Micronutrients and prevention of cervical pre-cancer in HPV vaccinated women: a cross-sectional study.

Objectives: Prophylactic vaccines against high-risk human papillomaviruses (HR-HPVs) hold promise to prevent the development of higher grade cervical intraepithelial neoplasia (CIN 2+) and cervical cancer (CC) that develop due to HR-HPV genotypes that are included, in HPV vaccines, but women will continue to develop CIN 2+ and CC due to HR-HPV genotypes that are not included in the quadrivalent HPV vaccine (qHPV) and 9-valent HPV vaccine (9VHPV). Thus, the current vaccines are likely to decrease but not entirely prevent the development of CIN 2+ or CC. The purpose of the study was to determine the prevalence and determinants of CIN 2+ that develop due to HR-HPVs not included in vaccines. Methods: Study population consisted of 1476 women tested for 37 HPVs and known to be negative for qHPVs (6/11/16/18, group A, n = 811) or 9VHPVs (6/11/16/18/31/33/45/52/58, group B, n = 331), but positive for other HR-HPVs. Regression models were used to determine the association between plasma concentrations of micronutrients, socio-demographic, lifestyle factors and risk of CIN 2+ due to HR-HPVs that are not included in vaccines. Results: The prevalence of infections with HPV 31, 33, 35 and 58 that contributed to CIN 2+ differed by race. In group A, African American (AA) women and current smokers were more likely to have CIN 2 (OR = 1.76, P = 0.032 and 1.79, P = 0.016, respectively) while in both groups of A and B, those with higher vitamin B12 were less likely to have similar lesions (OR = 0.62, P = 0.036 and 0.45, P = 0.035, respectively). Conclusions: We identified vitamin B12 status and smoking as independent modifiable factors and ethnicity as a factor that needs attention to reduce the risk of developing CIN 2+ in the post vaccination era. Continuation of tailored screening programs combined with non-vaccine-based approaches are needed to manage the residual risk of developing HPV-related CIN 2+ and CC in vaccinated women.

Potential Effects of Age-Based Changes in Screening Guidelines on the Identification of Women at Risk for Developing Cervical Cancer.

Current American Cancer Society guidelines estimated that screening starting at the age of 25 years with Pap and/or human papillomavirus (HPV) testing is sufficient to prevent cervical cancer. The effect of having HPV infections without Pap-based care until age 25 on the prevalence of higher grades of cervical intraepithelial neoplasia (≥CIN 2) and their determinants are largely unknown. The objectives of the study were to document the potential effects of age-based changes in screening guidelines on the identification of ≥CIN 2 and their determinants. The study included 1,584 women diagnosed with abnormal Pap and tested for HPVs and histologic diagnoses of cervical lesions. The association between demographic/lifestyle factors and HPV status and risk of being diagnosed with ≥CIN 2 among younger (21-<25 years) or older (≥25 year) women was tested using unconditional multiple logistic regression models. We observed that younger women who are not screened have a similar or higher risk of developing specific high-risk HPV genotype-associated ≥CIN 2 lesions compared with older women who are screened according to the current guidelines. In addition, younger women who reported live births, smoking, contraceptive use, and a higher number of sexual partners were significantly at higher risk of being diagnosed with ≥CIN 2. Targeted screening of younger women at risk for developing ≥CIN 2 will address the concern of overtreatment while providing the recommended care to those who require such care to prevent the development of cervical cancer. PREVENTION RELEVANCE: This study documents the concerns of the age-based changes in screening guidelines on the identification of higher grades of cervical intraepithelial neoplasia and their determinants in women diagnosed with abnormal Pap smear and emphasize the need for targeted screening of younger women to prevent cervical cancer.

Potential effects of age-based changes in screening guidelines on the identification of women at risk for developing cervical cancer.

