Shahnaz Begum

Publications (43)244.27 Total impact

• Article: GATA3 Immunohistochemical Expression in Salivary Gland Neoplasms.

  Lauren E Schwartz, Shahnaz Begum, William H Westra, Justin A Bishop
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  ABSTRACT: GATA3 is a zinc finger transcription factor that regulates the normal development of many tissues and cell types. Recent studies have shown that immunohistochemical nuclear staining for GATA3 among tumors is highly restricted to carcinomas of breast and urothelial origin; however salivary gland tumors have not been tested. Given that breast and salivary gland tissues are very similar with respect to embryologic development and structure, we performed GATA3 staining on a spectrum of salivary gland neoplasms. GATA3 immunohistochemistry was performed on a diverse collection of 180 benign and malignant salivary gland neoplasms including 10 acinic cell carcinomas, 2 adenocarcinomas not otherwise specified, 41 adenoid cystic carcinomas, 2 epithelial-myoepithelial carcinomas, 1 low grade cribriform cystadenocarcinoma, 15 mammary analogue secretory carcinomas, 7 metastatic squamous cell carcinomas, 27 mucoepidermoid carcinomas, 2 oncocytic carcinomas, 5 oncocytomas, 34 pleomorphic adenomas, 4 polymorphous low grade adenocarcinomas, 25 salivary duct carcinomas, and 5 Warthin tumors. Staining for GATA3 was observed in 92/180 (51 %) of salivary gland tumors. GATA3 staining was observed in most of the tumor types, but diffuse immunolabeling was consistently seen in salivary duct carcinoma (25 of 25) and mammary analogue secretory carcinoma (15 of 15)-the two tumor types that most closely resemble breast neoplasia. Background benign salivary gland tissue was also usually weakly positive in both acini and ducts. GATA3 immunostaining is not restricted to tumors of breast and urothelial origin. Rather, it is expressed across many different types of salivary gland neoplasms. As a result, salivary gland origin should be considered in the differential diagnosis of a GATA3-positive carcinoma, particularly in the head and neck. Although GATA3 immunohistochemistry is not helpful in resolving the differential diagnosis between a primary salivary gland neoplasm and metastatic breast cancer, it may have some utility in subtyping salivary gland tumors, particularly salivary duct carcinoma and mammary analogue secretory carcinoma.
  Head and Neck Pathology 04/2013;
• Article: Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling.

  Semra Demokan, Alice Y Chuang, Xiaofei Chang, Tanbir Khan, Ian M Smith, Kavita M Pattani, Santanu Dasgupta, Shahnaz Begum, Zubair Khan, Nanette J Liegeois, William H Westra, David Sidransky, Wayne Koch, Joseph A Califano
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  ABSTRACT: Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2'-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.
  International Journal of Oncology 04/2013; 42(4):1427-36. · 2.40 Impact Factor
• Article: Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma.

  Mariana Brait, Enrico Munari, Cynthia Lebron, Maartje G Noordhuis, Shahnaz Begum, Christina Michailidi, Nilda Gonzalez-Roibon, Leonel Maldonado, Tanusree Sen, Rafael Guerrero-Preston, Leslie Cope, Paola Parrella, Vito Michele Fazio, Patrick K Ha, George J Netto, David Sidransky, Mohammad O Hoque
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  ABSTRACT: Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 mo of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.
  Cell cycle (Georgetown, Tex.) 02/2013; 12(7). · 5.36 Impact Factor
• Article: SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling.

  Santanu Dasgupta, Jin Sung Jang, Chunbo Shao, Nitai D Mukhopadhyay, Upneet K Sokhi, Swadesh K Das, Mariana Brait, Conover Talbot, Rex C Yung, Shahnaz Begum, William H Westra, Mohammad Obaidul Hoque, Yang Ping, Joanne E Yi, Stephan Lam, Adi F Gazdar, Paul B Fisher, Jin Jen, David Sidransky
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  ABSTRACT: The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p = 0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced overexpression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p = 0.0015-0.030) and in vivo (p = 0.016) cellular growth, invasion (p = 0.029-0.049), and colony formation (p = 0.023-0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser(473)), STAT3 (Tyr(705)), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated ß-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
  Journal of Molecular Medicine 09/2012; · 4.67 Impact Factor
• Article: AIM1 promoter hypermethylation as a predictor of decreased risk of recurrence following radical prostatectomy.

