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Network statistics for network constructed from 89 genes with/near integration events.

The incidence of human papillomavirus (HPV)–related oropharynx cancer has steadily increased over the past two decades and now represents a majority of oropharyngeal cancer cases. Integration of the HPV genome into the host genome is a common event during carcinogenesis that has clinically relevant effects if the viral early genes are transcribed. Understanding the impact of HPV integration on clinical outcomes of head and neck squamous cell carcinoma (HNSCC) is critical for implementing deescalated treatment approaches for HPV+ HNSCC patients. RNA sequencing (RNA-seq) data from HNSCC tumors (n = 84) were used to identify and characterize expressed integration events, which were overrepresented near known head and neck, lung, and urogenital cancer genes. Five genes were recurrent, including CD274 (PD-L1). A significant number of genes detected to have integration events were found to interact with Tp63, ETS, and/or FOX1A. Patients with no detected integration had better survival than integration-positive and HPV− patients. Furthermore, integration-negative tumors were characterized by strongly heightened signatures for immune cells, including CD4+, CD3+, regulatory, CD8+ T cells, NK cells, and B cells, compared with integration-positive tumors. Finally, genes with elevated expression in integration-negative specimens were strongly enriched with immune-related gene ontology terms, while upregulated genes in integration-positive tumors were enriched for keratinization, RNA metabolism, and translation.Implications: These findings demonstrate the clinical relevancy of expressed HPV integration, which is characterized by a change in immune response and/or aberrant expression of the integration-harboring cancer-related genes, and suggest strong natural selection for tumor cells with expressed integration events in key carcinogenic genes. Mol Cancer Res; 16(1); 90–102. ©2017 AACR.

Supplemental Methods, Tables S5-S14, and Figures S1-S3

Enriched GO terms for genes differentially expressed between HPV(+) and HPV(-) HNC tumors in the UM cohort.

Supplementary methods and figures. from Subtypes of HPV-Positive Head and Neck Cancers Are Associated with HPV Characteristics, Copy Number Alterations, PIK3CA Mutation, and Pathway Signatures

Purpose: There is substantial heterogeneity within human papillomavirus (HPV)-associated head and neck cancer (HNC) tumors that predispose them to different outcomes; however, the molecular heterogeneity in this subgroup is poorly characterized due to various historical reasons.Experimental Design: We performed unsupervised gene expression clustering on deeply annotated (transcriptome and genome) HPV+ HNC samples from two cohorts (84 total primary tumors), including 18 HPV− HNC samples, to discover subtypes and characterize the differences between subgroups in terms of their HPV characteristics, pathway activity, whole-genome somatic copy number alterations, and mutation frequencies.Results: We identified two distinct HPV+ subtypes (namely HPV-KRT and HPV-IMU). HPV-KRT is characterized by elevated expression of genes in keratinocyte differentiation and oxidation–reduction process, whereas HPV-IMU has strong immune response and mesenchymal differentiation. The differences in expression are likely connected to the differences in HPV characteristics and genomic changes. HPV-KRT has more genic viral integration, lower E2/E4/E5 expression levels, and higher ratio of spliced to full-length HPV oncogene E6 than HPV-IMU; the subgroups also show differences in copy number alterations and mutations, in particular the loss of chr16q in HPV-IMU and gain of chr3q and PIK3CA mutation in HPV-KRT.Conclusions: Our characterization of two subtypes of HPV+ HNC tumors provides valuable molecular level information that point to two main carcinogenic paths. Together, these results shed light on stratifications of the HPV+ HNCs and will help to guide personalized care for HPV+ HNC patients. Clin Cancer Res; 22(18); 4735–45. ©2016 AACR.

Head and neck cancer is often diagnosed late and prognosis for most head and neck cancer patients remains poor. To aid early detection, we developed a risk prediction model based on demographic and lifestyle risk factors, human papillomavirus (HPV) serological markers, and genetic markers. A total of 10,126 head and neck cancer cases and 5,254 controls from 5 North American and European studies were included. HPV serostatus was determined by antibodies for HPV16 early oncoproteins (E6, E7) and regulatory early proteins (E1, E2, E4). The data were split into a training set (70%) for model development and a hold-out testing set (30%) for model performance evaluation, including discriminative ability and calibration. The risk models including demographic, lifestyle risk factors and polygenic risk score showed a reasonable predictive accuracy for head and neck cancer overall. A risk model that also included HPV serology showed substantially improved predictive accuracy for oropharyngeal cancer (AUC=0.94, 95%CI=0.92–0.95 in men and AUC=0.92, 95%CI=0.88–0.95 in women). The 5-year absolute risk estimates showed distinct trajectories by risk factor profiles. Based on the UK Biobank cohort, the risks of developing oropharyngeal cancer among 60 years old and HPV16 seropositive in the next 5 years ranged from 5.8% to 14.9% with an average of 8.1% for men, 1.3% to 4.4% with an average of 2.2% for women. Absolute risk was generally higher among individuals with heavy smoking, heavy drinking, HPV seropositivity, and those with higher polygenic risk score. These risk models may be helpful for identifying people at high risk of developing head and neck cancer.

Recently, ovarian cancer research has evolved considerably because of the emerging recognition that rather than a single disease, ovarian carcinomas comprise several different histotypes that vary by etiologic origin, risk factors, molecular profiles, therapeutic approaches and clinical outcome. Despite significant progress in our understanding of the etiologic heterogeneity of ovarian cancer, as well as important clinical advances, it remains the eighth most frequently diagnosed cancer in women worldwide and the most fatal gynecologic cancer. The International Agency for Research on Cancer and the United States National Cancer Institute jointly convened an expert panel on ovarian carcinoma to develop consensus research priorities based on evolving scientific discoveries. Expertise ranged from etiology, prevention, early detection, pathology, model systems, molecular characterization and treatment/clinical management. This report summarizes the current state of knowledge and highlights expert consensus on future directions to continue advancing etiologic, epidemiologic and prognostic research on ovarian carcinoma.