Current American Cancer Society (ACS) guidelines estimated that screening starting at the age of 25 years with Pap and/or human papillomavirus (HPV) testing is sufficient to prevent cervical cancer (CC). The effect of having HPV infections without Pap-based care until age 25 on the prevalence of higher grades of cervical intraepithelial neoplasia (≥ CIN 2) and their determinants are largely unknown. The objectives of the study were to document the potential effects of age-based changes in screening guidelines on the identification of ≥ CIN 2 and their determinants. The study included 1584 women diagnosed with abnormal Pap and tested for HPVs and histological diagnoses of cervical lesions. The association between demographic/lifestyle factors and HPV status and risk of being diagnosed with ≥ CIN 2 among younger (21-<25 years) or older (≥ 25 year) women was tested using unconditional multiple logistic regression models. We observed that younger women who are not screened have a similar or higher risk of developing specific high risk (HR)-HPV genotype-associated ≥ CIN 2 lesions compared to older women who are screened according to the current guidelines. In addition, younger women who reported live births, smoking, contraceptive use and a higher number of sexual partners were significantly at higher risk of being diagnosed with ≥ CIN 2. Targeted screening of younger women at risk for developing ≥ CIN 2 will address the concern of overtreatment while providing the recommended care to those who require such care to prevent the development of CC.

HPV E1 qPCR, a Low-Cost Alternative Assay to Roche Diagnostic Linear Array is Effective in Identifying Women at Risk for Developing Cervical Cancer.

OBJECTIVE: Since a quantitative polymerase chain reaction (qPCR) assay targeting the E1 region of HPV genome is cost-effective/simple to perform, we evaluated the agreement between the Roche Diagnostics Linear Array (RDLA) genotyping test and qPCR-based E1 assay to detect HR-HPV genotypes that are included or not included in HPV vaccines and compared their accuracy to detect CIN 2+. METHODS: Study population included 257 African American (AA) and 266 Caucasian American (CA) diagnosed with intraepithelial neoplasia (CIN) grades ≤CIN 1 or ≥CIN 2 (CIN 2+) and tested for HPV by the RDLA and E1 assay. The concordance was determined using Gwet's AC1. The calculated positive predictive value (PPV) and negative predictive value (NPV) of the two assays were used to determine their suitability to detect CIN lesions. RESULTS: Overall, the E1 assay showed substantial agreement with the RDLA assay to detect any HR-HPV genotype and the agreement was higher in women diagnosed with CIN 2+ than ≤CIN 1. The concordance was largely higher in Cas than in Aas. The NPV and PPV values to detect CIN lesions were similar between the two assays. CONCLUSION: Utilization of the HPV E1 assay as a tool for CC screening could be a cost-effective approach that applies to both vaccinated and unvaccinated populations.

The dataset for the assessment of the inflammatory potential of the overall diet consumed by women of childbearing age.

The data presented in this article is related to the research article titled "Racial differences in dietary choices and their relationship to inflammatory potential in childbearing age women at risk for exposure to COVID-19". This data article provides details of dietary intake data from 509 women (African American, n = 327 and Caucasian American, n = 182) who are residents of Birmingham, AL. All women were characterized for demographic and lifestyle factors and indicators of excess body weight (EBW) that are likely to influence overall dietary habits. Dietary intake data was collected by administering the modified version of the NCI validated Block food frequency questionnaire (98.2-isoflav version) that includes 110 food items of the original version (98.2 version) and an additional 24 phytochemical rich food items. The data article describes our approach to derive the dietary inflammatory score using a validated empirical dietary inflammatory index based on the frequency and the amount of consumption of each food item with minor modifications. This data will allow researchers to understand the composition of a Southern-style diet consumed by women of childbearing age and its relationship to inflammatory potential, EBW, dietary guidelines, dietary reference intakes or diet quality indices.

Human papillomavirus sequencing reveals its usefulness for the management of HIV-infected women at risk for developing cervical cancer.

