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1.  Human Endogenous Retrovirus Type K Antibodies and mRNA as Serum Biomarkers of Early-Stage Breast Cancer 
A simple and accurate test to detect early-stage breast cancer has not been developed. Previous studies indicate that the level of human endogenous retrovirus type K (group HERV-K(HML-2)) transcription may be increased in human breast tumors. We hypothesized that HERV-K(HML-2) reactivation can serve as a biomarker for early detection of breast cancer. Serum samples were collected from women without cancer (controls) and patients with ductal carcinoma in situ (DCIS) and invasive breast cancer. ELISA assays were employed to detect serum anti-HERV-K(HML-2) antibody titers. RNA was extracted from sera and analyzed by real-time RT-PCR to quantitate the level of HERV-K(HML-2) mRNA. We measured significantly higher serum mRNA and serum antibody titers against HERV-K(HML-2) proteins in women with DCIS and stage I disease than in women without cancer. At optimized cutoffs for the antibody titers, the assay produced an area under the ROC curve (AUC) of 0.89 (95% confidence interval 0.77 to 1.00) for DCIS and of 0.95 (95% confidence interval 0.89 to 1.00) for invasive breast cancer. These AUCs are comparable to those observed for mammograms. We also found that serum HERV-K(HML-2) mRNA tended to be higher in breast cancer patients with a primary tumor who later on developed the metastatic disease than in patients who did not develop cancer metastasis. Our results show that HERV-K(HML-2) antibodies and mRNA are already elevated in the blood at an early stage of breast cancer, and further increase in patients who are at risk of developing a metastatic disease.
doi:10.1002/ijc.28389
PMCID: PMC4191919  PMID: 23873154
HERV-K; endogenous retroviruses; early detection; biomarkers; breast cancer; metastasis
2.  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 
PLoS ONE  2014;9(10):e110093.
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.
doi:10.1371/journal.pone.0110093
PMCID: PMC4193871  PMID: 25302494
3.  The Protective Association of MTHFR Polymorphism on Cervical Intraepithelial Neoplasia is Modified by Riboflavin Status 
Objectives
We previously reported that women polymorphic for the methylenetetrahydrofolate reductase (MTHFR) gene were less likely to have cervical intraepithelial neoplasia (CIN) 2 or 3 (odds ratio [OR] = 0.40, 95 % confidence interval [CI]: 0.21, 0.78, p=0.007). In the current study, we tested whether this protective association is modified by the circulating riboflavin status in the same study population.
Methods
Riboflavin status was assessed by the erythrocyte glutathione reductase (EGR) assay and expressed in terms of an EGR activity coefficient (EGRAC). The status of MTHFR polymorphism, riboflavin, and circulating concentrations of folate, vitamins B-12, A, E, C and total carotene were ascertained in 170 White and 265 African-American women positive for the cervical presence of high-risk human papilloma virus (HR-HPV). Presence/absence of CIN 2 or 3 was determined histologically, and associations with risk factors were examined using multiple logistic regression. Eighty women with CIN 2 or 3 lesions were compared to 355 women without cervical lesions. Based on the median EGRAC of 1.4, women were grouped into low (> 1.4) and high (≤1.4) riboflavin status.
Results
Women with MTHFR polymorphism and low riboflavin status were significantly less likely to have CIN 2 or 3 than the referent group of women without the polymorphism and high riboflavin status (OR = 0.35, 95% CI: 0.13, 0.92, p= 0.034). MTHFR polymorphism was not associated with CIN 2 or 3 in women with high riboflavin status (OR = 0.51, 95% CI: 0.22, 1.19, p = 0.119), nor were any of the associations influenced by folate levels.
Conclusions
A further inactivation of polymorphic MTHFR by low riboflavin status and a resulting shift in the folate metabolic pathway toward DNA synthesis may explain these observations. The practical implications of this complex gene-nutrient-disease interaction will require further investigation.
doi:10.1016/j.nut.2006.12.006
PMCID: PMC2025704  PMID: 17303386
MTHFR; riboflavin; cervical; neoplasia
4.  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 
Nutrition Research and Practice  2013;7(4):315-325.