Background The change over time of distribution of breast cancer subtypes using population-based data has not been reported. Objective To describe the change over time of the distribution of female breast cancer by clinical subtype among the population in Chiang Mai, Thailand. Methods Data of breast cancer patients from Chiang Mai Cancer Registry, diagnosed from 2004 to 2013 were combined with immunohistochemical status from medical record, and used to describe the proportions of clinical breast cancer subtypes: (1) luminal A-like (ER+/PR+ and HER2-), (2) luminal B-like (ER+/PR+ and HER2+), (3) HER2 (ER- and PR- and HER2+), (4) triple-negative (ER- and PR- and HER2-). The distribution of breast cancer subtypes by age group was also described. Results Among 3,228 female breast cancer cases diagnosed during 2004–2013, the median age was 52 years and most patients presented at the regional stage. The unknown tumor subtype was lower than 25% in the periods 2008– 2009, 2010–2011, and 2012–2013. In those periods, the proportions of luminal A-like were 33%, 36%, and 48%; the proportions of luminal B-like were 14%, 20%, and 16%, the proportions of HER2 were 15%, 14%, and 13%; and the proportions of triple-negative were 16%, 14%, and 13%, respectively. In comparison with other groups, women aged ≥60 years had a significantly higher proportion of luminal A-like (P = 0.001), while women aged P = 0.10). Conclusions The proportion of breast cancer with luminal subtypes is increasing. Thus, in the future, treatment protocols with a variety of hormone therapies should be provided in order to improve efficacy and coverage of treatment for this population.

Purpose Prostate cancer is the second most common malignancy among men worldwide, and it poses a significant public health burden that has traditionally been limited mostly to developed countries. However, the burden of the disease is expected to increase, affecting developing countries, including Thailand. We undertook an analysis to investigate current and future trends of prostate cancer in the province of Songkhla, Thailand, using data from the Songkhla Cancer Registry from 1990 to 2013. Methods Joinpoint regression analysis was used to examine trends in age-adjusted incidence and mortality rates of prostate cancer and provide estimated annual percent change (EAPC) with 95% CIs. Age-period-cohort (APC) models were used to assess the effect of age, calendar year, and birth cohort on incidence and mortality rates. Three different methods (Joinpoint, Nordpred, and APC) were used to project trends from 2013 to 2030. Results Eight hundred fifty-five cases of prostate cancer were diagnosed from 1990 to 2013 in Songkhla, Thailand. The incidence rates of prostate cancer significantly increased since 1990 at an EAPC of 4.8% (95% CI, 3.6% to 5.9%). Similarly, mortality rates increased at an EAPC of 5.3% (95% CI, 3.4% to 7.2%). The APC models suggest that birth cohort is the most important factor driving the increased incidence and mortality rates of prostate cancer. Future incidence and mortality of prostate cancer are projected to continue to increase, doubling the rates observed in 2013 by 2030. Conclusion It is critical to allocate resources to provide care for the men who will be affected by this increase in prostate cancer incidence in Songkhla, Thailand, and to design context-appropriate interventions to prevent its increasing burden.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC., Genetic susceptibility loci for oropharyngeal cancer have been reported but these studies have not always examined human papillomavirus (HPV) status. Here, the authors perform genome-wide analysis taking into account HPV16 serology status and report two independent loci in the HLA region, suggesting the protective role of HLA variants against HPV infection.

Our study aimed to investigate the effect of Pap smear screening on stage at diagnosis of cervical cancer in a heterogeneous population of Thai women. Data was merged from the population-based cancer registry and screening registry based on unique identification numbers from 2006 to 2014. Patients being screened had lower odds to be diagnosed at late stage. After adjustment, married women had reduced risk of late stage cancer compared to single women. Muslim women had almost twice the risk of being diagnosed late stage compared to Buddhist women. The odds of being diagnosed at late stage decreased with increased number of screening. The probability of being diagnosed at late stage increased rapidly among females aged 40 to 55 years. Pap smear screening is a protective factor in diagnosis of late stage cervical cancer. Patients were more likely to be diagnosed at early stage with more frequent screening. For future screening programs, it will be beneficial to shorten screening intervals and take more concern for vulnerable population: women aged between 40 and 55 years, and women who are single or Muslim.

Purpose: There is substantial heterogeneity within human papillomavirus (HPV)-associated head and neck cancer (HNC) tumors that predispose them to different outcomes; however, the molecular heterogeneity in this subgroup is poorly characterized due to various historical reasons. Experimental Design: We performed unsupervised gene expression clustering on deeply annotated (transcriptome and genome) HPV+ HNC samples from two cohorts (84 total primary tumors), including 18 HPV− HNC samples, to discover subtypes and characterize the differences between subgroups in terms of their HPV characteristics, pathway activity, whole-genome somatic copy number alterations, and mutation frequencies. Results: We identified two distinct HPV+ subtypes (namely HPV-KRT and HPV-IMU). HPV-KRT is characterized by elevated expression of genes in keratinocyte differentiation and oxidation–reduction process, whereas HPV-IMU has strong immune response and mesenchymal differentiation. The differences in expression are likely connected to the differences in HPV characteristics and genomic changes. HPV-KRT has more genic viral integration, lower E2/E4/E5 expression levels, and higher ratio of spliced to full-length HPV oncogene E6 than HPV-IMU; the subgroups also show differences in copy number alterations and mutations, in particular the loss of chr16q in HPV-IMU and gain of chr3q and PIK3CA mutation in HPV-KRT. Conclusions: Our characterization of two subtypes of HPV+ HNC tumors provides valuable molecular level information that point to two main carcinogenic paths. Together, these results shed light on stratifications of the HPV+ HNCs and will help to guide personalized care for HPV+ HNC patients. Clin Cancer Res; 22(18); 4735–45. ©2016 AACR.