  Eli Rosenbaum, Shahnaz Begum, Mariana Brait, Marianna Zahurak, Leonel Maldonado, Leslie A Mangold, Mario A Eisenberger, Jonathan I Epstein, Alan W Partin, David Sidransky, Mohammad O Hoque
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  ABSTRACT: To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3, and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy (RP). Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and a minimum follow-up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed using the Log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. At a median follow-up time of 10 years, 48 patients (50.5%) had evidence of recurrence: Biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: Older age, HR = 0.95 (95% CI: 0.91, 1.0) (P = 0.03), positive lymph nodes HR = 2.11 (95% CI: 1.05, 4.26) (P = 0.04), and decreased hypermethylation of AIM1 HR = 0.45 (95% CI: 0.2, 1.0) (P = 0.05). Methylation status of AIM1 in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.
  The Prostate 11/2011; 72(10):1133-9. · 3.48 Impact Factor
• Article: An epigenetic marker panel for detection of lung cancer using cell-free serum DNA.

  Shahnaz Begum, Mariana Brait, Santanu Dasgupta, Kimberly L Ostrow, Marianna Zahurak, André L Carvalho, Joseph A Califano, Steven N Goodman, William H Westra, Mohammad Obaidul Hoque, David Sidransky
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  ABSTRACT: We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients. 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. 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). 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.
  Clinical Cancer Research 05/2011; 17(13):4494-503. · 7.74 Impact Factor
• Article: A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma.

  Myriam Loyo, Mariana Brait, Myoung S Kim, Kimberly L Ostrow, Chunfa C Jie, Alice Y Chuang, Joseph A Califano, Nanette J Liégeois, Shahnaz Begum, William H Westra, Mohammad O Hoque, Qian Tao, David Sidransky
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  ABSTRACT: Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In our study, we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumor's biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation-specific polymerase chain reaction (PCR) in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fisher's exact test and quantitative values were optimized using cut off values derived from receiver-operator characteristic curves. The methylation status of AIM1, APC, CALCA, deleted in colorectal carcinomas (DCC), DLEC, deleted in liver cancer 1 (DLC1), estrogen receptor alpha (ESR), FHIT, KIF1A and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.
  International Journal of Cancer 03/2011; 128(6):1393-403. · 5.44 Impact Factor
• Article: DeltaNp63alpha confers tumor cell resistance to cisplatin through the AKT1 transcriptional regulation.

  Tanusree Sen, Nilkantha Sen, Mariana Brait, Shahnaz Begum, Aditi Chatterjee, Mohammad Obaidul Hoque, Edward Ratovitski, David Sidransky
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  ABSTRACT: Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.
  Cancer Research 02/2011; 71(3):1167-76. · 7.86 Impact Factor
• Article: Genetic and epigenetic analysis of erbB signaling pathway genes in lung cancer.

  Mohammad Obaidul Hoque, Mariana Brait, Eli Rosenbaum, Maria Luana Poeta, Prodipto Pal, Shahnaz Begum, Santanu Dasgupta, Andre L Carvalho, Steven A Ahrendt, William H Westra, David Sidransky
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  ABSTRACT: Prognosis for patients with non-small cell lung cancer (NSCLC) is poor. The potential value of modulating epidermal growth factor receptor (EGFR) for treatment is reflected by the recent approval of specific drugs that inhibit its activity. Mutations in EGFR were reported in lung cancer and generated interest, once they enable the identification of lung cancers likely to respond to various targeted small molecules. We tested three key genetic and epigenetic alterations (EGFR, RASSF1A, and BRAF) of this pathway on a series of primary NSCLC (total 111; adenocarcinoma 49, squamous cell carcinoma [SCC] 48, and others 14). The mutational status of KRAS (and p53) was known for these samples. The purpose of this study was to define the pattern of erbB pathway alterations in NSCLC and to test for associations with clinical parameters. Five EGFR mutations were identified: three in adenocarcinoma (6%), one in SCC (2%), and one in adenocarcinoma with bronchoalveolar component tumor (7%). EGFR mutations included three in-frame deletions in exon 19 and two point mutations in exon 21. Promoter methylation of RASSF1A was detected in 25 of 45 adenocarcinomas and 18 of 46 SCC. Mutations of EGFR, BRAF, and KRAS in adenocarcinoma were mutually exclusive and inversely correlated with RASSF1A methylation (p = -0.394; p = 0.007). Overall, genetic and/or epigenetic alterations of erbB pathway genes were detected in 80% (39/49) of adenocarcinomas.Approximately half of primary adenocarcinoma harbor molecular alterations of the erbB pathway. Careful characterization of these alterations and response to anti-EGFR therapies is warranted to determine better and accurate determinants of clinical response.
  Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 12/2010; 5(12):1887-93. · 4.55 Impact Factor
• Source

