Lower LINE-1 Methylation Levels Research Articles

Correlation of LINE-1 hypomethylation with size and dysplasia in colorectal tubular adenomas, and risk for synchronous and metachronous CRC.

e16083 Background: Conventional adenomas (tubular or tubulovillous adenoma; TA) are frequently detected in patients undergoing average risk screening colonoscopy. Risk stratification of adenomas is currently limited to histologic features and size. Molecular features of TA could help further determine risk for development of CRC. Here, we report whether size of TA, high grade dysplasia, and synchronous or metachronous CRC associate with LINE-1 methylation. Methods: LINE-1 methylation was assessed by pyrosequencing of bisulfite-converted DNA. We compared LINE-1 methylation in TA among varying sizes, in the presence of high or low grade dysplasia, and between patients with synchronous and metachronous colorectal cancer. Results: LINE-1 methlyation was found to progressively decrease in TA of increasing size and with high grade dysplasia. TA < 5mm in size (n = 45) had higher LINE-1 methylation levels compared to TA 5-9mm in size (n = 42), and > = 10mm (n = 32) (72.31 ± 6.11 vs 67.50 ± 7.00 vs 66.75 ± 11.89, p = 0.013). TA with high grade dysplasia (n = 26) had lower LINE-1 methylation levels compared to low grade dysplasia (n = 135) (59.86±7.93 vs 67.16±9.20, p < 0.001). Tumor tissue (n = 36) had the lowest levels of LINE-1 methylation at 50.36±8.40. There were lower levels of LINE-1 methylation in TA of patients with synchronous CRC compared to those without (53.07+4.5 vs 59.95+5.4 vs, p < 0.001). LINE-1 methylation was lower in normal tissue from cancer patients compared to that from patients without any neoplasia (50.36 ± 8.40 vs 71.50 ±6.47, p < 0.001). LINE-1 methylation levels were higher in patients with initial low risk TA who developed metachronous high risk TA (n = 31) compared to those who did not (n = 35) (75.78±2.45 vs 64.84±4.58, p = 0.001). LINE-1 methylation levels were not different in patients with initial TA who developed metachronous CRC (n = 2) compared to those who did not (n = 89) (70.00±5.65 vs 66.85±8.20, p = 0.69). Conclusions: High-grade dysplasia and increasing size of conventional TA were associated with greater LINE-1 hypomethylation. This is supportive of a hypothesis that greater LINE-1 hypomethylation of tubular adenomas indicates advancement along the CRC tumorigenesis pathway. Greater LINE-1 hypomethylation in TA was also seen in patients with synchronous CRC compared to those without, though no difference was found in those who developed metachronous CRC. Higher LINE-1 methylation was seen in patients with initial low risk TA, who developed metachronous high risk TA compared to those who did not.

Association between serum 25-hydroxyvitamin D and global DNA methylation in visceral adipose tissue from colorectal cancer patients

BackgroundVisceral adipose tissue (VAT) has been identified as the essential fat depot for pathogenetic theories that associateobesity and colon cancer. LINE-1 hypomethylation has been mostly detected in tumor colon tissue, but less is known about the epigenetic pattern in surrounding tissues. The aim was to analyze for the first time the potential relationship between serum vitamin D, obesity and global methylation (LINE-1) in the visceral adipose tissue (VAT) from patients with and without colorectal cancer.MethodsA total of 55 patients with colorectal cancer and 35 control subjects participated in the study. LINE-1 DNA methylation in VAT was measured by pyrosequencing. Serum 25(OH)D levels were determined by ELISA.ResultsCancer patients had lower levels of LINE-1 methylation in VAT compared with the control group. In the subjects with colorectal cancer, LINE-1 DNA methylation levels were associated positively with vitamin D levels (r = 0,463; p < 0.001) and negatively with BMI (r = − 0.334, p = 0.01) and HOMA insulin resistance index (r = − 0.348, p = 0.01). Serum vitamin D was the main variable explaining the LINE-1% variance in the cancer group (β = 0.460, p < 0.001). In a multivariate analysis, subjects with higher LINE-1 methylation values had lower risk of developing colorectal cancer (OR = 0.53; IC95% =0.28–0.99) compared with the control group.ConclusionsWe showed for the first time an association between LINE-1 DNA methylation in VAT and vitamin D levels in subjects with colorectal cancer, highlighting the importance of VAT from cancer patients, which could be modified epigenetically compared to healthy subjects.