Background To better understand the associations between a history of tobacco use and survival outcomes, cigarette use was prospectively surveyed in 687 previously untreated patients with cancer of the oral cavity (n = 271), oropharynx (n = 257), larynx (n = 135), or hypopharynx (n = 24). Methods Kaplan–Meier and Cox models explored the associations of tobacco use intensity (packs/day), duration (years of use), and timing before diagnosis with overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS). Results Cigarette use duration, timing, and intensity were significant predictors for all outcomes in univariate analysis. Never smoking and pack-years were not significantly associated with outcomes after adjustment for prognostic factors, such as stage, comorbidities, and human papillomavirus (HPV) status, which were strongly associated with clinical outcomes. Conclusion The findings confirm the association between smoking history and survival and the importance of clinical variables in evaluating smoking as a prognostic factor. Timing, intensity, and duration of cigarette use should be considered with other prognostic factors when considering risk stratification for treatment planning. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1810–1820, 2016

Because immune responses within the tumor microenvironment may be important predictors of tumor biology, correlations of specific types of tumor infiltrating lymphocytes (TILs) with clinical variables and outcomes were determined in 278 previously untreated patients with head and neck cancer (HNSCC). Methods Infiltrating levels of CD4 (helper T cells), CD8 (cytotoxic/suppressor T cells), FoxP3, regulatory T cells), CD68 (myeloid derived suppressor cells) and CD1a (Langerhan) cells were retrospectively measured by immunohistology in tissue mircoarrays. Cox models tested associations with patient outcomes after adjusting for all known prognostic factors. Median follow up was 36.6 months.

Background As cancer progresses, methylation patterns change to promote the tumorigenic phenotype. However, stability of methylation markers over time and the extent that biopsy samples are representative of larger tumor specimens are unknown. This information is critical for clinical use of such biomarkers. Methods Ninety-eight patients with tumor specimens from 2 timepoints were measured for DNA methylation in the promoter regions across 4 genes. Results There were no significant differences in overall methylation of CCNA1 (cyclin A1), NDN (necdin), deleted in colorectal carcinoma (DCC), and cluster of differentiation 1a (CD1A) within paired specimens (p values = .56, .17, .66, and .58, respectively). All genes showed strong correlations between paired specimens across time. Methylation was most consistent for CCNA1 and NDN over time. Conclusion This report provides the first evidence that methylation markers measured in biopsy samples are representative of gene methylation in later specimens and suggests that biopsy markers could be representative biomarkers for use in defining personalized treatment utilizing epigenetic changes. © 2015 Wiley Periodicals, Head Neck, 2015

BACKGROUND:Maternal mortality (MM) was persistently high for several decades in Ethiopia though it has declined in recent years. The roles of time-varying elements in this decrease are unknown. Analyzing MM with age-period-cohort analysis will provide evidence to policymakers to re-direct resources towards vulnerable age groups. The aim of this analysis was to determine the role of age effect, period effect and birth cohort effect on the trend of age-specific maternal mortality in Ethiopia. METHODS:Age-period-cohort (APC) analysis was applied to examine the effect of age, period and birth cohort on MM in Ethiopia using data from the Ethiopian Demographic and Health Survey (EDHS) from years 2000, 2005, 2011 and 2016. Age-specific maternal mortality rates were calculated using standardized maternal death compared to age-specific population per 100,000 woman-years of exposure and the trend was analyzed. RESULT:In most age groups, the MM rate decreased in 2015 compared with the previous years except for older women. According to the APC analysis, the age-cohort effect explains the MM rate better than age-period effect. The period effect shows the risk ratio of MM after 2005 decreased compared with before. The cohort effect illustrates women born after 1980 has lower risk ratio compared with the older one. CONCLUSION:Maternal mortality in Ethiopia declined overall in recent years. However, certain age groups still face high maternal mortality rates. A national policy on MM reduction interventions for the identified high-risk age groups is required.

BackgroundCervical cancer rates are higher in low-resourced countries than high, partly due to lower rates of screening. Incidence in Thailand is nearly three times higher than in the USA (16.2 vs 6.5 age-standardised incidence), even with Thailand’s universal health coverage, which includes screening, suggesting that alternative methods are needed to reduce the burden. We investigated barriers to screening, as well as acceptability of self-collection human papillomavirus (HPV) testing as a primary form of cervical cancer screening among Buddhist and Muslim communities in Southern Thailand.Methods267 women from the Buddhist district of Ranot and Muslim district of Na Thawi, Songkhla were recruited to complete a survey assessing knowledge and risk factors of HPV and cervical cancer. Participants were offered an HPV self-collection test with a follow-up survey assessing acceptability. Samples were processed at Prince of Songkhla University and results were returned to participants.Results267 women participated in the study (132 Buddhist, 135 Muslim), 264 (99%) self-collecting. 98% reported comfort and ease, and 70% preferred it to doctor-facilitated cytology. The main predictor of prior screening was religion (92% Buddhist vs 73% Muslim reporting prior Pap). After adjustment with multivariate logistic models, Muslim women had an OR of prior Pap of 0.30 compared with Buddhist (95% CI: 0.12 to 0.66).ConclusionsSelf-collection HPV testing was highly acceptable across religious groups, suggesting that it could be beneficial for cervical cancer reduction in this region. Focus should be put into educating women from all backgrounds about the importance of screening to further improve screening rates among Thai women.

