Mariana Brait

Quantitative detection of Merkel cell virus in human tissues and possible mode of transmission.

Merkel Cell Virus (MCV) is a newly discovered polyomavirus, recently found in a rare skin cancer, Merkel cell carcinoma (MCC). However, MCV has also been detected in some normal tissue samples. We tested and compared the relative quantity of the MCV in a set of diverse human tissue samples with the MCC samples. The levels of MCV in MCCs were over 60 times higher than the highest values in all other tissues. Low quantities of MCV were detected in diverse tissue samples independently of malignant or benign histologic status. Higher levels of the virus were found in the upper aerodigestive tract, digestive system, and saliva compared to the lung and genitourinary system samples. These results confirm that MCV is widespread in the human body and suggest a possible fecal‐oral transmission route similar to the Hepatitis A virus. Despite widespread presence of the virus, it appears that only neuroendocrine skin cells are susceptible to transformation by MCV.

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.

An Epigenetic Marker Panel for Detection of Lung Cancer Using Cell-Free Serum DNA

Purpose: We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients. Experimental Design: To determine the analytic sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of 15 gene promoters from 10 patients with primary lung tumors by using quantitative methylation-specific PCR. We then tested this 15-gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A, and AIM1) for further elucidation of the diagnostic application of this panel of markers. Results: Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI: 25–47) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64–84) but decreased the specificity from 100% to 73% (95% CI: 54–88). Conclusion: This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer. Clin Cancer Res; 17(13); 4494–503. ©2011 AACR.

Cancer epigenetics: above and beyond

Epigenetics refers to the study of mechanisms that alter gene expression without altering the primary DNA sequence. Epigenetic mechanisms are heritable and reversible. Over the last few decades, epigenetics has obtained a large importance in cancer research. Epigenetic alterations are widely described as essential players in cancer progression. They comprise DNA methylation, histone modifications, nucleosome positioning, and small, noncoding RNAs (miRNA, siRNA). They are involved in transcriptional changes and decisive events that will determine cell fate and phenotype. Epigenetics not only offers light into cancer biological processes, but also represents an attractive opportunity of reverting cancer-specific alterations, which may lead, in the future, to a possibility of stopping this disease. Epigenetic changes have been identified as putative cancer biomarkers for early detection, disease monitoring, prognosis, and risk assessment. Other epigenetic alterations are promising therapeutic targets and even therapeutic agents. Emerging discoveries in this area are already contributing to cancer management and monitoring, and a lot more progresses are expected in the future.

Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer.

Purpose: The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features. Experimental Design: We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and χ2 or Fishers exact tests as appropriate. All statistical tests were two-sided. Results: Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion. Conclusion: Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon. (Cancer Epidemiol Biomarkers Prev 2008;17(10):2786–94)

Changes in CpG Islands Promoter Methylation Patterns during Ductal Breast Carcinoma Progression

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

Detection of Promoter Hypermethylation in Salivary Rinses as a Biomarker for Head and Neck Squamous Cell Carcinoma Surveillance

Purpose: Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP). Experimental Design: Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1. Results: We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8–80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2–6.5; P = 0.016). Conclusions: We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients. Clin Cancer Res; 17(14); 4782–9. ©2011 AACR.

The TGFβ–miR200–MIG6 Pathway Orchestrates the EMT-Associated Kinase Switch That Induces Resistance to EGFR Inhibitors

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor β (TGFβ)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFβ-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFβ-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

KIF1A and EDNRB are differentially methylated in primary HNSCC and salivary rinses

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation‐specific PCR (Q‐MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.