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Purpose: A major limitation of studies reporting a lower prevalence rate of human papilloma virus (HPV) in African American patients with oropharyngeal squamous cell cancer (OPSCC) than Caucasian Americans, with corresponding worse outcomes, was adequate representation of HPV-positive African American patients. This study examined survival outcomes in HPV-positive and HPV-negative African Americans with OPSCC.Experimental Design: The study cohort of 121 patients with primary OPSCC had 42% African Americans. Variables of interest included age, race, gender, HPV status, stage, marital status, smoking, treatment, and date of diagnosis.Results: Caucasian Americans are more likely to be HPV positive (OR = 3.28; P = 0.035), as are younger age (age < 50 OR = 7.14; P = 0.023 compared with age > 65) or being married (OR = 3.44; P = 0.016). HPV positivity and being unmarried were associated with being late stage (OR = 3.10; P = 0.047 and OR = 3.23; P = 0.038, respectively). HPV-negative patients had 2.7 times the risk of death as HPV-positive patients (P = 0.004). Overall, the HPV-race groups differed (log-rank P < 0.001), with significantly worse survival for HPV-negative African Americans versus (i) HPV-positive African Americans (HR = 3.44; P = 0.0012); (ii) HPV-positive Caucasian Americans (HR = 3.11; P = < 0.049); and (iii) HPV-negative Caucasian Americans (HR = 2.21; P = 0.049).Conclusions: HPV has a substantial impact on overall survival in African American patients with OPSCC. Among African American patients with OPSCC, HPV-positive patients had better survival than HPV negative. HPV-negative African Americans also did worse than both HPV-positive Caucasian Americans and HPV-negative Caucasian Americans. This study adds to the mounting evidence of HPV as a racially linked sexual behavior life style risk factor impacting survival outcomes for both African American and Caucasian American patients with OPSCC. Clin Cancer Res; 19(9); 2486–92. ©2013 AACR.

PDF file - 68K, Supplemental Table 1: Multivariable associations with late stage.

The incidence of thyroid cancer (TC) is increasing. Cytology by itself cannot distinguish TC from some benign nodules especially in certain subtypes of TC. Our immediate goal is to identify DNA methylation markers for early detection of TC and to molecularly differentiate TC subtypes from benign nodules. Promoter methylation status of 21 candidate genes was examined on formalin-fixed paraffin-embedded tissue (FFPE) utilizing quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients (56% white, 29% African American, 61% female) comprising 71 normal thyroid, 83 benign nodules [follicular adenomas (FA)], 90 follicular TC (FTC) and 85 papillary TC (PTC). All genes were analyzed individually (Kruskal–Wallis and Wilcoxon rank sum tests) and in combination (logistic regression models) to identify genes whose methylation levels might best separate groups. Combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA, and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%). The six gene panel of TIMP3, RARB2, SERPINB5, RASSF1, TPO and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue. Aberrant gene methylation used in combination panels may be useful clinically in differentiating FTC and PTC from benign nodules. If confirmed in additional studies, these findings could help reduce the over diagnosis of thyroid cancer and surgeries related to over diagnosis.

Background: There has been relatively little advancement in changing the management of women with estrogen receptor (ER) negative breast cancer (BC), mainly due to a dearth of actionable therapeutic targets. Therefore, understanding the underlying biology of such a complex disease is necessary for bringing new therapeutic treatments to light. A key question in cancer genomics is how to distinguish 'driver' or essential alterations, which contribute to tumorigenesis, from functionally neutral or 'passenger' alterations that go along for the ride. The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on mutations has overshadowed consideration of inclusion of epigenetic information. This study illustrates network integration of epigenomic data to prioritize ER negative specific methylated genes as potential epigenetic drivers of aggressive disease. Methods: Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. A master regulator is a gene or drug positioned as the central or master hub that has the ability to command or influence downstream events. Causal Network Analysis (CNA) was used to find networks that connect upstream master regulators with a 16 candidate methylation gene signature differentiating ER negative from ER positive BC. The 16 ER-negative specific gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) was identified via our drill-down approach starting from a discovery approach (Illumina 450k BeadChip) followed by expression verification, significant rankings in biological pathways (Ingenuity Pathway Analysis), confirmation by targeted sequencing using Illumina MiSeq, and additional filtering in 450K TCGA data sets. Results: CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, transcription regulator SP1, MSH2, and 15-ketoprotaglandin E2. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. Conclusion: In this study, direct regulatory influence, noted for 5/16 candidate genes indicates additional rationale for further consideration and validation of ALPL, CCND1, CIRBP, EGFR, ESR1 as potential epigenetic driver targets in ER negative BC. As cancer therapies become increasingly more specific and begin to move past cytotoxic agents, determining the molecular features of a tumor that predict response to a given drug has become increasingly essential to match patients with optimal therapy. Currently epigenetic therapy in the form of hypomethylating agents (e.g: decitabine) exhibit clinical efficacy in patients with AML and MDS including those patients not responding to cytotoxic therapy. Support: Komen Foundation: KG110218. Citation Format: Worsham MJ, Chen KM, Datta I, Stephen JK, Chitale D, Divine G. Network integration of epigenomic data: Leveraging the concept of master regulators in ER negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-06.

Background: Micro RNAs (miRNA) are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Compared to mRNA signatures, miRNAs have better and stronger biomarker properties with 20 times more power in biomarker studies as compared to mRNAs (when comparing 20,000 mRNAs to ∼1,000 miRNAs). Emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer, representing a novel class of potential biomarkers for diagnosis, prediction of treatment, or prognosis. ER-negative breast cancer (BC) is an aggressive histological subtype with limited treatment options and very poor prognosis. Our long term objective is to derive a diagnostic, prognostic, and predictive ER-negative specific miRNA panel for detection of early cancer, recurrence/metastasis, and as potential therapeutic targets for better management of ER-negative BC. Methods: The initial discovery step profiled 39 primary ER negative and 40 ER positive BC cases using the Illumina Infinium HumanMethylation450 BeadChip followed by a subanalysis focusing on 2249 miRNA CpGs assigned to 615 unique miRNAs. T-tests were used to compare the means of the M-values for the ER-positive and ER-negative groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER positive and ER negative of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). Because miRNAs perform their important functions via their targets, the targets of miRNAs were assessed for functional enrichment analysis in IPA for biologic involvement. Results: Over half of the miRNA CpGs (1224/2249, 54%) were differentially methylated between ER negative and ER positive BC with significant aFDR levels. The 1224 CpGs at Tier 1 were associated with 379 miRNAs; the 24 and 2 CpGs for Tiers 2 and 3 with 22 and 2 miRNAs, respectively. The 22 miRNA genes were assigned to 4621 targets using online databases that predict miRNA targets. The degree of confidence that a target gene is associated with a miRNA is characterized in these databases as either "experimentally observed", or just as "high" (predicted). Of these 4621 targets, 87 were designated as experimentally observed and were examined in IPA. Top pathways and networks designated by miRNA targets included the cell cycle G1/S checkpoint regulation canonical pathway, and the cell-to-cell interaction/cancer networks among others. MiRNA targets in top pathways and networks were circled back to their respective miRNAs revealing cooperatively mediated pathway dysregulation of ER negative BC. Conclusion: Aberrantly methylated miRNAs showed perturbation of biologically significant pathways and networks, suggesting that miRNAs mediate pathway dysregulation in a coordinated manner, strengthening the case for utility of miRNAs as viable biomarkers in ER negative BC. Support: Komen Foundation: KG110218. Citation Format: Worsham MJ, Chen KM, Datta I, Stephen JK, Chitale D, Divine G. Epigenetically altered microRNA mediated pathway dysregulation in ER negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-10.