While whole-exome DNA sequencing is the most common technology to study genetic variants in tumors in known exonic regions, RNA-seq is cheaper, covers most of the same exonic regions, and is often more readily available. In this study, we show the utility of mRNA-seq-based variant analysis combined with targeted gene sequencing performed on both tumor and matched blood as an alternative when exome data is unavailable. We use the approach to study expressed variant profiles in the well-characterized University of Michigan (UM) head and neck squamous carcinoma (HNSCC) cohort (n = 36). We found that 441 out of 455 (~97%) identified cancer genes with an expressed variant in the UM cohort also harbor a somatic mutation in TCGA. Fourteen (39%) patients had a germline variant in a cancer-related Fanconi Anemia (FA) pathway gene. HPV-positive patients had more nonsynonymous, rare, and damaging (NRD) variants in those genes than HPV-negative patients. Moreover, the known mutational signatures for DNA mismatch repair and APOBEC activation were attributive to the UM expressed NRD variants, and the APOBEC signature contribution differed by HPV status. Our results provide additional support to certain TCGA findings and suggest an association of expressed variants in FA/DNA repair pathways with HPV-associated HNSCC tumorigenesis. These results will benefit future studies on this and other cohorts by providing the genetic variants of key cancer-related genes.

Background The northeast has the lowest incidence of breast cancer of all regions in Thailand, although national rates are increasing. The heterogeneity in subnational trends necessitates a comprehensive evaluation of breast cancer incidence trends and projections to provide evidence for future region-specific strategies that may be employed to attenuate this growing burden. Methods Joinpoint regression and age-period-cohort modeling were used to describe trends from 1988-2012. Data was projected from three separate models to provide a range of estimates of incidence to the year 2030 by age group. Results Age-standardized rates (ASRs) increased significantly for all women from 1995-2012 by 4.5% per year. Rates for women below age 50 increased by 5.1% per year, while women age 50 years and older increased by 6% per year from 1988-2012. Projected rates show that women age 50 years and older have the largest projected increase in ASRs by 2030 compared to younger women and all women combined. Conclusions Breast cancer trends in Khon Kaen are presently lower than other regions but are expected to increase and become comparable to other regions by 2030, particularly for women ages 50 years and older.

Background. The incidence of liver and bile duct cancer continues to rise, especially in Thailand. We aimed to project the trends in incidence of this rare but lethal cancer in southern Thailand in order to determine its future disease burden. Methods. Gender-specific trends in age-standardized incidence rates per 100,000 person-years for hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) cases in Songkhla province of southern Thailand diagnosed between 1989 and 2013 were estimated and projected up to year 2030 using three different modeling techniques: a joinpoint model, an age-period-cohort model, and a modified age-period-cohort model. Results. Of 2,676 liver and bile duct (LBD) cancer cases identified, 73% were males, 51% were aged between 50 and 69 years, and HCC (44.4%) was slightly more common than CCA (38.1%). The models all predicted an increase in the incidence rate of CCA up to 2025 for both sexes whereas the incidence of HCC is expected to decrease among males and stabilize among females. The incidence rates of HCC and CCA among males in 2030 could reach 6.7 and 9.4 per 100,000 person-years, respectively, whereas the expected rates of HCC and CCA among females are expected to be around 1.5 and 3.9 per 100,000 person-years, respectively. Conclusions. The incidence of cholangiocarcinoma is expected to increase in Songkhla and will contribute a larger proportion of LBD cancers in the future. Future public health efforts and research studies should focus on this increasing trend.

Background Breast cancer has the highest incidence in women of all cancers and its burden is expected to continue to increase worldwide, especially in middle-income countries such as Thailand. The southern region of Thailand is unique in that it is comprised of 30% Muslims, whereas the rest of Thailand is 95% Buddhist. Breast cancer incidence and survival differ between these religious groups, but the association between clinical subtype of breast cancer and survival has not yet been assessed. Methods Here we characterized differences in breast cancer survival with consideration to clinical subtype by religious group (Muslim Thai and Buddhist Thai women). We compared distributions of age, stage and clinical subtype and assessed overall survival by religion. Results Our findings show that Muslim Thai women with breast cancer are diagnosed at a younger age, at later stages and have shorter overall survival times compared to Buddhist Thai women with breast cancer. We also observe a higher proportion of triple negative tumors characterized in Muslim Thai women. Conclusions Our findings confirm previous studies that have shown lower survival rates in Muslim Thai women compared to Buddhist women with breast cancer and offer novel information on subtype distribution. To date, this is the first study assessing clinical subtypes in southern Thailand by religious status. Impact Our findings are critical in providing information on the role of clinical subtype in cancer disparities and provide evidence from the Southeast Asian region for global studies on breast cancer survival.

In Thailand, five cancer types-breast, cervical, colorectal, liver and lung cancer-contribute to over half of the cancer burden. The magnitude of these cancers must be quantified over time to assess previous health policies and highlight future trajectories for targeted prevention efforts. We provide a comprehensive assessment of these five cancers nationally and subnationally, with trend analysis, projections, and number of cases expected for the year 2025 using cancer registry data. We found that breast (average annual percent change (AAPC): 3.1%) and colorectal cancer (female AAPC: 3.3%, male AAPC: 4.1%) are increasing while cervical cancer (AAPC: -4.4%) is decreasing nationwide. However, liver and lung cancers exhibit disproportionately higher burdens in the northeast and north regions, respectively. Lung cancer increased significantly in northeastern and southern women, despite low smoking rates. Liver cancers are expected to increase in the northern males and females. Liver cancer increased in the south, despite the absence of the liver fluke, a known factor, in this region. Our findings are presented in the context of health policy, population dynamics and serve to provide evidence for future prevention strategies. Our subnational estimates provide a basis for understanding variations in region-specific risk factor profiles that contribute to incidence trends over time.