  Available from: Satyendra Chandra Tripathi

  Article: Promoter hypermethylation in Indian primary oral squamous cell carcinoma.

  Jatinder Kaur, Semra Demokan, Satyendra Chandra Tripathi, Muzafar Ahmad Macha, Shahnaz Begum, Joseph A Califano, Ranju Ralhan
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  ABSTRACT: We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian-origin and compare with North-American head and neck squamous cell carcinomas (HNSCC). Quantitative-methylation-specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16(INK4a) and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at-least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16(INK4a) and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty-two of 72 node positive cases harbored p16(INK4a) and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16(INK4a) methylation (47.8%) in this Indian cohort in comparison with a North-American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16(INK4a) genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers.
  International Journal of Cancer 11/2010; 127(10):2367-73. · 5.44 Impact Factor
• Article: NKX3.1 as a marker of prostatic origin in metastatic tumors.

  Bora Gurel, Tehmina Z Ali, Elizabeth A Montgomery, Shahnaz Begum, Jessica Hicks, Michael Goggins, Charles G Eberhart, Douglas P Clark, Charles J Bieberich, Jonathan I Epstein, Angelo M De Marzo
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  ABSTRACT: NKX3.1 is a prostatic tumor suppressor gene located on chromosome 8p. Although most studies have shown that staining for NKX3.1 protein is positive in the majority of primary prostatic adenocarcinomas, it has been shown to be downregulated in many high-grade prostate cancers, and completely lost in the majority of metastatic prostate cancers (eg, in 65% to 78% of lesions). A recent study showed that NKX3.1 staining with a novel antibody was highly sensitive and specific for high-grade prostatic adenocarcinoma when compared with high-grade urothelial carcinoma. This raised the question that this antibody may perform better than earlier used antibodies in metastatic prostate tumors. However, the sensitivity and specificity for prostate carcinomas for this antibody in metastatic lesions was not determined. Although prostate-specific antigen (PSA) and prostatic-specific acid phosphatase (PSAP) are excellent tissue markers of prostate cancer, at times they may be expressed at low levels, focally, or not at all in poorly differentiated primary and metastatic prostatic adenocarcinomas. The purpose of this study was to determine the performance of NKX3.1 as a marker of metastatic adenocarcinoma of prostatic origin. Immunohistochemical staining against NKX3.1, PSA, and PSAP was carried out on a tissue microarray (TMA) (0.6-mm tissue cores) of hormone naïve metastatic prostate adenocarcinoma specimens from lymph nodes, bone, and soft tissue. To determine the specificity of NKX3.1 for prostatic adenocarcinoma, we used TMAs that contained cancers from various sites including the urinary bladder, breast, colon, salivary gland, stomach, pancreas, thyroid, and central nervous system, and standard paraffin sections of cancers from other sites including the adrenal cortex, kidney, liver, lung, and testis. Overall 349 nonprostatic tumors were evaluated. Any nuclear staining for NKX3.1 was considered positive and the percentage of cells with nuclear staining and their mean intensity level were assessed visually. Sensitivity was calculated by considering a case positive if any TMA core was positive. The sensitivity for identifying metastatic prostatic adenocarcinomas overall was 98.6% (68/69 cases positive) for NKX3.1, 94.2% (65/69 cores positive) for PSA, and 98.6% (68/69 cores positive) for PSAP. The specificity of NKX3.1 was 99.7% (1/349 nonprostatic tumors positive). The sole positive nonprostatic cancer case was an invasive lobular carcinoma of the breast. NKX3.1 seems to be a highly sensitive and specific tissue marker of metastatic prostatic adenocarcinoma. In the appropriate clinical setting, the addition of IHC staining for NKX3.1, along with other prostate-restricted markers, may prove to be a valuable adjunct to definitively determine prostatic origin in poorly differentiated metastatic carcinomas.
  The American journal of surgical pathology 08/2010; 34(8):1097-105. · 4.06 Impact Factor
• Article: Bcl2 and human papilloma virus 16 as predictors of outcome following concurrent chemoradiation for advanced oropharyngeal cancer.