AIM: Next-generation sequencing (NGS) is able to describe the composition of human papillomaviruses (HPVs) as percent (%) reads rather than positive/negative results. Therefore, we used this unique approach to assess the prevalence of cervical HPVs of HIV infected (HIV+) in order to understand the determinants of being infected with higher % reads of high risk (HR)-HPVs and cervical abnormalities of atypical squamous cells of unknown significance or higher (ASCUS+). METHODS: Study included 66 women characterized for relevant risk factors/cytology. Receiver-operating curve curve was used to derive the optimal % read cut point to identify ASCUS+ in relation to any HR-HPV genotype or other specific HPV genotypes. The determinants of ASCUS+ and HR-HPVs were tested using logistic regression. RESULTS: Women with >20% reads of any HR-HPV or >12% any HR-HPV other than HPV 16/18 were 5.7 and 12.6 times more likely to be diagnosed with ASCUS+, respectively. Lower CD4 count was a significant determinant of >20% reads of HR-HPV (odds ratio [OR] = 4.1) or >12% any HR-HPV other than HPV 16/18 (OR = 4.5). CONCLUSION: We envision that the NGS-based HPV detection will be more accurate for screening and management of HIV+ at risk for developing cervical cancer (CC). We raise concerns regarding the limitations of 16/18-based HPV testing for triage and the efficacy of current HPV vaccines for preventing CC in HIV+.

The accuracy of HPV genotyping in isolation and in combination with CD4 and HIV viral load for the identification of HIV-infected women at risk for developing cervical cancer.

BACKGROUND: Human papillomavirus (HPV) genotype testing has limited utility to identify human immunodeficiency virus-infected (HIV+) women's risk for developing cervical cancer (CC) due to high positivity rate of high-risk (HR) HPVs. We investigated the accuracy of HPV testing in isolation/in combination with CD4 and HIV viral load (VL) to identify HIV+ women at risk for developing CC. METHODS: Study consisted of 344 HIV+ women on combination antiretroviral therapy (cART), tested for cervical cytology/HPV using the Cobas test and had data on absolute CD4 count and VL measurements. We calculated the positive predictive value (PPV) and negative predictive value (NPV) of HPV testing, pre-, post-cART, and current CD4 and VL in isolation and in combinations to identify those with or free of higher than atypical squamous cells of unknown significance (ASCUS+) or low-grade intraepithelial lesions (LSIL+). RESULTS: HPV test in combination with pre-/post-cART or current CD4 counts and VL had higher PPVs compared to HPV test alone for identifying ASCUS+ or LSIL+. PPV of HPV-CD4 combinations yielded higher PPVs compared to HPV-VL combinations. The NPVs with pre-, post-cART, or current CD4 count and VL in isolation or in combinations were comparable to that of HPV test alone. CONCLUSIONS: Our results provide a more accurate tool for managing HIV+ women by combining Cobas HPV with CD4 and VL, especially those who had an undesirable pre-cART CD4 and VL status. Our results also indicate the usefulness of CD4 and VL measurements to identify those at lower risk in the absence of HPV testing.

A rigorous exploration of anal HPV genotypes using a next-generation sequencing (NGS) approach in HIV-infected men who have sex with men at risk for developing anal cancer.

BACKGROUND: There are no HPV-based measures for managing anal cancer (AC) in HIV-infected (HIV+) men who have sex with men (MSM) because of the high positivity of high-risk (HR)-HPVs. As next-generation sequencing (NGS) is able to describe the composition of HPVs as percent (%) reads rather than positive vs negative results, we used NGS approach to detect HPVs in anal samples of HIV+ MSM to test its ability to differentiate those who are diagnosed with atypical squamous cells of unknown significance or greater (ASCUS+) from those who are free of such lesions and to understand the burden of HPV infections in relation to HPV vaccines. METHODS: Study included 81 HIV+ MSM characterized for demographics, patient-reported outcome measures, HIV related laboratory measures and anal cytology. We summarized NGS HPV data using % read cut points (>0%->30%) and tested the relationship between % reads of HR-HPVs and risk of ASCUS+ using logistic regression. RESULTS: Forty-six HPVs were detected at the >0% read cut point. The prevalence of any HR-HPVs varied from 100% to 40% with >0% to >30% reads while ≥99% were infected with HR-HPVs included or not included in the 9 valent HPV vaccine at the >0% read cut point. MSM with >30% HR-HPV reads were 4.5 times more likely to be diagnosed with ASCUS+ compared to ≤30% reads (P = .033). CONCLUSION: NGS-based approach is more accurate than PCR-based HPV testing for identifying HIV+ MSM at risk for developing AC. We raise the concern regarding the efficacy of current HPV vaccines for preventing AC in this high-risk population.