Infection is a serious complication of hematopoietic stem cell transplantation (SCT). However, the optimum oral agent for antibacterial prophylaxis in SCT recipients remains uncertain. Different antibiotics might affect incidence of blood stream infections, resistance, Clostridium difficile, gut microbiome, GVHD and relapse. To explore this we started this first clinical trial of levofloxacin (Levo) versus ciprofloxacin (Cipro) at our center. Methods: This is a single center prospective randomized study. Patients who meet the SCT program criteria to undergo autologous or allogeneic hematopoietic stem cell transplantation are eligible. Results: We present interim results. 111 consecutive patients were randomized since June 2018. At the time of the present report 102 of these patients have follow up for 60 days or more. There were 62 males and 40 females. Median age at time of SCT was 61. Fifty received Cipro and 52 received Levo. Fifty-six patients had autologous (27 Cipro and 29 Levo) while 46 patients had allogeneic SCT (23 Cipro and 23 Levo). For allogeneic SCT, 12 patients in each group had ATG. Time to engraftment and length of stay were similar in both groups. In the Cipro group 6 patients (12%) had bacteremia while in the Levo group 7 (13%) had bacteremia. Of patients with bacteremia, in the Cipro group none had Gram negative bacteremia while in the Levo group 4 had gram negative bacteremia (OR 9.37, p = 0.137). In the Cipro group 6 patients had gram positive bacteremia while in the Levo group 3 patients had gram positive bacteremia (OR 2.22, p = 0.277). In the Cipro group, 6 (12%) had clostridium difficile while in the Levo group only 1 (1.9%) had Clostridium difficile (OR 6.95, p = 0.078). Acute GVHD of all grades accrued in 17 patients (34%) in Cipro group and in 16 patients (30.7%) in the Levo group. Grade III-IV aGVHD occurred in 6 patients (12%) in Cipro group and in 3 patients (5.8%) in the Levo group (OR 2.23, p = 0.277). The hazard ratio for progression free survival was 0.67 (95% CI: 0.27, 1.67), p=0.395 for Levo vs. Cipro. The hazard ratio for overall survival, adjusted for auto/allo and age at transplant was 0.29 (95% CI: 0.09, 0.95), p = 0.041, for Levo vs. Cipro. However, 3 of deaths in the Cipro group were not related to type of antibacterial prophylaxis (listeriosis from contaminated food, fungal pneumonia in a wooden-house builder and stroke from intraventricular thrombus). So, unadjusted hazard ratio for overall survival (Levo vs. Cipro): 0.41 (95% CI: 0.13, 1.34), p = 0.142. Hazard ratio for overall survival (Levo vs. Cipro), adjusting for allo/auto and age at transplant: 0.39 (95% CI: 0.11, 1.33), p = 0.132. Conclusion: At this point, in this interim analysis, there seems to be a trend for Cipro to protect patients from gram negative bacteremia compared to levo. As a trade-off, there is higher trend for Clostridium difficile in the Cipro group compared to Levo group, but none is statistically significant so far.

Thyroid cancer has the fastest rising incidence rates and is the fifth most common cancer in women. There are four main types of which the papillary and follicular types together account for >90%, followed by medullary cancers (3%-5%) and anaplastic carcinomas (

A tissue field of somatic genetic alterations precedes the histopathological phenotypic changes of carcinoma. Genomic changes could be of potential use in the diagnosis and prognosis of pre-invasive squamous head and neck carcinoma (HNSCC) lesions and as markers for cancer risk assessment. Studies of sequential molecular alterations and genetic progression of pre-invasive HNSCC have not been clearly defined. Studies have shown recurring alterations at chromosome 9p21 (location of the CDKN2A) and TP53 mutations in the early stages of HNSCC. However, gene silencing via hypermethylation is still a relatively new idea in the development of HNSCC and little is known about the contribution of epigenetics to the development of neoplasia, its transformation, progression, and recurrence in HNSCC. This review examines the role of promoter hypermethylation of tumor suppressor genes in the progression continuum from benign papillomas to malignancy in HNSCC.

ObjectiveThe goal was to determine the prevalence of high-risk HPV16 using saliva in a screening population in Detroit, Michigan.Materials and MethodsReal-time quantitative polymerase chain reactio...

In recent years, studies have suggested that promoter methylation in human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) has a mechanistic role and has the potential to improve patient survival. The present study aimed to replicate key molecular findings from previous analyses of the methylomes of HPV positive and HPV negative HNSCC in an independent cohort, to assess the reliability of differentially methylated markers in HPV-associated tumors. HPV was measured using real-time quantitative PCR and the biological significance of methylation differences was assessed by Ingenuity Pathway Analysis (IPA). Using an identical experimental design of a 450K methylation platform, 7 of the 11 genes were detected to be significantly differentially methylated and all 11 genes were either hypo- or hypermethylated, which was in agreement with the results of a previous study. IPA's enriched networks analysis identified one network with msh homeobox 2 (MSX2) as a central node. Locally dense interactions between genes in networks tend to reflect significant biology; therefore MSX2 was selected as an important gene. Sequestration in the top four canonical pathways was noted for 5-hydroxytryptamine receptor 1E (serotonin signaling), collapsin response mediator protein 1 (semaphorin signaling) and paired like homeodomain 2 (bone morphogenic protein and transforming growth factor-β signaling). Placement of 9 of the 11 genes in highly ranked pathways and bionetworks identified key biological processes to further emphasize differences between HNSCC HPV positive and negative pathogenesis.

ObjectiveThere is a lack of consensus regarding the causes of the differences in the higher incidence of and the mortality from head and neck squamous cell carcinoma (HNSCC) in African Americans (AA) versus Caucasian Americans (CA). We examined a comprehensive array of risk factors influencing health and disease in an access-to-care, racially diverse, primary HNSCC cohort.Study DesignCross-sectional study.SettingPrimary care academic health care system.Subjects and MethodsThe cohort of 673 patients comprised 391 CA and 282 AA (42%). Risk variables included demographic, histopathology, and clinical/epidemiologic factors. Tumor DNA was interrogated for loss and gain of 113 genes with known involvement in HNSCC/cancer. Logistic regression for univariate analysis was followed by multivariate modeling with determination of model predictability (c-index).ResultsOf the 39 univariate differences between AA and CA, multivariate modeling (c-index = 0.81) retained 7 differences (P < .05). AA were less likely to be m...