  Anthony C Nichols, Dianne M Finkelstein, William C Faquin, William H Westra, Edmund A Mroz, Peter Kneuertz, Shahnaz Begum, William A Michaud, Paul M Busse, John R Clark, James W Rocco
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  ABSTRACT: Oropharyngeal squamous cell carcinoma (OPSCC) associated with human papilloma virus (HPV) is rapidly growing in incidence. Despite better prognosis than OPSCC associated with traditional risk factors, treatment failure still occurs in a significant proportion of patients. We had identified the antiapoptotic protein Bcl2 as a marker for poor outcome in advanced OPSCC treated with concurrent chemoradiation. To determine whether Bcl2 and HPV together might further characterize treatment response, we examined whether the prognostic value of Bcl2 was independent of HPV status. Pretreatment tumor biopsies from 68 OPSCC patients were tested for HPV by in situ hybridization and were immunostained for Bcl2 to evaluate relations with disease-free (DFS) and overall survival following platin-based concurrent chemoradiation. Median follow-up among surviving patients was 47 months (range, 10-131 months). Bcl2 and HPV independently predicted DFS and overall survival. Hazard ratios (with 95% confidence interval) for positive versus negative status in bivariate Cox proportional hazard analysis of DFS were 6.1 (1.8-21) for Bcl2 and 0.11 (0.035-0.37) for HPV. Only 1 of 32 HPV-positive/Bcl2-negative tumors recurred. Pretreatment Bcl2 expression was specifically associated with distant metastasis; five of six distant metastases occurred in the <40% of patients whose primary tumors were Bcl2 positive. Independent of HPV status, pretreatment Bcl2 expression identifies a subset of OPSCC patients having increased risk of treatment failure, particularly through distant metastasis, after concurrent chemoradiation. Considering HPV and Bcl2 together should help in devising better personalized treatments for OPSCC.
  Clinical Cancer Research 03/2010; 16(7):2138-46. · 7.74 Impact Factor
• Article: Expression of p16 in benign and malignant cystic squamous lesions of the neck.

  Dengfeng Cao, Shahnaz Begum, Syed Z Ali, William H Westra
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  ABSTRACT: Metastatic cystic squamous cell carcinomas of the neck often harbor human papillomavirus 16 and, in turn, overexpress p16. P16 immunohistochemistry could be useful in the evaluation of patients who present with cystic squamous lesions of the neck, particularly when the distinction between a benign lymphoepithelial cyst and a metastatic squamous cell carcinoma cannot be easily resolved on clinical or pathologic grounds. Implementation of this strategy, however, awaits a description of p16 expression in benign lymphoepithelial cysts. The purpose of this study was to evaluate p16 staining in cystic squamous lesions of the neck with an emphasis on benign lymphoepithelial cysts. P16 immunohistochemistry was performed on tissue sections and fine needle aspirates of benign (n = 49) and malignant (n = 16) squamous lesions of the neck. P16-positive cases were further evaluated by human papillomavirus 16 in situ hybridization. P16 staining was seen in the tissue sections of 16 of 37 (43%) benign lymphoepithelial cysts. P16 staining tended to localize to regions of the squamous epithelium penetrated by interdigitating lymphocytes. In the aspirates, p16 staining was noted in 5 of 12 (42%) benign lymphoepithelial cysts and in 3 of 16 (19%) cystic squamous cell carcinomas. Human papillomavirus 16 was detected in the 3 p16-positive cystic squamous cell carcinomas but in none of the p16-positive benign lymphoepithelial cysts. P16 overexpression is not always linked to high-risk human papillomavirus integration, but may be intrinsic to the reticulated epithelium that lines benign lymphoepithelial cysts. This observation limits the role of p16 staining as a surrogate marker of human papillomavirus 16 infection and as a diagnostic tool in separating benign from malignant cystic squamous lesions of the neck.
  Human pathology 12/2009; 41(4):535-9. · 3.03 Impact Factor
• Article: Association between lifestyle factors and CpG island methylation in a cancer-free population.

