cases. Conclusions: Liver, lung and LN metastases showed similar L1 methylation level with primary tumors. In addition, L1 methylation level of superficial area was roughly in accordance with that of invasive area; thus supporting the absence of heterogeneity of L1 methylation level within one tumor. These results may suggest that cancer cells acquire “global DNA hypomethylation” at the early stage of tumor development, and it is preserved through the process of cancer invasion and metastasis. Citation Format: Asuka Murata, Yoshifumi Baba, Masayuki Watanabe, Hironobu Shigaki, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Hideo Baba. Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer. [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 648. doi:10.1158/1538-7445.AM2013-648