Cervical cancer has posed a serious problem in Thailand for decades. In 2002, a systematic screening program was implemented under universal healthcare coverage for all Thai women. However, there has been little research on how screening affected particular aspects of cervical cancer, such as stage distribution. This screening program has a target group; therefore, it is necessary to assess stage and incidence trends by age of those within and outside the screening target group. Using trend analysis, we assess in situ and malignant cervical cancers in Northern Thailand to measure changes after implementation of the national screening program. While incidence of malignant cancers is decreasing and incidence of in situ tumors is increasing across all age groups, women above age 60 still experience a high incidence of malignant tumors. The screening program is successful in the target group at downshifting the stage distribution of malignant tumors and reducing incidence of malignant tumors with in situ cases being captured. However, the high incidence of malignant tumors in women over age 60 will continue to be clinically relevant for cervical cancer management until younger generations undergoing screening enter this age group.

The incidence of human papillomavirus (HPV)–related oropharynx cancer has steadily increased over the past two decades and now represents a majority of oropharyngeal cancer cases. Integration of the HPV genome into the host genome is a common event during carcinogenesis that has clinically relevant effects if the viral early genes are transcribed. Understanding the impact of HPV integration on clinical outcomes of head and neck squamous cell carcinoma (HNSCC) is critical for implementing deescalated treatment approaches for HPV+ HNSCC patients. RNA sequencing (RNA-seq) data from HNSCC tumors (n = 84) were used to identify and characterize expressed integration events, which were overrepresented near known head and neck, lung, and urogenital cancer genes. Five genes were recurrent, including CD274 (PD-L1). A significant number of genes detected to have integration events were found to interact with Tp63, ETS, and/or FOX1A. Patients with no detected integration had better survival than integration-positive and HPV− patients. Furthermore, integration-negative tumors were characterized by strongly heightened signatures for immune cells, including CD4+, CD3+, regulatory, CD8+ T cells, NK cells, and B cells, compared with integration-positive tumors. Finally, genes with elevated expression in integration-negative specimens were strongly enriched with immune-related gene ontology terms, while upregulated genes in integration-positive tumors were enriched for keratinization, RNA metabolism, and translation. Implications: These findings demonstrate the clinical relevancy of expressed HPV integration, which is characterized by a change in immune response and/or aberrant expression of the integration-harboring cancer-related genes, and suggest strong natural selection for tumor cells with expressed integration events in key carcinogenic genes. Mol Cancer Res; 16(1); 90–102. ©2017 AACR.

DNA methylation changes with age, and may serve as a biomarker of aging. Cadmium (Cd) modifies cellular processes that promote aging and disrupts methylation globally. Whether Cd modifies aging processes by influencing establishment of age-associated methylation marks is currently unknown. In this pilot study, we characterized methylation profiles in > 450 000 CpG sites in 40 non-smoking women (age 40–80) differentially exposed to environmental Cd from Thailand. Based on specific gravity adjusted urinary Cd, we classified them as high (HE) and low (LE) exposed and age-matched within 5 years. Urinary Cd was defined as below 2 µg/l in the LE group. We predicted epigenetic age (DNAm-age) using two published methods by Horvath and Hannum and examined the difference between epigenetic age and chronologic age (Δage). We assessed differences by Cd exposure using linear mixed models adjusted for estimated white blood cell proportions, BMI, and urinary creatinine. We identified 213 age-associated CpG sites in our population (P < 10−4). Counterintuitively, the mean Δage was smaller in HE vs. LE (Hannum: 3.6 vs. 7.6 years, P = 0.0093; Horvath: 2.4 vs. 4.5 years, P = 0.1308). The Cd exposed group was associated with changes in methylation (P < 0.05) at 12, 8, and 20 age-associated sites identified in our population, Hannum, and Horvath. From the results of this pilot study, elevated Cd exposure is associated with methylation changes at age-associated sites and smaller differences between DNAm-age and chronologic age, in contrast to expected age-accelerating effects. Cd may modify epigenetic aging, and biomarkers of aging warrant further investigation when examining Cd and its relationship with chronic disease and mortality.

Objective: Thailand is undergoing a period of rapid economic growth, which has led to an epidemiologic transition from infectious to chronic diseases such as cancer. Preliminary research shows a difference in rates of breast cancer incidence and mortality between Buddhist and Muslim women in southern Thailand. This study examines differences in breast cancer and potential explanations for this disparity. Methods: Demographic, diagnostic, and outcome data from breast cancer patients diagnosed from 2001-2015 were collected from a population-based cancer registry in southern Thailand and matched with formalin-fixed paraffin-embedded blocks from Songklanagarind Hospital. This region was selected due to the relatively high proportion of Muslim women. Of 424 cases selected, 369 were Buddhist and 55 were Muslim. Formalin-fixed paraffin-embedded blocks were sent to Michigan Medicine and histological and subtype analyses were performed by two pathologists. Statistical analysis was performed in RStudio using chi-square tests of independence, logistic regression, and Cox proportional hazards models. Results: Muslim women were generally diagnosed at a younger age than Buddhist women (Muslim mean: 46.6 years, Buddhist mean: 52.7 years) and with later stage cancers (Muslim: 36% stage 3 and 14% stage 4, Buddhist: 20% stage 3 and 8% stage 4). Buddhist and Muslim women had similar distributions of Luminal A and B subtypes, but Muslim women were more likely to be diagnosed with Triple Negative breast cancer (Muslim: 30%, Buddhist: 18%), the histological subtype with the worst prognosis. Invasive ductal carcinoma was most common in both groups, but Buddhists had more heterogeneity in subtypes. There were no significant differences in tumor histology or subtype by religion. Conclusions: These analyses show differences in the distribution of breast cancer between Buddhist and Muslim women. Muslim women are diagnosed younger with later stage breast cancers, and with histological differences, which generally lead to lower survival rates. Lack of statistical significance may be due to small sample size, necessitating further studies. Citation Format: Kali Defever, Katie M. Rentschler, Ana Khazan, Elizabeth Pinkerton, Shama Virani, Hutcha Sriplung, Judy Pang, Celina G. Kleer, Carlos F. Mendes de Leon, Justin A. Colacino, Laura S. Rozek. Breast cancer distribution and survival among Buddhist and Muslim women in southern Thailand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4219.