  Mariana Brait, Jean G Ford, Srinivas Papaiahgari, Mary A Garza, Jin I Lee, Myriam Loyo, Leonel Maldonado, Shahnaz Begum, Lee McCaffrey, Mollie Howerton, David Sidransky, Mark R Emerson, Saifuddin Ahmed, Carla D Williams, Mohammad Obaidul Hoque
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  ABSTRACT: Many risk factors have been associated with cancer, such as age, family history, race, smoking, high-fat diet, and poor nutrition. It is important to reveal the molecular changes related to risk factors that could facilitate early detection, prevention, and overall control of cancer. We selected six cancer-specific methylated genes that have previously been reported in primary tumors and have also been detected in different bodily fluids of cancer patients. Here, we used quantitative fluorogenic real-time methylation-specific PCR in plasma DNA samples for the detection of methylation changes from an asymptomatic population who do not have any known cancer. The promoter methylation frequencies of the studied genes were as follows: APC (7%), CCND2 (22%), GSTP1 (2%), MGMT (9%), RARbeta2 (29%), and P16 (3%). Promoter methylation of at least one of the genes analyzed was observed in approximately 46% (72 of 157) of the samples by binary dichotomization. Promoter hypermethylation of at least two genes was detected in 17% (26 of 157) of the samples. RARbeta2 methylation was observed in 45% of subjects who had a high-fat diet in contrast with those who had a low-fat diet (23%; P = 0.007). Our findings may help to elucidate early methylation changes that may lead to cancer development. These methylation changes could be due to exposure to risk factors and may be useful for cancer prevention measures such as changes in lifestyle. Longitudinal follow-up of a high-risk population is needed to understand the association of methylation of candidate genes in cancer development.
  Cancer Epidemiology Biomarkers &amp Prevention 11/2009; 18(11):2984-91. · 4.12 Impact Factor
• Article: Prognostic significance of human papillomavirus in oropharyngeal squamous cell carcinomas.

  Ahmad R Sedaghat, Zhe Zhang, Shahnaz Begum, Robert Palermo, Simon Best, Karen M Ulmer, Marshall Levine, Eva Zinreich, Barbara P Messing, Dorothy Gold, Annie A Wu, Kevin J Niparko, Jeanne Kowalski, Richard M Hirata, John R Saunders, William H Westra, Sara I Pai
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  ABSTRACT: The human papillomavirus (HPV) has been identified as a causative factor in 20% to 25% of all head and neck squamous cell carcinomas (HNSCC). Ongoing research suggests that the presence of HPV DNA in HNSCC predicts a positive prognosis with respect to disease-free and overall survival. However, most studies have been limited by the heterogeneity in treatment regimens and/or anatomic subsites of tumor origin. In this study, we correlate clinical outcomes with HPV status for patients with oropharyngeal carcinomas who were uniformly treated with a concurrent chemoradiation treatment protocol. Retrospective study. Demographic and clinicopathologic parameters, including age at diagnosis, gender, race, smoking and alcohol history, tumor stage and grade, locoregional recurrence, metastatic spread, recurrence-free survival, overall survival and disease-specific death, were obtained from medical charts and established databases. These parameters were correlated with HPV status of the tumors established by in situ hybridization analysis. HPV positivity correlated with improved clinical outcomes regarding locoregional control (P = .042), recurrence-free survival (P = .009), overall survival (P = .017), and disease-specific death (P = .09). Advanced T stage was a significant risk factor for recurrence and death independent of HPV status. In patients with oropharyngeal carcinoma uniformly treated with chemoradiation, the presence of HPV is a favorable prognostic indicator with respect to recurrence and overall survival. However, advanced T stage was an independent risk factor for recurrence and death that can to some degree offset this benefit.
  The Laryngoscope 07/2009; 119(8):1542-9. · 1.75 Impact Factor
• Article: Novel integrative methods for gene discovery associated with head and neck squamous cell carcinoma development.