IntroductionMicroRNAs (miRNAs) are endogenous, small, noncoding RNAs of 17 to 25 nucleotides that regulate approximately 30% of human genes. They are differentially expressed in various types of cancers compared with noncancerous tissues, suggesting that they may have crucial roles in tumorigenesis. The objective of this study was to identify laryngeal squamous cell cancer (LSCC)-specific miRNAs.Materials and methodsA retrospective cohort of 10 LSCC and five normal laryngeal squamous epithelium samples were examined using a global miRNA profiling approach (HTG, Tucson, AZ, USA, 800 human miRNAs plus 10 endogenous control miRNAs). The expression status of selected dysregulated miRNAs that were significantly different from normal were verified by real-time quantitative PCR (qPCR).ResultsTwenty-three of the 800 human miRNAs had significantly different expression levels (p < 0.05) between LSCC and normal tissues. Fifteen of the 23 have not been previously reported in HNSCC and include: miR-663b, miR-663, miR-193b, miR-1291, miR-720, miR-191, miR-1224-3p, miR-214, miR- 1285, miR-1207-5p, miR-483-5p, miR-1225-3p, miR-1228, miR-1280 and miR-638. Consistently upregulated miR-31 and miR- 193b and differentially expressed miR-663b in LSCC were verified by qPCR.ConclusionThe 15 novel miRNAs identified in this exploratory study, pending further confirmation and validation, may have clinical utility as LSCC-specific markers.How to cite this articleChen KM, Stephen JK, Havard S Shah V, Gardner G, Schweitzer VG, Worsham MJ. Novel Dysregulated MicroRNAs in Primary Laryngeal Squamous Cell Cancer. Int J Head Neck Surg 2012;3(2):76-81.

Genetic events specific to the pathogenesis of malignancy can offer clues to the tumorigenesis process. The objective of this study was to identify gene alterations that differentiate tumor and nontumor lesions in squamous head and neck cancer (HNSCC). DNA from 220 primary HNSCC with concurrently present tumor and nontumor lesions from the same patient was interrogated for genomic alterations of loss or gain of copy. Conditional logistic regression dealt with tumor and non-tumor records within a patient. Of 113 genes, 53 had univariate effects (P<0.01), of which 16 genes remained in the multivariable model withP<0.01. The model had a C-index (ROC) of 0.93. Loss ofCDKN2Band gain ofBCL6, FGF3, andPTP4A3predicted tumor. Loss ofBAK1andCCND1and gain ofSTCHpredicted nontumor. This highly powered model assigned alterations in 16 genes specific for malignant versus nonmalignant lesions, supporting their contribution to the pathogenesis of HNSCC as well as their potential utility as relevant targets for further evaluation as markers of early detection and progression.

Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up < 3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has overshadowed consideration of inclusion of epigenetic information. Epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for DNA methylation with potential to impact improved survival outcomes. The purpose of this study was to illustrate network integration of epigenomic data in head and neck cancer to prioritize elements that drive biological states. Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. To further establish the driver potential of 11 genes: C14orf162/ CCDC177, CDH8, CRMP1, ELMO1, HTR1E, MEI1, MSX2, PCDH10, PCDHB11, PITX2, SYN2 previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples, and subsequently validated by our group in two independent sample sets, their master regulatory networks were identified utilizing Causal Network Analysis (CNA) software from Ingenuity Pathway Analysis. To reflect expected gene expression direction implied by methylation changes, the inverse of the methylation ratio from HPV positive vs. HPV negative HNSCC was used for CNA. CNA identified 23 top hierarchical networks (significant z score of absolute 2) associated with HNSCC and their corresponding master regulatory molecules characterized as transcription regulators and kinase inhibitors among others. Of the 11 target genes, 7 had representation in multiple networks; CRMP1 in 21/23, MSX2 and SYN2 in 20/23, CDH8 and PITX2 in 17/23, PCDH10 in 10/23, and ELMO1 in 5/23 networks. Of the 23 networks, 12 indicated activation and 11 inhibition by at least 4 or more of the 7 target genes. Master regulators were within 2 or 3 hops (intermediate regulators) to the target genes. CNA raised the profile of CDH8, CRMP1, ELMO1, MSX2, PCDH10, SYN2, and PITX2 (7/11 target genes) for further consideration as epigenetic drivers of HPV-associated HNSCC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, Tamer Ghanem, Lamont Jones, Laura Garcia-Rodriquez, George Divine. Network integration of epigenomic data in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A26.

Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.

Currently, the human papilloma virus (HPV), in addition to tobacco and alcohol, is considered another independent risk factor for oropharyngeal squamous head and neck cancer (OPSCC), where the prevalence of HPV-16 increases to 50-90 % for the oropharynx. Also, incidence and mortality in head and neck SCC (HNSCC) continue to be higher in African Americans (AA) than in Caucasian Americans (CA). A recent study found that poorer survival outcomes for AA versus CA with oropharyngeal tumors were attributable to racial differences in the prevalence of HPV positive (+) tumors; HPV negative (-) AA and CA patients had similar outcomes (Settle et al., Cancer Prev Res (Phila) 2:776-781, 2009). Evidence indicates that a HPV+ diagnosis has significant prognostic implications; these patients have at least half the risk of death when compared with the HPV- patient, due in part to a better response to chemoradiotherapy (Fakhry et al., J Natl Cancer Inst 100:261-269, 2008).Epigenetic events of promoter hypermethylation are emerging as promising molecular strategies for cancer detection, representing tumor-specific markers occurring early in tumor progression. HPV infection is now recognized to play a role in the pathogenesis of OPSCC, where HPV+ and HPV- patients appear to be clinically and biologically distinct with reported genome-wide hypomethylation and promoter hypermethylation in HPV+ HNSCC tumors. A recent study from our group applying pathway analysis to investigate the biological role of the differentially methylated genes in HPV+ and HPV- HNSCC reported 8 signal transduction pathways germane to HNSCC (Worsham et al., Otolaryngol Head Neck Surg 149:409-416, 2013).

Currently, the human papilloma virus (HPV), in addition to tobacco and alcohol, is considered another independent risk factor for oropharyngeal squamous head and neck cancer (OPSCC), where the prevalence of HPV-16 increases to 50-90 % for the oropharynx. Also, incidence and mortality in head and neck SCC (HNSCC) continue to be higher in African Americans (AA) than in Caucasian Americans (CA). A recent study found that poorer survival outcomes for AA versus CA with oropharyngeal tumors were attributable to racial differences in the prevalence of HPV positive (+) tumors; HPV negative (-) AA and CA patients had similar outcomes (Settle et al., Cancer Prev Res (Phila) 2:776-781, 2009). Evidence indicates that a HPV+ diagnosis has significant prognostic implications; these patients have at least half the risk of death when compared with the HPV- patient, due in part to a better response to chemoradiotherapy (Fakhry et al., J Natl Cancer Inst 100:261-269, 2008).Epigenetic events of promoter hypermethylation are emerging as promising molecular strategies for cancer detection, representing tumor-specific markers occurring early in tumor progression. HPV infection is now recognized to play a role in the pathogenesis of OPSCC, where HPV+ and HPV- patients appear to be clinically and biologically distinct with reported genome-wide hypomethylation and promoter hypermethylation in HPV+ HNSCC tumors. A recent study from our group applying pathway analysis to investigate the biological role of the differentially methylated genes in HPV+ and HPV- HNSCC reported 8 signal transduction pathways germane to HNSCC (Worsham et al., Otolaryngol Head Neck Surg 149:409-416, 2013).