A higher degree of expression of DNA methyl transferase 1 in cervical cancer is associated with poor survival outcome.

BACKGROUND: Even though novel therapies based on aberrant DNA methylation could be of particular importance for the treatment of cervical cancer (CC) because the oncoproteins E6/E7 of high-risk human papillomaviruses, the causative agents for developing CC, have the capacity to bind and upregulate DNA methyltransferases (DNMTs), to our knowledge, no previous studies have evaluated the expression of this enzyme in CC in relation to survival outcomes. The purpose of the study was to evaluate the expression of DNMT1 in CC and its association with survival outcomes. METHODS: The study population consisted of 76 women treated for primary CC and followed up by the University of Alabama at Birmingham (UAB) cancer registry. The expression of DNMT1 was examined using immunohistochemistry, and the degree of expression of DNMT1 was expressed as a percentage of cells positive for DNMT1 and its intensity. Cox proportional hazards model was used to assess the relationship between the degree of expression of DNMT1 and overall survival after adjusting for relevant covariates. RESULTS: The expression of DNMT1 was significantly higher in CC cells compared to that in the normal cervical epithelium. A higher percentage of cells positive for DNMT1 and a higher intensity score for DNMT1 were significantly associated with poor survival outcome (hazard ratio [HR] =4.3, P=0.03 and HR =4.9, P=0.02, respectively). CONCLUSION: Our findings suggested that the degree of expression of DNMT1 could be considered as a target in the epigenetic treatment of CC. Replication of our results in other study populations with CC could create the opportunity of using DNMT inhibitors to treat CC.

Expression of p16INK4A in cervical precancerous lesions that is unlikely to be preventable by human papillomavirus vaccines.

BACKGROUND: Whether higher grade cervical intraepithelial neoplasia (CIN grade 2 or greater [CIN ≥ 2]) that develops because of human papillomavirus (HPV) genotypes not included in vaccines may progress to cervical cancer is largely unknown. The objectives of this study were to document expression of the cyclin-dependent kinase inhibitor 2A (p16) tumor-suppressor protein p16INK4A as a biomarker of cervical carcinogenesis or of malignant potential and to evaluate whether its expression differs between lesions associated with vaccine and nonvaccine high-risk (HR) human papillomavirus (HPV) genotypes. METHODS: The study population consisted of 371 women who had not received HPV vaccines. Women were categorized into vaccine and nonvaccine HR-HPV genotypes and lesions associated with those types. Logistic regression analyses were used to determine the association between positive expression p16INK4A and the risk of being diagnosed with CIN 2 or CIN 3. Differences in the proportion of CIN ≥2 lesions that were positive for p16INK4A expression by vaccine-related or nonvaccine-related HR-HPV genotype were determined using the Pearson chi-square test. RESULTS: Specimens that were positive for p16INK4A expression were 5.3 and 16.6 times more likely to be diagnosed as CIN 2 and CIN 3 lesions, respectively, compared to CIN 1 lesions. CIN ≥ 2 lesions that were negative for the bivalent and 9-valent HR-HPV genotypes had similar rates of positive p16INK4A expression compared with lesions that were positive for those HR-HPV genotypes. CONCLUSIONS: Lesions that may develop because of HR-HPV genotypes not included in HPV vaccines are likely to have similar malignant potential, suggesting that well developed screening programs combined with nonvaccine-based approaches may be needed to manage the residual risk of developing cervical cancer in the post-HPV vaccination era. Cancer 2016;122:3615-23. © 2016 American Cancer Society.

Accuracy of urinary human papillomavirus testing for the presence of cervical human papillomaviruses and higher grades of cervical intraepithelial neoplasia.