  Ian M Smith, Suhail K Mithani, Chunyan Liu, Steven S Chang, Shahnaz Begum, Mousiumi Dhara, William Westra, David Sidranksy, Joseph A Califano
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  ABSTRACT: To find head and neck squamous cell carcinoma (HNSCC) specific genetic changes using integrative genetics. Genetic analysis. Three separate cohorts of patients with primary HNSCC were evaluated for expression-microarray of 33 000 genes (8 patients), quantitative real-time polymerase chain reaction (qRT-PCR) (36 patients), and quantitative DNA/qRT-PCR (12 patients). Controls with normal upper-aerodigestive mucosa were evaluated for expression microarray (6 patients) and qRT-PCR (7 patients). We utilized (1) prior reports of DNA loss and gain HNSCC accompanied by comparative genomic hybridization high-definition array data of the entire human genome, (2) a genome-wide survey of cancer-specific DNA mutations from the consensus cancer coding sequence (13 023 genes), and (3) our RNA expression microarray data of 33 000 genes to define candidate oncogenes activated by amplification or candidate tumor suppressor genes inactivated by deletion. Gene expression in tissue measured by quantitative reverse transcriptase PCR. Gene copy number was measured by quantitative PCR. We found 20 genes that were in areas of demonstrated amplification or deletion overlapping with the somatic mutants from genome-wide screening of the consensus DNA cancer coding sequence reported by Sjöblom et al. Three were chosen for further study based on expression differences and proof of cancer causation from in silico study: RUNX1T1, RFC4, and DLEC1. From 12 patients with HNSCC, matched tumor DNA/RNA and leukocyte-derived DNA were studied. Six of 12 (50%) of the tumors demonstrated amplification of the RUNX1T1 locus (P = .01), and 4 of those 6 (67%) demonstrated upregulated transcription of this gene (P = .02). Five of 12 (42%) of the tumors demonstrated amplification of the RFC4 locus (P = .03), and 1 of those 5 (20%) demonstrated upregulated messenger RNA (mRNA) transcription of the gene (P = .60). Four of 12 of the tumors (33%) (P = .05) demonstrated deletion in the DLEC locus (consistent with previously published 3p22 loss of 40%), and 3 of those 4 (75%) demonstrated reductions in mRNA expression (P = .06). With the advent of high-throughput techniques to study cancer genetics, novel comparisons of large data sets using integrative methods may elucidate genetic alterations in HNSCC cancer.
  Archives of otolaryngology--head & neck surgery 06/2009; 135(5):487-95. · 1.92 Impact Factor
• Article: Bcl-2 blocks cisplatin-induced apoptosis and predicts poor outcome following chemoradiation treatment in advanced oropharyngeal squamous cell carcinoma.