LINE-1 is preferentially hypomethylated within adenomatous polyps in the presence of synchronous colorectal cancer

BackgroundConventional tubular adenomas are frequently detected in patients undergoing average risk screening colonoscopy and are over-represented in patients who will develop colorectal cancer (CRC). Whether features of adenomas could serve as predictors of synchronous CRC is not known. Here, we investigate whether global methylation markers, including LINE-1, differ within adenomas in patients with and without synchronous CRC.MethodsColorectal tubular/tubulovillous adenomatous polyps in the absence (P group, n = 45) and in the presence of synchronous CRC (PC group, n = 32) were identified. Global methylation and demethylation by ELISA for 5-methylcytosine (5-mC) and 5-hydroxymethyl cytosine (5-hmC), respectively, were assessed in polyps and adjacent normal non-neoplastic tissue. LINE-1 hypomethylation was assessed by pyrosequencing of bisulfite-converted DNA as well.ResultsGlobal methylation (5-mC) showed no differences in overall methylation status in the adenomatous polyps in the two groups (5-mC relative to control %, PC group 0.117; P group 0.161, p = 0.148). Global hydroxymethylation 5-hmC was also not significantly different in adenomatous polyps of the PC group than in those of the P group (0.0059 vs 0.0097, p = 0.681). Similarly, global 5-hmC was not different between normal tissues from patients without neoplasia in comparison to those from CRC patients (0.0461 ± 0.080 vs 0.039 ± 0.159, p = 0.215). In contrast, adenomatous polyps of the PC group had lower levels of LINE-1 methylation compared to the adenomas in the P group (53.07 ± 4.5 vs 59.95 ± 5.4, p < 0.001). LINE-1 methylation was also significantly lower in the normal tissue from cancer patients compared to that from patients without any neoplasia (58.07 ± 3.78 vs 71.50 ± 6.47, p < 0.001).ConclusionsLINE-1 hypomethylation of precancerous adenomas correlates with the presence of synchronous CRC. Measurement of DNA hypomethylation levels of colorectal adenomas by LINE-1 could have future implications in approaches to defining CRC risk in screening programs.

Repetitive element hypermethylation in multiple sclerosis patients.

BackgroundMultiple sclerosis (MS) is a complex disorder of the central nervous system whose cause is currently unknown. Evidence is increasing that DNA methylation alterations could be involved in inflammatory and neurodegenerative diseases and could contribute to MS pathogenesis. Repetitive elements Alu, LINE-1 and SAT-α, are widely known as estimators of global DNA methylation. We investigated Alu, LINE-1 and SAT-α methylation levels to evaluate their difference in a case–control setup and their role as a marker of disability.ResultsWe obtained blood samples from 51 MS patients and 137 healthy volunteers matched by gender, age and smoking. Methylation was assessed using bisulfite-PCR-pyrosequencing. For all participants, medical history, physical and neurological examinations and screening laboratory tests were collected. All repetitive elements were hypermethylated in MS patients compared to healthy controls. A lower Expanded Disability Status Scale (EDSS) score was associated with a lower levels of LINE-1 methylation for ‘EDSS = 1.0’ and ‘1.5 ≤ EDSS ≤ 2.5’ compared to an EDSS higher than 3, while Alu was associated with a higher level of methylation in these groups: ‘EDSS = 1.0’ and ‘1.5 ≤ EDSS ≤ 2.5’.ConclusionsMS patients exhibit an hypermethylation in repetitive elements compared to healthy controls. Alu and LINE-1 were associated with degree of EDSS score. Forthcoming studies focusing on epigenetics and the multifactorial pathogenetic mechanism of MS could elucidate these links further.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0395-0) contains supplementary material, which is available to authorized users.

LINE-1 Methylation Status Correlates Significantly to Post-Therapeutic Recurrence in Stage III Colon Cancer Patients Receiving FOLFOX-4 Adjuvant Chemotherapy.

BackgroundMethylation levels of long interspersed nucleotide elements (LINE-1) are representative of genome-wide methylation status and crucial in maintaining genomic stability and expression. Their prognostic impact on colon cancer patients receiving adjuvant chemotherapy has not been well established. We evaluated the association between LINE-1 methylation status and clinicopathologic features and postoperative oncological outcomes in stage III colon cancer patients.Materials and Methods129 UICC stage III colon cancer patients who had received radical resection and FOLFOX adjuvant chemotherapy were enrolled. Global methylation was estimated by analyzing tumor LINE-1 methylation status using bisulfite-polymerase chain reaction (PCR) and pyrosequencing assay. Demographics, clinicopathological data, and postoperative outcomes were recorded by trained abstractors. Outcome measurements included postoperative recurrence and disease-free survival. Univariate, multivariate, and survival analyses were conducted to identify prognostic factors of oncological outcomes.ResultsThe LINE-1 methylation of all 129 patients was measured on a 0–100 scale (mean 63.3; median 63.7, standard deviation 7.1), LINE-1 hypomethylation was more common in patients aged 65 years and above (61.7%±7.6% vs. 64.6±6.4, p=0.019) and those with post-therapeutic recurrence (61.7±7.4 vs 64.3±6.7, p=0.041). Considering risk adjustment, LINE-1 hypomethylation was found to be an independent risk factor of post-therapeutic recurrence (Adjusted OR=14.1, p=0.012). Kaplan-Meier analysis indicated that patients in the low methylation group had shorter period of disease free survival (p=0.01). In a stratified analysis that included 48 patients with post-therapeutic recurrence, it was found that those who experienced shorter period of disease free survival (≦6 months) appeared to have lower LINE-1 methylation levels than patients who reported of recurrence after 6 months (56.68±15.75 vs. 63.55±7.57, p=0.041)ConclusionThere was a significantly greater risk of early postoperative recurrence and a shorter period of disease-free survival in Stage III colon cancer patients exhibiting LINE-1 hypomethylation status after being treated with radical resection and FOLFOX chemotherapy.