BACKGROUND: Although urine-based testing for human papillomavirus (HPV) is being explored as a practical approach for cervical cancer screening, whether the results differ by age, race, or indicators of excess body weight or in populations exposed to HPV vaccines has not been documented by previous studies. The purpose of this study was to determine the accuracy of urinary HPV testing for the presence of cervical HPVs and high-grade cervical intraepithelial lesions (grade 2 and 3 cervical intraepithelial neoplasia [CIN]) by the aforementioned population characteristics. METHODS: The study population consisted of 502 women diagnosed with different grades of CIN. HPV testing was performed with paired urine and cervical cell DNA with the Roche Diagnostics Linear Array test. Agreement coefficient 1 and probabilities were calculated to determine the accuracy of urinary HPV testing for the presence of cervical HPVs and CIN lesions. RESULTS: Substantial to almost perfect agreement (0.66-0.83) was observed in the detection of any HPV genotype in urine specimens versus cervical specimens, regardless of the population characteristics. Although the positive predictive value for the detection of CIN lesions was relatively low, the negative predictive value for CIN-3 was high (≥90%) among women positive for any of the urinary or cervical high-risk human papillomavirus (HR-HPV) genotypes or HPV genotypes not included in currently available HPV vaccines. CONCLUSIONS: The results demonstrate that urinary HPV testing provides highly satisfactory results for excluding the possibility of any cervical HPV infections, including HPV types not included in vaccines and CIN lesions associated with any HR-HPV, regardless of a woman's age, race, or excess body weight. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2836-2844. © 2016 American Cancer Society.

An examination of racial differences in 5-year survival of cervical cancer among African American and white American women in the southeastern US from 1985 to 2010.

Disparities in Cervical Cancer (CC) mortality outcomes between African American (AA) and White women have been studied for decades. However, conclusions about the effect of race on CC survival differ across studies. This study assessed differences in CC survival between AA and White women diagnosed between 1985 and 2010 and treated at two major hospitals in the southeastern US. The study sample included 925 AA and 1192 White women diagnosed with cervical adenocarcinoma, adenosquamous cell carcinoma, or squamous cell carcinoma. Propensity score adjustment and matching were employed to compare 5-year survival between the two racial groups. Crude comparisons suggested relevant racial differences in survival. However, the racial differences became of small magnitude after propensity-score adjustment and in matched analyses. Nonlinear models identified age at diagnosis, cancer stage, mode of treatment, and histological subtype as the most salient characteristics predicting 5-year survival of CC, yet these characteristics were also associated with race. Crude racial differences in survival might be partly explained by underlying differences in the characteristics of racial groups, such as age at diagnosis, histological subtype, cancer stage, and the mode of treatment. The study results highlight the need to improve access to early screening and treatment opportunities for AA women to improve posttreatment survival from CC.

Homocysteinemia is Associated with a Lower Degree of PBMC LINE-1 Methylation and a Higher Risk of CIN 2C in the U.S. Post-Folic Acid Fortification Era.

The primary aim of the study was to determine whether plasma concentrations of homocysteine (Hcy), a functional indicator of methyl donor nutrients, are associated with altered risk of higher grades of cervical intraepithelial neoplasia (CIN 2+) and the degree of methylation in long interspersed nucleotide elements (LINE-1s) of peripheral blood mononuclear cells, a potential biomarker of CIN 2+ in a population of women exposed to the United States folic acid fortification program. The secondary aim was to assess the determinants of plasma Hcy in the same population. The study included 457 women diagnosed with either CIN 2+ (cases, n = 132) or ≤ CIN 1 (non-cases, n = 325). Unconditional logistic regression models were used to test the associations after adjusting for relevant risk factors of cervical cancer. Women with higher Hcy concentrations were at a greater risk of being diagnosed with CIN 2+ [odds ratio (OR) = 1.86, P = 0.005]. Higher plasma folate concentrations were a significant determinant of lower Hcy (OR = 0.40, P = 0.0002). Women with higher Hcy concentrations were more likely to have a lower degree of LINE-1 methylation (OR = 2.30, P = 0.0007). These results suggested that further improvement in folate status in this population may be beneficial for lowering Hcy and improving the degree of LINE-1 methylation.