Background: The unique structure and function of normal tissues is known to be regulated by epigenetic mechanisms. Understanding how normal cells in their respective tumor milieus might affect their susceptibility to become not only malignant but acquire breast cancer (BC) subtype-specific phenotypes, may determine tumor clinical behavior outcomes. The goal was to compare genome wide methylation profiles of non-coding miRNAs of breast cancer tissue and normal breast epithelium, respectively, from ER negative and ER positive tumors, and assess their miRNA methylomes in the context of tumor ER phenotypes as ER negative vs ER positive. Methods: Breast cancer tissue from 79 patients (40 ER-positive and 39 ER-negative) and normal tissue from 39 of these patients (19 ER-negative and 20-ER-positive) were assayed using the Illumina 450K bead array. A sub analysis focused on 2249 miRNA CpGs assigned to 615 unique miRNAs. M-values were computed as a logit function [(log (beta/ (1-beta))] of the methylation beta values. T-tests were used to compare the means of the M-values for the ER-positive and ER-negative groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER-positive and ER-negative of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). Results: In the tumor cohort, 1224/2249 (54%) CpGs were differentially methylated between ER negative and ER positive BC at Tier 1 (aFDR 0.05 or lower). Of the 1224, 963 (78.7%) were hypermethylated, and 1035 (84.6%) were associated with the promoter region. The 1224, 24 and 2 CpGs were associated with 379, 22 and 2 genes for Tiers 1, 2 and 3, respectively. When the same analysis was performed on normal tissue only (19 ER-negative and 20-ER-positive) 76 of the 2249 CpGs had significant aFDR values and none of those met the Tier 2 or Tier 3 criteria. Seventy-one of the 76 (93.4%) where hypermethylated, and 65 (85.5%) were associated with the promoter region. The 76 significant Tier 1 (aFDR) differentially methylated CpGs were associated with 48 genes of which 43 were common to tumor Tier 1 differentially methylated miRNA genes, 10 were common to tumor Tier 2 genes, and 5 were restricted to normal tissue only. Conclusions Normal epithelial tissues demonstrated similar differential methylation directionality as their respective tumor counterparts (although to a lesser extent), favoring promoter region localization. Accordingly, the recognition of normal breast tissue-specific epigenetic propensities that align with their tumor phenotypes, suggest the possibility of progression markers specific for estrogen receptor status as well as markers not associated with progression. This provides insights into our view of possible links between epigenetic programming, progression continuums, and how hormonal receptor subtypes may be determined. Support: Komen Foundation: KG110218. Citation Format: Worsham MJ, Chen KM, Chitale D, Stephen JK, Divine G. Differentially methylated miRNA methylomes of normal breast tissue from ER negative and ER positive breast cancer mimic their respective tumor phenotypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-03-01.

Many recent studies examining aberrant DNA methylation in thyroid cancer are restricted to either candidate genes or genome-wide methylation in specific thyroid tumor subtypes. The goal of this study was to identify differentially methylated genes globally and their association with molecular pathways and signaling networks. Common mutations in papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), follicular adenoma (FA) and normal thyroid were also examined. Genome-wide methylation profiling using the Infinium HumanMethylation450FFPE (formalin fixed paraffin embedded) BeadChip Array was performed on 24 thyroid cases (8 PTC, 8 FTC, 4 FA and 4 normal thyroid). Ingenuity Pathway Analysis (IPA) was utilized to assess the roles of significantly differentially methylated genes in biological functions, signaling/metabolic pathways, and networks. Common mutations in 4 genes (BRAF, NRAS, HRAS, and KRAS) were assessed using TaqMan Mutation Detection assay. Twelve genes were significantly differentially methylated among 4 comparison groups: cancer vs normal, cancer vs adenoma, PTC vs normal, and PTC follicular variant (PTC-FV) vs PTC-Classic. CTU1 and HLA-DPB1 were significantly hypermethylated and AARS2, TMSB10, RNF216L, KIF15, KIAA1143, and SLC2A13 were significantly hypomethylated between cancer and normal. Significant differential hypermethylation was noted for PNPLA7 and NPC1L1 in cancer vs adenoma and SNX6 in PTC-FV vs PTC-Classic. NT5C1B was hypermethylated and AARS2 was hypomethylated in PTC vs normal. IPA identified 2 gene networks, involving 11/12 genes, characterized by 1) Cellular development, Cellular growth and proliferation, Connective tissue development and function and 2) Drug metabolism, Cell-mediated immune response, Cellular development. NT5C1B was involved in all 4 highly ranked canonical nucleotide degradation pathways. Several significant bio-functions involved NPC1L1. Mutations of NRAS codon 61 were identified in 1 sample each of FTC-Classic, PTC-FV and FA and BRAF V600E in one PTC-Classic sample. Differential methylation of AARS2, CTU1, HLA-DPB1, SLC2A13, PNPLA7, NPC1L1, NT5C1B and SNX6 suggest potential markers for discriminating thyroid cancers from adenomas and normal. NT5C1B was noted in highly ranked canonical pathways, suggesting a role in nucleotide degradation. Pathway analysis of differentially methylated genes support important biological processes in thyroid cancer pathogenesis. Supported by JFCI/CRAG A20038 Citation Format: Josena K. Stephen, Kang Mei Chen, Jason Merritt, Indrani Datta, Dhananjay Chitale, George Divine, Maria J. Worsham. Methylome differences in differentiated thyroid cancers and benign adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2017-3361