Abstract 408: The relationship between LINE-1 hypomethylation and fibrosis status in noncancerous liver tissues of hepatocellular carcinoma patients

Abstract Background: The DNA methylation alterations that occur in human cancers include global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation plays an important role in genomic instability, leading to cancer development. Since LINE-1 or the L1 retrotransposon constitutes a substantial portion of the human genome, LINE-1 methylation status reflects global DNA methylation. Our previous study has revealed that LINE-1 methylation level is a potential surrogate marker for epigenetic field defects induced by tobacco smoking in esophageal cancer patients. Given that liver fibrosis can be one phenomena of field cancerization theory in hepatocellular carcinoma (HCC), we examined the relationship between LINE-1 methylation level and fibrosis status in noncancerous liver tissues of HCC patients. Methods: Using histologically normal liver tissue samples and matched tumor tissue samples from 89 HCC cases, we measured LINE-1 methylation levels by pyrosequencing technology. Results: LINE-1 methylation of noncancerous liver tissues range was 35.8-97.3 of a 0-100 scale (mean: 78.1; median: 81.0; standard deviation: 11.9). HCC tissues showed significantly lower levels of LINE-1 methylation than matched normal tissue (P&amp;lt;0.01). LINE-1 methylation of noncancerous liver tissues was not associated with age, sex, Hepatitis B Virus infection, or Hepatitis C Virus infection. Interestingly, LINE-1 methylation level of noncancerous liver tissues with severe fibrosis (score 2-4) was significantly lower than that with mild fibrosis (score 0-1) (P=0.013). Conclusion: Liver fibrosis in noncancerous liver tissues of HCC patients was significantly associated with LINE-1 hypomethylation. This result supports the potential of LINE-1 methylation level as an indicator of epigenetic field for HCC cancerization. Citation Format: Yoshifumi Baba, Kazuto Harada, Keisuke Kosumi, Hiromitsu Hayashi, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. The relationship between LINE-1 hypomethylation and fibrosis status in noncancerous liver tissues of hepatocellular carcinoma patients. [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 408. doi:10.1158/1538-7445.AM2014-408

1133 Development and Clinical Performance of a Novel Low Coherence Enhanced Backscattering Spectroscopy (LEBS) Fiberoptic Probe for Duodenal Sensing of Pancreatic Cancer Risk

Genome-wide hypomethylation and CpG island hypermethylation play a role in human cancer. We evaluated the utility of measuring epigenetic alterations in pancreatic and biliary fluids as molecular markers for pancreatobiliary cancers. As a surrogate of genome-wide hypomethylation, levels of LINE-1methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of tumor-associated genes was analyzed using MethyLight. Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease. However, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids. CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the ubiquitin carboxylterminal esterase L1 (UCHL1) gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancer, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancer. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines. The results suggest that hypermethylation of the UCHL1 gene plays a key role in the pathogenesis of pancreatobiliary cancers and that detection of hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluids is useful for the diagnosis of these malignancies.

1132 Detection of Pancreatobiliary Cancer by Hypermethylation of the Ubiquitin Carboxyl-Terminal Esterase L1 Gene in Pancreatobiliary Fluids

Genome-wide hypomethylation and CpG island hypermethylation play a role in human cancer. We evaluated the utility of measuring epigenetic alterations in pancreatic and biliary fluids as molecular markers for pancreatobiliary cancers. As a surrogate of genome-wide hypomethylation, levels of LINE-1methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of tumor-associated genes was analyzed using MethyLight. Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease. However, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids. CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the ubiquitin carboxylterminal esterase L1 (UCHL1) gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancer, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancer. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines. The results suggest that hypermethylation of the UCHL1 gene plays a key role in the pathogenesis of pancreatobiliary cancers and that detection of hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluids is useful for the diagnosis of these malignancies.