Introduction: Cadmium (Cd) exposure initiates cellular processes that promote aging and disrupts methylation globally. Aging is associated with DNA methylation changes, and age-associated methylati...

Table S1. Primer Sets and PCR Conditions for Methylation Analysis. Table S2. CpG Locations. Table S3. Quartile and Interquartile Ranges. Figure S1. Comparison of survival and recurrence outcomes by HPV (+) oropharyngeal (OP) and HPV (+) non-OP patients. Significant difference between groups is determined by a univariable Cox Proportional Hazards model for each outcome with a p-value

Background The national cervical cancer screening program in Thailand has been successful in reducing overall burden from this disease. However, evaluation on spatial and temporal scales is needed to assess the efficacy of this program in smaller regions. Here, we geographically assess incidence in a province with a uniquely heterogeneous distribution of lifestyle factors associated with religiosity. Methods Cervical cancer cases were extracted from the provincial cancer registry from 1989 to 2013. Age-adjusted incidence rates were calculated using population statistics from the census bureau and adjusted to the Segi world standard population. Bayesian hierarchical modelling was employed to spatiotemporally map cervical cancer incidence trends in Songkhla province in 5-year period. Results Overall, the incidence of cervical cancer decreased in Songkhla province. The three districts with a Muslim population of greater than 70% had consistently lower cervical cancer rates from 1989 to 2013 compared with the rest of the predominantly Buddhist districts. Hotspots of incidence were identified in Sadao, Hat Yai and the juncture of Mueang Songkhla and Singhanakhon in each 5-year period. Conclusions Distinct cervical cancer incidence trends by religion over time indicate differences in sexual habits, lifestyle and religion-associated culture between Muslims and Buddhists, and suggest divergent risk factor profiles for these groups. The high incidence rates in Sadao and Hat Yai is likely explained by the main road to Malaysia, which runs across these two areas and has frequent commercial sex trade. Female sex workers should be targeted as a vulnerable population for screening efforts to address this continuing burden of cervical cancer.

Background: Prostate cancer is one of the most commonly diagnosed cancers in men worldwide. In Asia, the incidence of prostate cancer is expected to increase in the next decades as the population ages and as developing Asian countries undergo the epidemiologic transition. There is a scarcity of studies on the epidemiology of prostate cancer in Thailand. The characterization and projection of the burden of prostate cancer are important to design cost-effective strategies for the prevention and control of this disease. The purpose of this study is to examine incidence and mortality trends of prostate cancer in southern Thailand. Methods: Incident prostate cancer cases from the Songkhla cancer registry were used to analyze incidence and mortality trends; complete data on incidence was available from 1990-2013 and data on mortality was available from 1990-2010. Characteristics of the cases on sociodemographic factors and tumor characteristics were collected, as was vital status. Joinpoint analysis was used to examine the incidence and mortality trends and calculate the annual percent change (APC). The number of joinpoints, slope of the trends, and their significance was determined by permutation tests. Incidence and mortality rates were standardized to the Segi's world standard population. Results: Eight hundred fifty-five prostate cancer cases were diagnosed from 1990 to 2013. The median age at diagnosis was 74 and 89.6% of the diagnosed cases were Buddhist, with the remainder being Muslim. The majority (79.8%) of prostate cancers were unstaged. However, among those cases whose tumors were staged, the distribution was as follows: 3.5% stage I, 17.9% stage II, 2.9% stage III, 75.7% stage IV. The proportion of unstaged tumors decreased during the period 2005-2009 compared to earlier periods, and there was an increase in the proportion of stage II tumors for the same period. The overall prostate cancer incidence rates increased significantly at an APC of 5.1% (95%CI: 3.8, 6.4) from 1990 to 2013. Among Buddhist men, the APC was very similar to the overall incidence (APC: 5.2%, 95%CI: 4.0, 6.5), while we observed a very low incidence rate and no evidence of an increasing trend in Muslim men. As of 2010, 72.7% of the cases had died. Similar to incidence rates, prostate cancer mortality rates have significantly increased at and APC of 3.2% (95CI%: 1.7, 4.8) from 1990 to 2010. Conclusion: This analysis showed that there has been a significant increase in prostate cancer incidence and mortality in Southern Thailand during the period 1990-2013. Next steps include assessing the effects of age, calendar year, and birth-cohort on the incidence and mortality of prostate cancer and projection of the incidence and mortality rates over the next fifteen years in order to fully characterize this cancer and help to design strategies to reduce the future burden of prostate cancer in Thailand. Citation Format: Christian Alvarez, Shama Virani, Rafael Meza, Laura Rozek, Hutcha Sriplung, Alison M. Mondul. Incidence and mortality trends of prostate cancer in southern Thailand. [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 5210.