The unique structure and function of normal tissues is known to be regulated by epigenetic mechanisms. Understanding how normal cells in their respective tumor milieus might affect their susceptibility to become not only malignant but acquire breast cancer (BC) subtype-specific phenotypes, may determine tumor clinical behavior outcomes. The goal was to compare genome wide methylation profiles of non-coding miRNAs of breast cancer tissue and normal breast epithelium from estrogen receptor (ER) negative (-) and ER positive (+) tumors, and assess their miRNA methylomes in the context of tumor ER phenotypes as either ER- or ER+. BC tissue from 79 patients (39 ER- and 40 ER+) and normal tissue from 39 of these patients (19 ER- and 20-ER+) were assayed using the Illumina 450K bead array. A sub analysis focused on 2249 miRNA CpGs assigned to 615 unique miRNAs. M-values were computed as a logit function [(log (beta/ (1-beta))] of the methylation beta values. T-tests were used to compare the means of the M-values for the ER+ and ER- groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER+ and ER- of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). In the tumor cohort, 1224/2249 (54%) CpGs were differentially methylated between ER- and ER+ BC at Tier 1. Of the 1224, 963 (78.7%) were hypermethylated, and 1035 (84.6%) were in promoter regions. The 1224 CpGs at Tier 1, the 24 at Tier 2, and 2 CpGs at Tier 3 were associated with 379, 22 and 2 genes respectively. When the same analysis was performed on normal tissue only (19 ER- and 20-ER+), 76 of the 2249 CpGs had significant aFDR values and none of those met the Tier 2 or Tier 3 criteria. Seventy-one of the 76 (93.4%) were hypermethylated, and 65 (85.5%) were in promoter regions. The 76 significant Tier 1 (aFDR) differentially methylated CpGs were associated with 48 genes of which 43 were common to tumor Tier 1 differentially methylated miRNA genes, 10 were common to tumor Tier 2 genes, and 5 were restricted to normal tissue only. Normal epithelial tissues demonstrated similar differential methylation directionality as their respective tumor counterparts, favoring promoter region localization. Accordingly, the recognition of normal breast tissue-specific epigenetic propensities that align with their tumor phenotypes, suggest the possibility of progression markers specific for ER status as well as markers not associated with progression. This provides insights into our view of possible links between epigenetic programming, progression continuums, and how hormonal receptor subtypes may be determined. Support: Komen Foundation: KG110218 Citation Format: Kang Mei Chen, Josena K. Stephen, Indrani Datta, Dhananjay Chitale, George Divine, Maria J. Worsham. MicroRNA methylomes of normal breast tissue from ER negative and ER positive breast cancer identify progression markers specific for estrogen receptor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2017-4434

Human papilloma virus type 16 (HPV16) is a causative agent for some head and neck squamous cell carcinoma (HNSCC) and an independent risk factor for oropharyngeal SCC. The goal of this study was to examine HPV16 associated gene methylation in paired saliva and tumor DNA with assessment of the sensitivity, specificity, positive predictive, and negative predictive value for saliva HPV as a test for HNSCC. HPV16 status was determined by quantitative PCR (qPCR) in 35 primary HNSCC paired tumor and saliva specimens. Tumor cut points >=0.03 and >=0.1 and saliva cut points >0 and ?0.001 were used to classify results as HPV positive or negative. Aberrant methylation was determined by the methylation-specific multiplex ligation probe amplification (MS-MLPA) assay. The frequency of promoter hypermethylation in tumor samples was 66% (23/35) versus 17% (6/35) in saliva. Two of 35 paired tumor and saliva samples had commonly methylated genes. HPV and methylation were correlated for IGSF4 (p=0.01) in tumor samples (cut point ?0.03) and for ESR1 in saliva samples (cut point >0). Although the sensitivity of HPV detection in saliva was significantly reduced when saliva cut points were increased from >0 to >=0.001, the specificity and positive predictive values were 100% at saliva cut point of >=0.001, regardless of tumor cut points. Within clearly defined parameters, HPV detection in saliva DNA shows promise as a non invasive approach for tumor HPV status. Methylated genes detected in saliva may be useful in early detection and as potential predictive markers of HNSCC. Further confirmation and validation in larger cohorts is required.

Human papilloma virus (HPV) positive and HPV negative head and neck squamous cell cancer (HNSCC) are biologically distinct with a prognostic advantage for HPV positive patients compared to HPV negative cases. DNA promoter methylation is central to human diseases such as cancer, including HNSCC, with reported genome-wide hypomethylaton and promoter hypermethylation in HPV positive HNSCC tumors. The goal of this study was to identify differentially methylated genes in HPV positive versus HPV negative primary HNSCC genomes with clues to signaling networks.Laboratory-based study.Primary care academic health care system.DNA from 4 HPV positive and 4 HPV negative freshly frozen primary HNSCC were subject to comprehensive genome-wide methylation profiling. Differentially methylated gene lists were examined using the Signal Transduction Pathways (canonical) filter in the Genomatix Pathway System (GePS).Twofold methylation differences were observed between HPV positive and HPV negative cases for 1168 genes. Pathway analysis applied to investigate the biological role of the 1168 differentially methylated genes revealed 8 signal transduction pathways forming a network of 66 genes, of which 62% are hypermethylated.Our study reveals a predominant hypermethylation profile for genes in signal transduction pathways of HPV positive HNSCC tumor genomes. Because signaling events in the cell play a critical role in the execution of key biological functions, insights into how complex cellular signaling cascades and networks may be programmed in HNSCC are likely to be critical in the development of new biological agents designed to hit multiple targets.

Purpose: A major limitation of studies reporting a lower prevalence rate of human papilloma virus (HPV) in African American patients with oropharyngeal squamous cell cancer (OPSCC) than Caucasian Americans, with corresponding worse outcomes, was adequate representation of HPV-positive African American patients. This study examined survival outcomes in HPV-positive and HPV-negative African Americans with OPSCC. Experimental Design: The study cohort of 121 patients with primary OPSCC had 42% African Americans. Variables of interest included age, race, gender, HPV status, stage, marital status, smoking, treatment, and date of diagnosis. Results: Caucasian Americans are more likely to be HPV positive (OR = 3.28; P = 0.035), as are younger age (age < 50 OR = 7.14; P = 0.023 compared with age > 65) or being married (OR = 3.44; P = 0.016). HPV positivity and being unmarried were associated with being late stage (OR = 3.10; P = 0.047 and OR = 3.23; P = 0.038, respectively). HPV-negative patients had 2.7 times the risk of death as HPV-positive patients (P = 0.004). Overall, the HPV-race groups differed (log-rank P < 0.001), with significantly worse survival for HPV-negative African Americans versus (i) HPV-positive African Americans (HR = 3.44; P = 0.0012); (ii) HPV-positive Caucasian Americans (HR = 3.11; P = < 0.049); and (iii) HPV-negative Caucasian Americans (HR = 2.21; P = 0.049). Conclusions: HPV has a substantial impact on overall survival in African American patients with OPSCC. Among African American patients with OPSCC, HPV-positive patients had better survival than HPV negative. HPV-negative African Americans also did worse than both HPV-positive Caucasian Americans and HPV-negative Caucasian Americans. This study adds to the mounting evidence of HPV as a racially linked sexual behavior life style risk factor impacting survival outcomes for both African American and Caucasian American patients with OPSCC. Clin Cancer Res; 19(9); 2486–92. ©2013 AACR.