Changes in LINE-1 methylation level in cancers during metastasis

Background: Significantly lower LINE-1 methylation levels have been observed in several cancers compared to their normal cell counterparts. It is of interest to note that this epigenetic phenomenon is rather common to cancers with multistage oncogenesis. Objective: We compared LINE-1 methylation levels of cancer cells between the primary and matched metastatic sites. Methods: COBRA method was used to determine LINE-1 methylation levels of tumor cells at the primary and matched metastatic sites in five malignancies (10 cases each of head and neck squamous carcinoma, papillary thyroid carcinoma, non-small cell lung cancer, invasive mammary ductal carcinoma, and colorectal adenocarcinoma). LINE-1 methylation levels between the primary and matched metastatic site were compared. Results: Significantly lower LINE-1 methylation levels were observed at metastatic sites (as compared to primary sites) in head and neck squamous cell carcinoma (p = 0.002) and non-small cell lung cancer (p = 0.012). Conclusion: In head, neck, and pulmonary cancers, significantly lower LINE-1 methylation levels were found at metastatic sites, as compared to the matched primary tumor. Further studies are warranted to determine whether this altered methylation is the result or the cause of cancer metastasis. Keywords: LINE-1 methylation, metastasis, multi-step oncogenesis, primary tumor, tumor progression

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002-03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p=0.04), and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p=0.02) and lower levels of fasting HDL (p=0.009). The findings from this study confirm that DNA "global" hypomethylation, (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.

Differential LINE-1 Hypomethylation of Gastric Low-Grade Dysplasia from High Grade Dysplasia and Intramucosal Cancer

Background/AimsGastric epithelial dysplasia is considered a precancerous lesion with a variable clinical course. There is disagreement, however, regarding histology-based diagnoses, which has led to confusion in choosing a therapeutic plan. New objective markers are needed to determine which lesions progress to true malignancy. We measured LINE-1 methylation levels, which have been reported to strongly correlate with the global methylation level in gastric epithelial dysplasia and intramucosal cancer.MethodsA total of 145 tissue samples were analyzed by two histopathologists. All tissues were excised by therapeutic endoscopic mucosal resection and paired with adjacent normal tissue samples. A modified long interspersed nucleotide elements-combined bisulfite restriction analysis (COBRA-LINE-1) method was used.ResultsGastric epithelial dysplasia and intramucosal cancer tissues had significantly lower levels of LINE-1 methylation than adjacent normal gastric tissues. High-grade dysplasia and intramucosal cancer were distinguishable from low-grade dysplasia based on LINE-1 methylation levels. Furthermore, the distinction could be determined with high sensitivity and specificity, as shown by the receiver operating characteristic (ROC) curve (AUC, 0.82; 95% confidence interval, 0.74 to 0.88).ConclusionsLINE-1 methylation levels may provide a diagnostic tool for identifying high-grade dysplasia and intramucosal cancer.

A Novel Correlation between LINE-1 Hypomethylation and the Malignancy of Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens. LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations. Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations.

Abstract 156: Implications of LINE1 methylation for bladder cancer risk in women

Abstract Bladder cancer is the ninth most incident form of cancer in the United States and is an exposure-related disease which contributes significantly to morbidity and health care costs. Of concern is the fact that incidence rates have been stable in men in the last twenty years but have been increasing in women at a rate of 0.2% a year. Although tobacco use and occupational exposures have been linked to bladder cancer, much of the etiology of this disease remains unexplained. Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of this disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer. LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case control study of bladder cancer in New Hampshire. Cases included white, 25-74 year olds, with a histopathologically confirmed diagnosis of bladder cancer while controls were selected from records of the Department of Transportation and the Medicare Program. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation and for a number of different risk factors for bladder cancer including arsenic. Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer (95% CI, 1.12-2.90) in models controlling for gender, age and smoking, with the association being stronger in women than in men (ORs = 2.48, 95% CI 1.19-5.17 in women and 1.47, 95% CI 0.79-2.74 in men). Amongst non-invasive cancers, being in the lowest LINE1 methylation decile conferred an almost two fold risk of bladder cancer (OR 1.94, 95% CI, 1.17-3.22) while the risk was non-significant amongst invasive cancers after adjusting for gender, age and smoking. Amongst controls, women were more likely to have lower LINE1 methylation than men (p-value 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (p-value 0.04). This investigation has identified a novel epigenetic marker of bladder cancer which has the potential for utility as a screening strategy for this disease, and which may aid in providing a more comprehensive understanding of the etiology of bladder cancer. In addition, this study shows an increased risk of LINE1 hypomethylation with high levels of arsenic, raising important questions about the nature of the causal pathway for alterations in DNA repeat methylation to contribute to cancer risk. These results also suggest that women demonstrate lower levels of LINE1 methylation, which may also aid in explaining the disparate incidence of this disease in men and women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 156.