  William A Michaud, Anthony C Nichols, Edmund A Mroz, William C Faquin, John R Clark, Shahnaz Begum, William H Westra, Hiroshi Wada, Paul M Busse, Leif W Ellisen, James W Rocco
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  ABSTRACT: This study aimed to test the hypothesis that elevated expression of antiapoptotic Bcl-2 family proteins predicts a poor therapeutic response of oropharyngeal squamous cell carcinoma (OPSCC) to concurrent platinum-based chemoradiation therapy. Levels of Bcl-2, Bcl-XL, and Bcl-w were determined and correlated with resistance to cisplatin in a large panel of cell lines derived from squamous cell carcinoma of the head and neck (HNSCC). Univariate and multivariate analyses were used to evaluate the relationship between Bcl-2 and Bcl-XL expression and disease-free survival following chemoradiation therapy in a uniformly treated cohort of patients with OPSCC. In HNSCC cell lines, high endogenous Bcl-2 expression was associated with increased cisplatin resistance, and experimental overexpression of Bcl-2 promoted cisplatin resistance. In patients, tumors positive for Bcl-2 before treatment had greater risk of treatment failure (hazard ratio, 5.99; 95% confidence interval, 1.73-20.8; P=0.0014). In contrast, endogenous Bcl-XL showed no correlation either with cisplatin sensitivity in the cell line panel in vitro, or with risk of recurrence in vivo (hazard ratio, 1.28; 95% confidence interval, 0.39-4.19; P=0.68). Associations between Bcl-2 expression and other clinical characteristics did not account for the predictive value of Bcl-2. Immunohistochemical assessment of Bcl-2 in pretreatment biopsy specimens can predict response of advanced OPSCC to concurrent platinum-based chemoradiation. As treatments targeting Bcl-2 and its family members become available, this immunohistochemical assessment could help personalize therapy by identifying a subpopulation of patients with a poor prognosis who might benefit from such treatments.
  Clinical Cancer Research 03/2009; 15(5):1645-54. · 7.74 Impact Factor
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  Available from: PubMed Central

  Article: Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

  Ian M Smith, Chad A Glazer, Suhail K Mithani, Michael F Ochs, Wenyue Sun, Sheetal Bhan, Alexander Vostrov, Ziedulla Abdullaev, Victor Lobanenkov, Andrew Gray, Chunyan Liu, Steven S Chang, Kimberly L Ostrow, William H Westra, Shahnaz Begum, Mousumi Dhara, Joseph Califano
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  ABSTRACT: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.
  PLoS ONE 02/2009; 4(3):e4961. · 4.09 Impact Factor
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  Available from: PubMed Central

  Article: Integrative discovery of epigenetically derepressed cancer testis antigens in NSCLC.

  Chad A Glazer, Ian M Smith, Michael F Ochs, Shahnaz Begum, William Westra, Steven S Chang, Wenyue Sun, Sheetal Bhan, Zubair Khan, Steven Ahrendt, Joseph A Califano
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  ABSTRACT: Cancer/testis antigens (CTAs) were first discovered as immunogenic targets normally expressed in germline cells, but differentially expressed in a variety of human cancers. In this study, we used an integrative epigenetic screening approach to identify coordinately expressed genes in human non-small cell lung cancer (NSCLC) whose transcription is driven by promoter demethylation. Our screening approach found 290 significant genes from the over 47,000 transcripts incorporated in the Affymetrix Human Genome U133 Plus 2.0 expression array. Of the top 55 candidates, 10 showed both differential overexpression and promoter region hypomethylation in NSCLC. Surprisingly, 6 of the 10 genes discovered by this approach were CTAs. Using a separate cohort of primary tumor and normal tissue, we validated NSCLC promoter hypomethylation and increased expression by quantitative RT-PCR for all 10 genes. We noted significant, coordinated coexpression of multiple target genes, as well as coordinated promoter demethylation, in a large set of individual tumors that was associated with the SCC subtype of NSCLC. In addition, we identified 2 novel target genes that exhibited growth-promoting effects in multiple cell lines. Coordinated promoter demethylation in NSCLC is associated with aberrant expression of CTAs and potential, novel candidate protooncogenes that can be identified using integrative discovery techniques. These findings have significant implications for discovery of novel CTAs and CT antigen directed immunotherapy.
  PLoS ONE 01/2009; 4(12):e8189. · 4.09 Impact Factor
• Article: Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer.

  Mariana Brait, Shahnaz Begum, André L Carvalho, Santanu Dasgupta, André L Vettore, Bogdan Czerniak, Otávia L Caballero, William H Westra, David Sidransky, Mohammad Obaidul Hoque
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  ABSTRACT: 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. 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 chi(2) or Fisher's exact tests as appropriate. All statistical tests were two-sided. 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. 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 Epidemiology Biomarkers &amp Prevention 11/2008; 17(10):2786-94. · 4.12 Impact Factor