Laura Reimers

Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer

OBJECTIVE Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA-protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia. METHODS Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n=46), CIN2 (n=12), and CIN3+ (n=27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing. RESULTS Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+. CONCLUSIONS These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.

Sequence Imputation of HPV16 Genomes for Genetic Association Studies

Background Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity. Methods A reference set of 62 HPV16 complete genome sequences was established and used to examine patterns of evolutionary relatedness amongst variants using a pairwise identity heatmap and HPV16 phylogeny. A BLAST-based algorithm was developed to impute complete genome data from partial sequence information using the reference database. To interrogate the oncogenic risk of determined and imputed HPV16 SNPs, odds-ratios for each SNP were calculated in a case-control viral genome-wide association study (VWAS) using biopsy confirmed high-grade cervix neoplasia and self-limited HPV16 infections from Guanacaste, Costa Rica. Results HPV16 variants display evolutionarily stable lineages that contain conserved diagnostic SNPs. The imputation algorithm indicated that an average of 97.5±1.03% of SNPs could be accurately imputed. The VWAS revealed specific HPV16 viral SNPs associated with variant lineages and elevated odds ratios; however, individual causal SNPs could not be distinguished with certainty due to the nature of HPV evolution. Conclusions Conserved and lineage-specific SNPs can be imputed with a high degree of accuracy from limited viral polymorphic data due to the lack of recombination and the stochastic mechanism of variation accumulation in the HPV genome. However, to determine the role of novel variants or non-lineage-specific SNPs by VWAS will require direct sequence analysis. The investigation of patterns of genetic variation and the identification of diagnostic SNPs for lineages of HPV16 variants provides a valuable resource for future studies of HPV16 pathogenicity.

First-trimester 3-dimensional power Doppler of the uteroplacental circulation space: a potential screening method for preeclampsia

OBJECTIVE The objective of the study was to compare 3-dimensional power Doppler (3DPD) of the uteroplacental circulation space (UPCS) in the first trimester between women who develop preeclampsia (PEC) and those who do not and to assess the 3DPD method as a screening tool for PEC. STUDY DESIGN This was a prospective observational study of singleton pregnancies at 10 weeks 4 days to 13 weeks 6 days. The 3DPD indices, vascularization index (VI), flow index (FI), and vascularization flow index (VFI), were determined on a UPSC sphere biopsy with the virtual organ computer-aided analysis (VOCAL) program. RESULTS Of 277 women enrolled, 24 developed PEC. The 3DPD indices were lower in women who developed PEC. The area under the receiver-operating characteristics curve for the prediction of PEC was 78.9%, 77.6%, and 79.6% for VI, FI, and VFI, respectively. CONCLUSION Patients who develop PEC have lower 3DPD indices of their UPCS during the first trimester. Our findings suggest that this ultrasonographic tool has the potential to predict the development of PEC.

Outcomes after an excisional procedure for cervical intraepithelial neoplasia in HIV-infected women.

OBJECTIVE To determine predictors of treatment failure and recurrence after surgical excisional procedures for CIN in HIV-infected women. METHODS A retrospective cohort study was conducted in which 136 eligible HIV-infected women treated for CIN between 1999 and 2005 were included. Data were abstracted from charts and computer databases. Treatment failures were defined as the presence of CIN 1+ at initial follow-up. Recurrences were defined as the presence of CIN 1+ subsequent to initial normal follow-up. RESULTS Treatment failure at initial follow-up was common, occurring in 51% of CIN 1 and 55% of CIN 2+. Most lesions detected at treatment failure were high grade (>70%), regardless of the grade of initial lesion. Significant risk factors for treatment failure were loop electrosurgical excision procedure (LEEP) compared to cold knife conization (RR=1.76; 95% CI: 1.15-2.64), and low CD4+ count (p=0.04). Among those with an initial normal clinical evaluation, 55% eventually recurred. As with treatment failure, most lesions detected at recurrence were high grade. Risk factors for recurrence included use of LEEP (hazard ratio [HR]=3.38; 95% CI: 1.55-7.39), higher HIV RNA level, and the presence of positive margins at treatment (HR=6.12; 95% CI: 1.90-19.73). CONCLUSIONS Most CIN treatment of HIV-infected women studied either failed or resulted in recurrence. Of particular concern, many of these subsequent lesions were high grade. Conization, however, was associated with significantly less failure/recurrence than LEEP. Clinicians treating CIN in HIV-infected women should avoid raising expectations of cure and instead focus on the achievable goal of cancer prevention until there are better therapies for this patient population.

Risk factors for persistent cervical intraepithelial neoplasia grades 1 and 2: managed by watchful waiting.