A lower degree of PBMC L1 methylation in women with lower folate status may explain the MTHFR C677T polymorphism associated higher risk of CIN in the US post folic acid fortification era.

BACKGROUND: Studies in populations unexposed to folic acid (FA) fortification have demonstrated that MTHFR C677T polymorphism is associated with increased risk of higher grades of cervical intraepithelial neoplasia (CIN 2+). However, it is unknown whether exposure to higher folate as a result of the FA fortification program has altered the association between MTHFR C677T and risk of CIN, or the mechanisms involved with such alterations. The current study investigated the following in a FA fortified population: 1) The association between MTHFR C677T polymorphism and risk of CIN 2+; 2) The modifying effects of plasma folate concentrations on this association; and 3) The modifying effects of plasma folate on the association between the polymorphism and degree of methylation of long interspersed nucleotide elements (L1s), in peripheral blood mononuclear cell (PBMC) DNA, a documented biomarker of CIN risk. METHODS: The study included 457 US women diagnosed with either CIN 2+ (cases) or ≤ CIN 1 (non-cases). Unconditional logistic regression models were used to test the associations after adjusting for relevant risk factors for CIN. RESULTS: The 677CT/TT MTHFR genotypes were not associated with the risk of CIN 2+. Women with CT/TT genotype with lower folate, however, were more likely to be diagnosed with CIN 2+ compared to women with CT/TT genotype with higher folate (OR = 2.41, P = 0.030). Women with CT/TT genotype with lower folate were less likely to have a higher degree of PBMC L1 methylation compared to women with CT/TT genotype with higher folate (OR = 0.28, P = 0.017). CONCLUSIONS: This study provides the first evidence that the MTHFR 677CT/TT genotype-associated lower degree of PBMC L1 methylation increases the risk of CIN 2+ in women in the US post-FA fortification era. Thus, even in the post-FA fortification era, not all women have adequate folate status to overcome MTHFR 677CT/TT genotype-associated lower degree of L1 methylation.

Folate and vitamin B12 may play a critical role in lowering the HPV 16 methylation-associated risk of developing higher grades of CIN.

We previously reported that a higher degree of methylation of CpG sites in the promoter (positions 31, 37, 43, 52, and 58) and enhancer site 7862 of human papillomavirus (HPV) 16 was associated with a lower likelihood of being diagnosed with HPV 16-associated CIN 2+. The purpose of this study was to replicate our previous findings and, in addition, to evaluate the influence of plasma concentrations of folate and vitamin B12 on the degree of HPV 16 methylation (HPV 16m). The study included 315 HPV 16-positive women diagnosed with either CIN 2+ or ≤CIN 1. Pyrosequencing technology was used to quantify the degree of HPV 16m. We reproduced the previously reported inverse association between HPV 16m and risk of being diagnosed with CIN 2+. In addition, we observed that women with higher plasma folate and HPV 16m or those with higher plasma vitamin B12 and HPV 16m were 75% (P < 0.01) and 60% (P = 0.02) less likely to be diagnosed with CIN 2+, respectively. With a tertile increase in the plasma folate or vitamin B12, there was a 50% (P = 0.03) and 40% (P = 0.07) increase in the odds of having a higher degree of HPV 16m, respectively. This study provides initial evidence that methyl donor micronutrients, folate and vitamin B12, may play an important role in maintaining a desirably high degree of methylation at specific CpG sites in the HPV E6 promoter and enhancer that are associated with the likelihood of being diagnosed with CIN 2+.

Determinants of neural tube defect (NTD)-protective circulating concentrations of folate in women of child-bearing age in the US post-folic acid fortification era.