Expression of p16INK4A (p16 positive) is highly correlated with human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC), however, p16-positivity is not limited to HPV positive tumors and therefore, not a perfect surrogate for HPV. p16 survival outcomes are best documented for the oropharyngeal site (OP); non-OP sites such as the oral cavity (OC), larynx, and hypopharynx (HP) are understudied. The goal of this study was to evaluate p16 in the context of HPV16 and examine p16 survival outcomes in HPV16 positive and HPV16 negative site-specific HNSCC. p16 and HPV16 status were determined by immunohistochemistry and qPCR respectively, on 80 primary HNSCC from four sites: OC, OP, larynx and HP. p16 expression was different across sites (p

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. There has been relatively little advancement in changing the management of women with ER negative BC, mainly due to a dearth of actionable therapeutic targets. Our drill-down approach to identified an ER negative-specific 16 gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) starting from a discovery approach (Illumina Infinium HumanMethylation450 BeadChip, 40 ER negative vs. 40 ER positive BC) followed by expression verification, significant rankings in biological pathways (Ingenuity Pathway Analysis), and confirmation by targeted sequencing using Illumina MiSeq. Causal Networks are small hierarchical networks of regulators that control the expression/methylation of dataset targets. They can enhance understanding of the effect of master regulators on disease or function. The objective of this study was to identify regulatory networks utilizing IPA's Causal Network Analysis (CNA) in order to illuminate possible causes and mechanisms underlying the biological activities of the 16 candidate gene signature differentiating ER negative from ER positive BC. To reflect expected gene expression direction implied by methylation changes for the 16 candidate genes, the inverse of the methylation ratio from ER negative vs. ER positive tissue was used for CNA. CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, MSH2, 15-ketoprotaglandin E2, and transcription regulator SP1. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. CNA raised the profile of ALPL, CCND1, CIRBP, EGFR, ESR1 (5/16 candidate genes) for further consideration as potential epigenetic drivers of ER negative BC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, George Divine. Differentially methylation between ER negative and ER positive breast cancer identifies master regulators to expose potential epigenetic drivers of aggressive disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4484.

Objective: For optimal feasibility using saliva rinse screening of HPV for head and neck cancer (HNC), the prevalence should be 5% or higher in the population screened. The aim of our study was to determine the prevalence of HPV16 infection in a screening population in Detroit, MI.Method: Real time quantitative PCR was applied to detect HPV16 in saliva DNA from 349 screening subjects without HNC, 157 HNC, and 19 controls. Cut points for HPV positivity were: >0 and >0.001 copy/cell. Proportions were compared between groups using exact chi-square or Fisher’s exact tests as appropriate (set at P 0, each group had an overall HPV prevalence of over 5%, with a much higher prevalence of 31.2% in the HNC patient group. At cut point >0.001, the prevalence was lower in each group, 0% in the control, 1.2% in the screening, and 17.2% in the HNC group. At >0, females in the screening group had a higher prevalence of HPV than males (P = .010), and at >0.001, prevalence was higher for males...

In addition to genetic alterations of gains and losses, epigenetic events of promoter methylation appear to further undermine a destabilized genomic repertoire in squamous head and neck carcinoma (HNSCC). This chapter provides an overview of frequently methylated tumor suppressor genes in benign head and neck papillomas, primary HNSCC tumors, and HNSCC cell lines and their relevance as epigenetic markers in head and neck tumorigenesis.

Human papillomavirus (HPV), particularly HPV16, is a causative agent for 25% of head and neck squamous cell cancer, including laryngeal squamous cell cancer (LSCC). HPV-positive (HPV+ve) patients, particularly those with oropharyngeal SCC, have improved prognosis. For LSCC patients, this remains to be established. The goal was to determine stage and survival outcomes in LSCC in the context of HPV infection.Historical cohort study.Primary care academic health system.In 79 patients with primary LSCC, HPV was determined using real-time quantitative polymerase chain reaction. χ(2) or Fisher exact test was used to test the association of HPV+ve with 21 risk factors including race, stage, gender, age, smoking, alcohol, treatment, and health insurance. Kaplan-Meier and log-rank tests were used to study the association of HPV and LSCC survival outcome.HPV16 was detected in 27% of LSCC patients. Caucasian American (CA) patients had higher HPV prevalence (33%) than did African American (AA) LSCC patients (16%; P = .058). HPV was significantly associated with gender (P = .016) and insurance type (P = .001). There were no differences in survival between HPV+ve and HPV-negative (HPV-ve) patients. There was no association with HPV and other risk factors including stage (early vs late).We found a high prevalence of HPV in men and a lower prevalence of HPV infection in AA compared with CA. Despite the strikingly better survival of patients with HPV+ve oropharyngeal tumors, even when adjusted for smoking, this correlation does not seem to hold true in the larynx. Larger multiethnic LSCC cohorts are needed to more clearly delineate HPV-related survival across ethnicities.

In a study of genetic alterations, the Multiplex Ligation-dependent Probe Amplification (MLPA) assay was used to measure gain or loss of 113 gene-probes in tumor and non-tumor tissue samples collected from each of the 220 patients with squamous head and neck cancer (HNSCC). Conditional and marginal models were available; both models account for correlated data but have different aspects. The conditional logistic regression model was proposed to estimate the subject-specific risk of tumor based on the paired tumor and non-tumor data collection, which was in contrast with the marginal model to estimate population-average risk. The modeling process included rigorous variable selection, an initial multivariable model, a final model selection, and model validation. Genes with individual effect (p

Objective: MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs of 17 to 25 nucleotides that regulate approximately 30% of human genes. They are differentially expressed in various types of cancers compared with noncancerous tissues, suggesting that they may have crucial roles in tumorigenesis. The objective of this study was to discover LSCC-specific miRNAs.Method: Global miRNA profiling (800 human miRNAs plus 10 endogenous control miRNAs) was performed on 8 formalin-fixed archival LSCC samples and 5 normal laryngeal squamous epithelium (HTG, Tucson, AZ, USA). Quantitative real-time PCR (qRT-PCR) approach was employed to verify expression status of selected miRNAs that were significantly different from normal controls.Results: Thirty-one of the 800 human miRNAs were significantly differentially expressed (P < .05) between LSCC vs normal tissues. Selected miRNAs (miR-31, miR-193b, miR-663b, miR-923, and miR-1826), by qRT-PCR, verified expression of consistently upregulated miR-31 and miR-193b as well ...

Objective: Oncogenic human papillomavirus (HPV) has been established as a causative agent for 25% of head and neck squamous cell cancer (HNSCC), including laryngeal squamous cell cancer (LSCC). HPV-positive oropharyngeal patients have improved prognosis. We determined outcomes in LSCC in the context of HPV infection.Method: Seventy-nine primary LSCC were examined for 21 risk factors. HPV status was determined using real-time quantitative PCR. Chi-square/Fisher exact test were used to test association of HPV-positive with the other risk factors. Kaplan-Meier and log rank test were used to study the risk of HPV-positive status for overall survival.Results: HPV-16 was detected in 21 out of 77 (27%) primary LSCC, 16% (5/32) African American (AA) and 33% (15/45) Caucasian American (CA). Significance between race and HPV status was marginal (P = .058). HPV-positive status was significantly associated with male gender (P = .017) and insurance type (P = .001). HPV-positive LSCC had longer median survival (6.64 ye...