Soy intake has been associated with improved survival in HNSCC and soy isoflavones have been suggested as potential chemopreventive agents with a favorable therapeutic index and safety profile. Multiple in vivo and in vitro anti-cancer effects have been postulated including modulation of gene methylation. To determine if high dose soy isoflavone treatment changes methylation of genes associated with cancer outcomes, we measured genomic and gene-specific methylation in tumor tissue collected as a part of a multi-institutional neoadjuvant soy isoflavone clinical trial in HNSCC patients undergoing definitive surgical management. Methods: Thirty-nine patients participated in this clinical trial. Patients were scheduled to receive 2 weeks (range 7-39 days, median = 15.2) of soy isoflavone supplements (300 mg/day G-2535; DCP, NCI, NIH) orally prior to surgery. Levels of methylation of LINE-1 (global methylation), and 6 other candidate genes previously associated with HNSCC (p16, DCC, NDN, CD1a, CCNA1 and Gadd45α) were measured by pyrosequencing in biopsy, resection and whole blood specimens. Twelve patients were stage I/II, 27 were stage III/IV. Thirty one patients had cancer of the oral cavity, 4 had larynx and 4 had oropharynx cancer. Mean age of the patients was 60.1 (sd = 12.5). Changes in methylation were tested using paired t-tests and clinical associations explored using ANOVA. Results: LINE-1 methylation increased significantly (mean increase 4.9%; range -34.8% to +20.9%) in tumor specimens after soy isoflavone (p Conclusions: This is the first demonstration of significant increases in tissue-specific global methylation associated with soy isoflavone intake, indicating increased genomic stability. The association of hypomethylation of LINE-1 with genetic instability, carcinogenesis and poor treatment outcomes suggests that soy isoflavones should be studied further as a potential chemopreventive agent in HNSCC. Citation Format: Laura Rozek, Shama Virani, Emily Bellile, Jeremy Taylor, Maureen Sartor, Alisha Virani, Katie Rentschler, Claire Cote, Francis Worden, Lisa Peterson, Douglas Chepeha, Mark Prince, Scott McLean, George Yoo, Neil F. Saba, Dong M. Shin, Omer Kucuk, Gregory T. Wolf. Soy isoflavones modulate global methylation in head and neck squamous cell carcinoma (HNSCC). [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 3114.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, and an increasing percentage of HNSCC cases are associated with human papillomavirus (HPV) which have improved prognosis compared to HPV(-) HNSCC. Identifying distinctions between HPV(+) and HPV(-) HNSCC may lead to improved prognosis or new treatments, such as determining mutagen(s) that are driving accumulation of genetic variants in each tumor. A recent approach uses the fact that different mutagens cause different types of mutations, referred to as a mutagen's signature. By comparing the mutations in a tumor sample with the mutagen signatures, the mutagens responsible for most of the mutations in the tumor can be identified. We hypothesized that mutational signatures could distinguish HPV(+) from HPV(-) HNSCC tumors. Materials and Methods We performed RNA-sequencing on 18 HPV(+) and 18 HPV(-) tumor samples collected from HNSCC patients by the University of Michigan Head and Neck SPORE. Variant calling was performed on each sample using a GATK best practices for RNA-seq pipeline. These variants were then filtered to include only variants that were both rare ( Results In the studied HNSCC tumors, signatures related to aging and impaired DNA repair were responsible for the largest proportion of the mutations found. The signatures that were significantly different between HPV(+) and HPV(-) tumors were associated with APOBEC editing and defective DNA mismatch repair. Expression and mutational analysis validated that APOBEC is more active in HPV(+) than HPV(-) tumors. Interestingly, signatures associated with smoking were not among the most relevant. Conclusion The evaluation of mutational signatures in HNSCC tumors identified the main sources of mutations for HNSCC. The differences in mutational signatures between HPV(+) and HPV(-) identify distinct methods of carcinogenesis for these two subtypes of HNSCC. Citation Format: Pelle Hall, Shama Virani, Charles Warden, Yanxiao Zhang, Lada Koneva, Katie M. Rentschler, Alisha Virani, Laura S. Rozek, Maureen A. Sartor, The UM Head and Neck SPORE Investigators. Mutational signatures from RNA-seq data distinguish HPV(+) and HPV(-) HNSCC. [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 1511.

Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most incident cancer worldwide. Human papillomavirus (HPV) is implicated in at least 70% of oropharyngeal squamous cell carcinomas in the US. HPV genomic integration events are a likely critical step in progression to cancer and occur as a consequence of HPV oncogene-induced chromosomal instability. The majority of viral integration events in the human genome appear to occur within or near genic regions. Identified HPV integration events in HNSCC were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and inter-/intrachromosomal rearrangements. Materials and methods We analyzed 84 HPV-positive HNSCC samples collected at the University of Michigan (18 tumors) and from The Cancer Genome Atlas (66 tumors). We used VirusSeq software for detection of integration events. Integrations were evidenced by host-virus fusion transcripts in RNA-seq, considering only breakpoints supported by at least four discordant read pairs and at least one junction spanning read. Results We identified 50 integration-positive tumors, which consisted of 41 with HPV16, one tumor with HPV18, 5 tumors with HPV33 and 3 with HPV35. We found an overall 271 breakpoints of integration within or near 83 human genes. Fusion virus-host transcripts with breakpoints within E6 and E7 HPV oncogenes were more common (59.3%) compared with breakpoints into other viral genes: E1 and E2 (19%), E4 and E5 (16.8%), L1 and L2 (4.7%). The detected viral integration events were widespread across the human genome with a few hotspots of recurrent integrations in genic regions at 1p36, 9p24, and 13p22. After accounting for regions covered by the data, we did not find an association of integration sites with aphidicolin-induced common fragile sites (CFSs), as observed for HPV integration sites in cervical cancer. Analysis of the protein interaction network constructed from the 83 host genes harboring viral integrants showed that 56 of them were linked into a highly connected sub-network with direct interactions. ETS2, TP63, FOXA1, CTGF and KLF5 were hubs in this network. The genes were statistically enriched for cancer genes known as important in “Head and Neck Neoplasms”(p = 1.74E-11). Conclusion HPV integration events in HNSCC are largely different from those common to cervical cancers. While TP63 appears to be a recurrent target in both cervical cancer and HNSCC, HNSCCs have a surprisingly high percent of integration events in known HNSCC-related genes. Overall, our results suggest that there is strong selective pressure for integration events that occur in HNSCC relevant genes. Citation Format: Lada A. Koneva, Yanxiao Zhang, Pelle Hall, Shama Virani, Alisha Virani, Thomas E. Carey, Laura S. Rozek, Maureen A. Sartor. Identification and characterization of HPV-host fusion transcripts in HNSCCs. [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 1515.