We evaluated folate status of child-bearing age women diagnosed with abnormal pap smear in the US post-folic acid (FA) fortification era and assessed the determinants of NTD-protective and supra-physiologic (SP) concentrations of folate. The distribution of 843 women according to NTD-protective concentrations of RBC folate, plasma folate and SP concentrations of plasma folate were tested in relation to demographic and life-style factors. Logistic regression models specified NTD-protective concentrations of RBC and plasma folate or SP concentrations of plasma folate as dependent variables and demographic and life-style factors as independent predictors of interest. More than 82% reached NTD-protective concentrations of RBC and plasma folate and ~30% reached SP concentrations of plasma folate. FA supplement use was associated with having SP concentrations of plasma folate rather than NTD-protective concentrations of folate. African American (AA) women and smokers were significantly less likely to achieve NTD-protective concentrations of RBC and plasma folate. A large majority of women reached NTD-protective concentrations of folate with the current level of FA fortification without using supplementary FA. Therefore, the remaining disparities in AA women and in smokers should be addressed by targeted individual improvements in folate intake.

A dietary pattern associated with LINE-1 methylation alters the risk of developing cervical intraepithelial neoplasia.

There is a paucity of research examining the relationships between dietary patterns and risk of developing precancerous lesions as well as biomarkers associated with such dietary patterns. The purpose of the current study was to identify dietary patterns that are associated with higher grades of cervical intraepithelial neoplasia (CIN 2+) and to determine whether these dietary patterns are associated with the degree of DNA methylation in the long interspersed nucleotide elements (L1s) of peripheral blood mononuclear cells (PBMCs), a biomarker associated with risk of developing CIN 2+. Study population consisted of 319 child-bearing age women. Dietary patterns were derived by factor analysis. The degree of PBMC L1 methylation was assessed by pyrosequencing. Logistic regression models were used to evaluate the associations between dietary patterns and CIN 2+. Similar models were used to evaluate the associations between dietary patterns and degree of PBMC L1 methylation in women free of CIN 2+. Women with the unhealthiest dietary pattern were 3.5 times more likely to be diagnosed with CIN 2+ than women with the healthiest dietary pattern [OR = 3.5; 95% confidence interval (CI), 1.2-10.1; P = 0.02]. Women at risk for developing CIN 2+ with the healthiest dietary pattern were 3.3 times more likely to have higher PBMC L1 methylation than women with the unhealthiest dietary pattern (OR = 3.3; 95% CI, 1.0-10.6; P = 0.04). Our findings suggest that human papilloma virus associated risk of developing CIN 2+ may be reduced by improving dietary patterns. The degree of PBMC L1 methylation may serve as a biomarker for monitoring the effectiveness of dietary modifications needed for reducing the risk of CIN 2+.

A higher degree of LINE-1 methylation in peripheral blood mononuclear cells, a one-carbon nutrient related epigenetic alteration, is associated with a lower risk of developing cervical intraepithelial neoplasia.

OBJECTIVE: The objective of the study was to evaluate LINE-1 methylation as an intermediate biomarker for the effect of folate and vitamin B12 on the occurrence of higher grades of cervical intraepithelial neoplasia (CIN ≥ 2). METHODS: This study included 376 women who tested positive for high-risk human papillomaviruses and were diagnosed with CIN ≥ 2 (cases) or CIN ≤ 1 (non-cases). CIN ≥ 2 (yes/no) was the dependent variable in logistic regression models that specified the degree of LINE-1 methylation of peripheral blood mononuclear cells (PBMCs) and of exfoliated cervical cells (CCs) as the independent predictors of primary interest. In analyses restricted to non-cases, PBMC LINE-1 methylation (≥ 70% versus <70%) and CC LINE-1 methylation (≥ 54% versus <54%) were the dependent variables in logistic regression models that specified the circulating concentrations of folate and vitamin B12 as the primary independent predictors. RESULTS: Women in the highest tertile of PBMC LINE-1 methylation had 56% lower odds of being diagnosed with CIN ≥ 2 (odds ratio 0.44, 95% confidence interval 0.24-0.83, P = 0.011), whereas there was no significant association between degree of CC LINE-1 methylation and CIN ≥ 2 (odds ratio 0.86, 95% confidence interval 0.51-1.46, P = 0.578). Among non-cases, women with supraphysiologic concentrations of folate (>19.8 ng/mL) and sufficient concentrations of plasma vitamin B12 (≥ 200.6 ng/mL) were significantly more likely to have highly methylated PBMCs compared with women with lower folate and lower vitamin B12 (odds ratio 3.92, 95% confidence interval 1.06-14.52, P = 0.041). None of the variables including folate and vitamin B12 were significantly associated with CC LINE-1 methylation. CONCLUSION: These results suggest that a higher degree of LINE-1 methylation in PBMCs, a one-carbon nutrient-related epigenetic alteration, is associated with a lower risk of developing CIN.