Aberrant methylation of promoter CpG islands is a hallmark of human cancers and is an early event in carcinogenesis. We examined whether promoter hypermethylation contributes to the pathogenesis of benign breast lesions along a progression continuum to invasive breast cancer. The exploratory study cohort comprised 17 breast cancer patients with multiple benign and/or in situ lesions concurrently present with invasive carcinoma within a tumor biopsy. DNA from tumor tissue, normal breast epithelium when present, benign lesions (fibroadenoma, hyperplasia, papilloma, sclerosing adenosis, apocrine metaplasia, atypical lobular hyperplasia or atypical ductal hyperplasia), and in situ lesions of lobular carcinoma and ductal carcinoma were interrogated for promoter methylation status in 22 tumor suppressor genes using the multiplex ligation-dependent probe amplification assay (MS-MLPA). Methylation specific PCR was performed to confirm hypermethylation detected by MS-MLPA. Promoter methylation was detected in 11/22 tumor suppressor genes in 16/17 cases. Hypermethylation of RASSF1 was most frequent, present in 14/17 cases, followed by APC in 12/17, and GSTP1 in 9/17 cases with establishment of an epigenetic monocloncal progression continuum to invasive breast cancer. Hypermethylated promoter regions in normal breast epithelium, benign, and premalignant lesions within the same tumor biopsy implicate RASSF1, APC, GSTP1, TIMP3, CDKN2B, CDKN2A, ESR1, CDH13, RARB, CASP8, and TP73 as early events. DNA hypermethylation underlies the pathogenesis of step-wise transformation along a monoclonal continuum from normal to preneoplasia to invasive breast cancer.

The goal was to determine recurrent or second primary status for late stomal malignancies, 16 and 17 years post-total laryngectomy in two laryngeal squamous cell carcinoma (LSCC) patients, based on DNA methylation signatures and HPV typing. Adopting a literature review based definition of late stomal recurrences as new primaries at the site of the stoma or neopharynx occurring more than 5 years after total laryngectomy, we employed a multi-gene candidate approach to examine promoter methylation in 24 tumor suppressor genes and PCR-based assays for HPV status offered additional insights into whether the late stomal tumors post total laryngectomy were related or not. The primary tumor for Patient 1 was negative for HPV but had aberrant hypermethylation of APC, MLH1, and BRCA1. The stomal biopsy 17-years later showed presence of HPV-16 without any methylated genes. In Patient 2, HPV-11 and promoter methylation of APC identified in the primary tumor was also observed in the stomal malignancy 16 years post total laryngectomy. Additional information provided by molecular typing for HPV and methylation markers underscored Patient 1’s and 2’s late stomal presentation as most likely a second primary and recurrence, respectively. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable marker. Molecular marks to discern genetic heterogeneity or relatedness of stomal malignancies several years post-total laryngectomy can provide clues to their status as either second primaries or likely recurrences. Our results support the hypothesis that a subset of stomal recurrences after total laryngectomy represents second primary tumors.

IntroductionThis study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC).Materials and methodsA retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multigene (22 tumor suppressor genes) methylation-specific multiplex ligationdependent probe amplification assay and confirmed using methylation specific PCR.ResultsTwenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation ofCDKN2BandTIMP3was most frequent. Promoter hypermethylation ofBRCA2, APC, CDKN2AandCDKN2Bwas detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33.ConclusionConsistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

Objective Promoter hypermethylation is emerging as a promising molecular strategy for early detection of cancer. We examined promoter methylation status of 1143 cancer-associated genes to perform a global but unbiased inspection of methylated regions in head and neck squamous cell carcinoma (HNSCC). Study Design Laboratory-based study. Setting Integrated health care system. Subjects and Methods Five samples, two frozen primary HNSCC biopsies and three HNSCC cell lines, were examined. Whole genomic DNA was interrogated using a combination of DNA immunoprecipitation (IP) and Affymetrix whole-genome tiling arrays. Results Of the 1143 unique cancer genes on the array, 265 were recorded across five samples. Of the 265 genes, 55 were present in all five samples, and 36 were common to four of five samples, 46 to three of five, 56 to two of five, and 72 to one of five samples. Hypermethylated genes in the five samples were cross-examined against those in PubMeth, a cancer methylation database combining text mining and expert annotation ( http://www.pubmeth.org ). Of the 441 genes in PubMeth, only 33 are referenced to HNSCC. We matched 34 genes in our samples to the 441 genes in the PubMeth database. Of the 34 genes, eight are reported in PubMeth as HNSCC associated. Conclusion This pilot study examined the contribution of global DNA hypermethylation to the pathogenesis of HNSCC. The whole-genome methylation approach indicated 231 new genes with methylated promoter regions not yet reported in HNSCC. Examination of this comprehensive gene panel in a larger HNSCC cohort should advance selection of HNSCC-specific candidate genes for further validation as biomarkers in HNSCC.

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

Objective To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease. Design A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment. Setting Primary care medical center. Subjects We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors. Results Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB , APC , and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB , 7 (25%) for CHFR , and 6 (21%) for APC . Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR . Conclusions Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB , APC , and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

Epidemiological and laboratory evidence indicate the human papilloma virus (HPV) as a causative agent for some squamous head and neck cancer (HNSCC). The biologic significance of HPV as independent risk factor is also underscored by the improved prognosis for patients with HPV positive HNSCC relative to HPV negative HNSCC, due in part to a better therapeutic response to chemoradiotherapy. The mechanism for this improved prognosis remains underexplored. To assess the potential of HPV specific methylation biomarkers, we evaluated the status of 10 genes (CDH8, CRMP1, HTRE1, ELMO1, MEI1, MSX2, PCDH10, PCDHB11, C14orf162, and PITX2) in a cohort of 18 HPV positive and 14 HPV negative HNSCC using targeted sequencing (Illumina MiSeq). The genes were previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC using the Infinium HumanMethylation450 BeadChip (Lechner et al., 2013). In our sample, of the 10 genes, targeted sequencing confirmed significant differential methylation between HPV positive and negative for C14orf162 (p = 0.007), MEI1(p = 0.004), MSX2 (p = 0.002), and PCDHB11(p = 0.002). PCDHB11 and MSX2 were hypermethylated; MEI1 and C14orf162 were hypomethylated, concordant with the Lechner et al., study. In this independent sample set, confirmation of 4 of the 10 genes as differentially methylated in HPV positive vs HPV negative HNSCC with concordance of methylation direction (hypo- vs hypermethylated), validates their potential for differentiating HPV positive and HPV negative HNSCC. Cadherins are implicated in tumor progression and metastasis and are targets of the Polycomb group (PcG) proteins, which form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Our study further supports cadherin PCDHB11, together with C14orf162, MEI1, and MSX2 as likely biomarkers in HPV-associated HNSCC. Support: Komen KG110218 Citation Format: Kang Mei Chen, Josena K. Stephen, George Divine, Dhananjay Chitale, Tamer Ghanem, Steven Chang, Maria J. Worsham. Validation of potential methylation biomarkers in HPV-associated HNSCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2015-3834