Introduction: Human Papillomavirus (HPV) infection is recognized as a major cause for an increasing percent of oropharyngeal squamous cell carcinomas (OPSCC). Patients with HPV-induced OPSCC have a better prognosis than those who are HPV-negative, which implies distinct molecular mechanisms. However, predicting prognosis with the HPV-positive population remains elusive. In this study, we aimed to characterize molecular aspects of HPV expression, HPV integration, and host gene expression to later correlate with survival and recurrence as those data become available for our study population. Materials and methods: Thirty-six primary head and neck SCC (HNSCC) tumor samples were collected between 2011-2013 at University of Michigan hospital, of which 18 were determined to be HPV+ (type16, 18 or 35). Paired-end RNA-Seq libraries were generated using Illumina HiSeq for all samples. The sequences were aligned to both the human and HPV genome. Gene expression levels were quantified for both human and HPV genes using TopHat2 and HTSeq, and HPV integration events were determined using Virus-seq. Results: Hierarchical clustering of the gene expression levels showed three distinct clusters among the samples. The HPV- samples were first separated from the HPV+ samples, and then there were two robust subgroups within the HPV+ samples. Subsequent analysis revealed one subgroup had significantly higher HPV expression levels than the other (p-value: 0.03; Wilcoxon test). The subgroups were also highly correlated with HPV-integration status (8 of the 16 tumors had at least one identified HPV integration event). Differentially expressed genes between HPV-integration positive and negative samples were found to be enriched for interesting pathways, including “keratinocyte differentiation”, “cell adhesion” and “growth factor binding”, suggesting possible differences in carcinogenesis We also examined the HPV integration sites and found the majority of them were transcription factors. These also include several cancer-related genes, such as “BIRC3”, “CD274” and “PBX1”. One gene, KLF12, contained an integration event in one of our tumors and in one of the 36 HPV+ tumors from The Cancer Genome Atlas (TCGA). Network analysis of the genes harboring an integration event in our population or the TCGA HNSCC population (38 total genes) revealed interactions around p63 and ETS. Conclusion: Our data is an important addition to TCGA HNSCC cohort data, because as opposed to TCGA, we did not enforce selection bias towards large tumor size. With our data, we identified two distinct HPV+ subgroups characterized by RNA-Seq expression levels and strongly correlated with HPV viral expression levels and integration status. A significant portion of the HPV+ tumors (50%) had integrated HPV genome, implying important oncogenic potentials for integration events. Citation Format: Yanxiao Zhang, Lada A. Koneva, Shama Virani, Alisha Virani, Katie M. Rentschler, Thomas E. Carey, Laura S. Rozek, Maureen A. Sartor, the University of Michigan Head and Neck SPORE. Gene expression analysis of human papillomavirus positive head and neck cancer primary tumor samples reveals two distinct subgroups. [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 2184. doi:10.1158/1538-7445.AM2015-2184

Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world. HPV-positive HNSCC is considered a distinct molecular entity with a better prognosis than HPV-negative. To better understand the mechanisms underlying the clinical and molecular behavior between HPV-positive and HPV-negative HNSCC we applied bioinformatics approaches to transcriptomic data to identify signature patterns associated with 25 different cell types, and characterize cell type heterogeneity by HPV-status. Materials and methods We analyzed 18 HPV (+) and 18 HPV (-) samples collected at University of Michigan hospital from 36 individuals with HNSCC; all were determined to be >70% cellularity by a pathologist. Gene expression was evaluated using Illumina HiSeq RNA-seq. Differentially expressed genes were identified using TopHat, HTSeq, and then edgeR, resulting in 559 and 873 up in HPV(+) or HPV(-), respectively, with FDR < 0.05 and fold change > 3. Cell type-specific expression patterns were identified using > 700 microarray samples from 25 relevant cell types. Using this database and the list of genes differentially expressed by HPV status we identified which cell type(s) contributed the strongest expression pattern by calculating cell type-specific enrichment scores as a weighted Kolmogorov-Smirnov-like statistics against the top ranked 250 genes. Results Significantly higher estimates of cell type composition in HPV(+) samples were observed for T lymphocytes (CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, CD34+ T-cells, γδ T-cells, regulatory T-cells), NK-cells, B cells, and monocytes (p-value < 0.0004). Conversely, endothelial cells, epidermis, dermis, keratinocyte, and fibroblast all had significantly higher estimates of cell type proportion in HPV(-) samples (p-value < 0.0004). For validation, we compared these results to results calculated in an identical way using TCGA HNSC RNA-seq data (39 HPV(+) vs 277 HPV(-) samples) and found that the largest differences in cell types higher in HPV(+) were shown also in T lymphocytes, NK-cells & B cells, and that dermis, fibroblast, epidermis & keratinocyte cell types were again estimated to be higher in HPV(-) samples (p-value < 0.0004). Conclusion Differential expression in HNSCC by HPV status can partly be explained by differences in the tumor cellular composition, and this in turn will help to focus on differential expression within the tumor cells themselves. Our results predict that T lymphocytes and other cell types responsible for the adaptive immune response contribute a larger portion of the cellular composition in HPV(+) tumors compared to HPV(-). Conversely, keratinocytes and fibroblasts are predicted to make up a larger portion of cells in HPV(-) tumors. Surprisingly, the stem cell expression signature was not significantly different by HPV-status, even though HPV(+) tumors tend to be less differentiated than HPV(-) tumors. Citation Format: Lada A. Koneva, Yanxiao Zhang, William R. Swindell, Shama Virani, Alisha Virani, Thomas E. Carey, Laura S. Rozek, Maureen A. Sartor. Immune, keratinocyte, and fibroblast expression signatures distinguish HNSCC samples by HPV status. [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 2180. doi:10.1158/1538-7445.AM2015-2180