A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia.

BACKGROUND: Although HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with precancerous lesions of the cervix. Therefore, molecular changes in HPV 16, rather than infections per se, may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+). METHODS: The study included 75 HPV 16-positive women diagnosed with CIN 2+ or ≤CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer, and circulating concentrations of "cancer-protective" micronutrients. RESULTS: The odds of being diagnosed with CIN 2+ were 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites was ≥9.5% (OR = 0.21; 95% CI, 0.06-0.79; P = .02). CONCLUSIONS: Results suggested that CpG methylation is independently involved in the biology of HPV 16 as well as in the development of higher grades of CIN.

Mandatory fortification with folic acid in the United States appears to have adverse effects on histone methylation in women with pre-cancer but not in women free of pre-cancer.

OBJECTIVE: To evaluate whether mandatory fortification of grain products with folic acid in the US is associated with changes in histone methylation in cells involved in cervical carcinogenesis. METHODS: Cervical specimens obtained before (1990 to 1992) and after mandatory folic acid fortification (2000 to 2002) were used to examine the degree of histone methylation (H3 Lys-9) by immunohistochemistry. 91 women (51 before and 40 after fortification) were diagnosed with cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS) and sections utilized in the study also contained normal, reactive or metaplastic cervical epithelium, CIN 1 or CIN 2. 64 women (34 before and 30 after fortification) were free of CIN and these sections contained only normal or reactive cervical epithelium. Immunohistochemical staining for H3 Lys-9, its assessment in different cell or lesion types and data entry were blinded for fortification status. For each cell type or lesion category we used PROC MIXED in SAS with the specimen identifier as a random effect and the robust variance estimator to estimate age- and race-adjusted intensity score for H3 Lys-9 in the pre- and post-fortification periods. RESULTS: Degree of H3 Lys-9 methylation was significantly higher (P < 0.0001) in ≥CIN 2 lesions (CIN 2, CIN 3 and CIS) than in ≤CIN 1 lesions (CIN 1, normal, reactive and metaplastic), in both pre- and post-fortification CIN 3/CIS specimens. Age- and race-adjusted mean H3 Lys-9 score was significantly higher in all cell or lesion types in CIN 3/CIS specimens obtained in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). In contrast, in specimens obtained from women free of CIN, Lys-9 methylation in normal/reactive cervical epithelium was significantly lower in post-fortification specimens than in pre-fortification specimens (P = 0.03). CONCLUSIONS: Higher levels of Lys-9 methylation in ≥CIN 2 compared to ≤CIN 1 lesions suggest that higher Lys-9 methylation is associated with progression of lower grade CIN to higher grade CIN. Higher Lys-9 methylation in cervical tissues of women diagnosed with CIN 3 in the post-fortification period than in pre-fortification period suggest that fortification may adversely affect histone methylation in already initiated cells. Lower Lys-9 methylation in normal/reactive cervical cells of women free of CIN in the post-fortification period than pre-fortification on the other hand suggests that fortification is likely to protect against initiation of carcinogenic process in the cervix. These results suggest that mandatory fortification with folic acid in the US seems to have different effects on cancer depending on the stage of carcinogenesis. Because this is the first study to report folic acid fortification-associated differences in histone methylation and because of the limitations inherent to the approach we have taken to demonstrate these differences, validation of the results in other study populations or with other techniques for assessing histone methylation is necessary.