There has been relatively little advancement in changing the management of women with ER negative (ER-) breast cancer (BC). Our objective is to examine the contribution of aberrantly methylated genes to the pathogenesis of ER- BC, determine their utility in refining classification of ER- subtypes [basal-like (BL) and Her2-enriched], in differentiating ER- BC in African American (AA) and Caucasian American (CA) women, and as new prognostic and therapeutic targets for better management of ER- BC. The methylation status of 40 ER- and 40 ER positive (ER+) breast cancer samples was assessed using the Illumina 450K. A 3-tiered approach was used to call out genes most differentially methylated between ER- and ER+. The top 70 genes were further downsized using the TaqMan low-density array expression assays, Ingenuity Pathway Analysis (IPA) to assess their biologic significance, followed by targeted sequencing (Illumina Miseq). Further assessment of the genes in the 450K TCGA data sets (UCSC Brower 2015) was used for final downsizing. Of the 70 highly ranked differentially methylated genes, 56 were hypermethylated and 14 hypomethylated, with positive correlation with gene expression for the majority of genes. Expression verification and significant rankings in canonical pathways and bionetworks resulted in further downsizing to 41 genes upon confirmation by targeted sequencing. Further filtering of the 41 genes with 450K TCGA data sets provided additional refinement to a 10 gene candidate panel currently being vetted in a retrospective cohort of over 1000 ER negative BC (with 40% AA). Our iterative approach to identify an ER negative-specific gene signature has the potential to offer predictive and prognostic markers of improved classification for ER- BC in AA and CA women. Confirmation and validation of these methylation profiles in independent cohorts of BL BC defined by cDNA microarrays should further stratify poor prognosis breast cancers subgroups and serve as targets for epigenetic-modifying agents individualized to AA and CA women with ER- BC. Human therapeutic intervention trials to reverse deleterious epigenetic changes have shown promise in halting tumor growth by reactivation of the tumor suppressor genes or by reversing DNA hypomethylation. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Dhananjay Chitale, Kang Mei Chen, Indrani Datta, Josena K. Stephen, George Divine. A drill-down approach to identifying an ER negative-specific gene signature. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2015-4765

Importance Human papillomavirus (HPV) is a known causative agent for oropharyngeal squamous cell carcinoma (OPSCC). Whereas it is becoming more firmly established that HPV-positive head and neck squamous cell carcinoma is associated with better survival outcomes, believed to be because of better response to chemoradiation therapy, the specific mechanisms for these improved survival outcomes remain underexplored. Objective To examine the relationship between HPV status and promoter methylation in an OPSCC cohort. Design, Setting, and Participants Real-time quantitative polymerase chain reaction was used to examine oncogenic HPV type 16 in a retrospective cohort of 121 patients with primary OPSCC. Aberrant promoter methylation of IGSF4, DAPK1, and ESR1 genes, known to be methylated in head and neck squamous cell carcinoma, including OPSCC, was examined by means of quantitative methylation-specific polymerase chain reaction. Interventions Patients received standard therapy. Main Outcomes and Measures Univariate associations between HPV and methylation were analyzed using Fisher exact tests followed by multivariable logistic regression. Cox proportional-hazards regression was used to model the risk of death given age, race, sex, HPV status, methylation, stage, smoking, and treatment. Results In univariate logistic regression analyses, HPV-positive status was significantly associated with Caucasian race ( P = .02), treatment (radiotherapy only, P = .01; chemoradiotherapy, P = .007), and IGSF4 methylation ( P = .005). The final multivariate logistic model, after controlling for patient characteristics (sex, age, smoking, race, and treatment) with backward variable selection among genes, retained IGSF4 methylation (OR, 4.5 [95% CI, 1.6-12.8]; P = .005), Caucasian race (OR, 2.9 [95% CI, 1.0-8.3]; P = .053), treatment (radiotherapy only vs neither: OR, 11.62 [95% CI, 2.02-66.82]; P = .02; chemoradiotherapy vs neither: OR, 11.15 [95% CI, 1.92-64.65]; P = .01), male sex (OR, 4.7 [95% CI, 1.3-17.0]; P = .02), and younger age (OR, 0.9 [95% CI, 0.90-1.0]; P = .008) as independent predictors of HPV-positive status. Cox regression modeling indicated HPV-negative status, age, male sex, smoking, and radiation treatment as independent predictors of mortality. Conclusions and Relevance Methylation of IGSF4 is an independent predictor of HPV-positive status. DNA methylation in conjunction with HPV infection appears to play a role in OPSCC.

Human papilloma virus (HPV) is a known causative agent for oropharyngeal squamous cell cancer (OPSCC). The goal of our study was to determine the relationship of HPV type 16 (HPV16) infection and methylation status in an OPSCC cohort. HPV16 was detected using real-time quantitative PCR (qPCR) in a retrospective cohort of 111 primary OPSCC. A 24 tumor suppressor gene candidate panel was assessed for methylation using the methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) assay. Univariate associations with HPV and gene methylation, age, gender, race and smoking were analyzed using Fisher's exact tests and logistic regression. A final multiple logistic regression model, to include patient characteristics and genes significant on univariate analysis, was built using backwards selection of genes. Statistical significance was set at p Citation Format: Kang Mei Chen, Josena K. Stephen, Meredith Mahan, Shaleta Havard, George Divine, Glendon Gardner, Vanessa P. Schweitzer, Maria J. Worsham. IGSF4 methylation is an independent marker of HPV positive OPSCC. [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 650. doi:10.1158/1538-7445.AM2013-650

Expression of p16INK4A (p16+ve) is highly correlated with human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC), however, p16-positivityis not limited to HPV-positive (HPV+ve) tumors and therefore, not a perfect surrogate for HPV. Also, p16+ve status has a strong relationship with survival and treatment outcomes in HNSCC, which is best documented for the oropharyngeal site (OP); non-OP sites such as the oral cavity (OC), larynx, and hypopharynx (HP) are understudied. The goal of this study was to evaluate p16 in the context of HPV and examine p16 survival outcomes in HPV+ve and HPV-negative (HPV-ve) site-specific HNSCC. The cohort of 80 primary HNSCC consisted of 10 HPV16+ve and 10 HPV16-ve cases each for four sites: OC, OP, larynx and HP. Expression of p16 was significantly different across sites (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 722. doi:1538-7445.AM2012-722

Objective To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis. Design Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay. Subjects Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). Results Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases. Conclusions A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

This study examined molecular (DNA hypermethylation), clinical, histopathological, demographical, smoking, and alcohol variables to assess diagnosis (early versus late stage) and prognosis (survival) outcomes in a retrospective primary laryngeal squamous cell carcinoma (LSCC) cohort. The study cohort of 79 primary LSCC was drawn from a multi-ethnic (37% African American), primary care patient population, diagnosed by surgical biopsies in the Henry Ford Health System from 1991 to 2004 and followed from 5 to 18 years (through 2009). Of the 41 variables, univariate risk factors of p p ESR1; p = 0.01), race as African American (p = 0.04), and tumor necrosis (extensive; p = 0.02) were independent predictors of late stage LSCC. Independent predictors of poor survival included presence of vascular invasion (p = 0.0009), late stage disease (p = 0.03), and methylation of the hypermethylated in cancer 1 (HIC1) gene (p = 0.0002). Aberrant methylation of ESR1 and HIC1 signified independent markers of poorer outcome. In this multi-ethnic, primary LSCC cohort, race remained a predictor of late stage disease supporting disparate diagnosis outcomes for African American patients with LSCC.