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.
doi:10.4162/nrp.2013.7.4.315
PMCID: PMC3746167  PMID: 23964320
Folate; neural tube defect; child-bearing age
5.  Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors 
Background
The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer.
Methods
We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb–mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided.
Results
The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti–HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm3; difference = 972.89 mm3, 95% CI = 470.17 to 1475.61 mm3; P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb–treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003).
Conclusion
Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy.
doi:10.1093/jnci/djr540
PMCID: PMC3274512  PMID: 22247020
6.  A Lower Degree of PBMC L1 Methylation Is Associated with Excess Body Weight and Higher HOMA-IR in the Presence of Lower Concentrations of Plasma Folate 
PLoS ONE  2013;8(1):e54544.
Background
Identification of associations between global DNA methylation and excess body weight (EBW) and related diseases and their modifying factors are an unmet research need that may lead to decreasing DNA methylation-associated disease risks in humans. The purpose of the current study was to evaluate the following; 1) Association between the degree of peripheral blood mononuclear cell (PBMC) L1 methylation and folate, and indicators of EBW, 2) Association between the degree of PBMC L1 methylation and folate, and insulin resistance (IR) as indicated by a higher homeostasis model assessment (HOMA-IR).
Methods
The study population consisted of 470 child-bearing age women diagnosed with abnormal pap. The degree of PBMC L1 methylation was assessed by pyrosequencing. Logistic regression models specified indicators of EBW (body mass index–BMI, body fat–BF and waist circumference–WC) or HOMA-IR as dependent variables and the degree of PBMC L1 methylation and circulating concentrations of folate as the independent predictor of primary interest.
Results
Women with a lower degree of PBMC L1 methylation and lower plasma folate concentrations were significantly more likely to have higher BMI, % BF or WC (OR = 2.49, 95% CI:1.41–4.47, P = 0.002; OR = 2.49, 95% CI:1.40–4.51, P = 0.002 and OR = 1.98, 95%  = 1.14–3.48 P = 0.0145, respectively) and higher HOMA-IR (OR = 1.78, 95% CI:1.02–3.13, P = 0.041).
Conclusion
Our results demonstrated that a lower degree of PBMC L1 methylation is associated with excess body weight and higher HOMA-IR, especially in the presence of lower concentrations of plasma folate.
doi:10.1371/journal.pone.0054544
PMCID: PMC3554730  PMID: 23358786
7.  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
Study included 376 women who tested positive for HR-HPVs 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% vs. <70%) and CC LINE-1 methylation (≥54% vs. <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+ (OR = 0.44; 95% CI, 0.24-0.83; P = 0.011) while there was no significant association between degree of CC LINE-1 methylation and CIN 2+ (OR = 0.86; 95% CI, 0.51-1.46; P = 0.578). Among non-cases, women with supra-physiologic 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 to women with lower folate and lower vitamin B12 (OR = 3.92; 95% CI, 1.06-14.52; P = 0.041). None of the variables including folate and vitamin B12 were significantly associated with CC LINE-1 methylation.
Conclusions
These results suggest that 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.
doi:10.1016/j.nut.2010.08.018
PMCID: PMC3070926  PMID: 21463750
methylation; cervical; neoplasia
8.  A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia 
Cancer  2010;117(5):957-963.
Background
Even though 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 pre-cancerous 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+ was 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites were ≥9.5% (OR= 0.21; 95% CI, 0.06–0.79; P=0.02).
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.
doi:10.1002/cncr.25511
PMCID: PMC3023831  PMID: 20945322
HPV 16; methylation; cervical; neoplasia
9.  Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer 
Genes & Cancer  2011;2(9):914-922.
We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, χ2 log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer.
doi:10.1177/1947601911431841
PMCID: PMC3352153  PMID: 22593804
human endogenous retrovirus K (HERV-K); breast cancer; envelope (env) protein; prognosis; survival

Results 1-9 (9)