Objective: This study examines risk factors for persistent cervical intraepithelial neoplasia (CIN) and examines whether human papillomavirus (HPV) testing predicts persistent lesions. Materials and Methods: Women with histologically diagnosed CIN 1 or CIN 2 (n = 206) were followed up every 3 months without treatment. Human papillomavirus genotyping, plasma levels of ascorbic acid, and red blood cell folate levels were obtained. Cervical biopsy at 12 months determined the presence of CIN. Relative risk (RR) was estimated by log-linked binomial regression models. Results: At 12 months, 70% of CIN 1 versus 54% of CIN 2 lesions spontaneously regressed (p < .001). Levels of folate or ascorbic acid were not associated with persistent CIN at 12 months. Compared with HPV-negative women, those with multiple HPV types (RRs ranged from 1.68 to 2.17 at each follow-up visit) or high-risk types (RRs range = 1.74-2.09) were at increased risk for persistent CIN; women with HPV-16/18 had the highest risk (RRs range = 1.91-2.21). Persistent infection with a high-risk type was also associated with persistent CIN (RRs range = 1.50-2.35). Typing for high-risk HPVs at 6 months only had a sensitivity of 46% in predicting persistence of any lesions at 12 months. Conclusions: Spontaneous regression of CIN 1 and 2 occurs frequently within 12 months. Human papillomavirus infection is the major risk factor for persistent CIN. However, HPV testing cannot reliably predict persistence of any lesion.

Human leucocyte antigen class I and II imputation in a multiracial population

Human leucocyte antigen (HLA) genes play a central role in response to pathogens and in autoimmunity. Research to understand the effects of HLA genes on health has been limited because HLA genotyping protocols are labour intensive and expensive. Recently, algorithms to impute HLA genotype data using genome‐wide association study (GWAS) data have been published. However, imputation accuracy for most of these algorithms was based primarily on training data sets of European ancestry individuals. We considered performance of two HLA‐dedicated imputation algorithms – SNP2HLA and HIBAG – in a multiracial population of n = 1587 women with HLA genotyping data by gold standard methods. We first compared accuracy – defined as the percentage of correctly predicted alleles – of HLA‐B and HLA‐C imputation using SNP2HLA and HIBAG using a breakdown of the data set into an 80% training group and a 20% testing group. Estimates of accuracy for HIBAG were either the same or better than those for SNP2HLA. We then conducted a more thorough test of HIBAG imputation accuracy using five independent 10‐fold cross‐validation procedures with delineation of ancestry groups using ancestry informative markers. Overall accuracy for HIBAG was 89%. Accuracy by HLA gene was 93% for HLA‐A, 84% for HLA‐B, 94% for HLA‐C, 83% for HLA‐DQA1, 91% for HLA‐DQB1 and 88% for HLA‐DRB1. Accuracy was highest in the African ancestry group (the largest group) and lowest in the Hispanic group (the smallest group). Despite suboptimal imputation accuracy for some HLA gene/ancestry group combinations, the HIBAG algorithm has the advantage of providing posterior estimates of accuracy which enable the investigator to analyse subsets of the population with high predicted (e.g. >95%) imputation accuracy.

Performance of Implementing Guideline Driven Cervical Cancer Screening Measures in an Inner City Hospital System

Objective: In 2006, the American Society for Colposcopy and Cervical Pathology updated evidence-based guidelines recommending screening intervals for women with abnormal cervical cytology diagnosis. In our low-income inner-city population, we sought to improve performance by uniformly applying the guidelines to all patients. We report the prospective performance of a comprehensive tracking, evidence-based algorithmically driven call back, and appointment scheduling system for cervical cancer screening in a resource-limited inner-city population. Materials and Methods: Outreach efforts were formalized with algorithm-based protocols for triage to colposcopy, with universal adherence to evidence-based guidelines. During implementation from August 2006 to July 2008, we prospectively tracked performance using the electronic medical record with administrative and pathology reports to determine performance variables such as the total number of Pap tests, colposcopy visits, and the distribution of abnormal cytology and histology results, including all cervical intraepithelial neoplasia 2, 3 diagnoses. Results: A total of 86,257 gynecologic visits and 41,527 Pap tests were performed system-wide during this period of widespread and uniform implementation of standard cervical cancer screening guidelines. The number of Pap tests performed per month varied little. The incidence of CIN 1 significantly decreased from 117 (68.4%) of 171 during the first tracked month to 52 (54.7%) of 95 during the last tracked month (p = 0.04). The monthly incidence rate of CIN 2, 3, including incident cervical cancers, did not change. The total number of colposcopy visits declined, resulting in a 50% decrease in costs related to colposcopy services and approximately a 12% decrease in costs related to excisional biopsies. Conclusions: Adherence to cervical cancer screening guidelines reduced the number of unnecessary colposcopies without increasing numbers of potentially missed CIN 2, 3 lesions, including cervical cancer. Uniform implementation of administrative-based performance initiatives for cervical cancer screening minimizes differences in provider practices and maximizes performance of screening while containing cervical cancer screening costs.

Simultaneous endometrial aspiration and sonohysterography for the evaluation of endometrial pathology in women aged 50 years and older.

OBJECTIVE: To evaluate the performance of simultaneous endometrial aspiration at the time of sonohysterography for screening postmenopausal women at risk for endometrial cancer. METHODS: A retrospective cohort study of women older than 50 years who underwent saline-infusion sonohysterography for the evaluation of their endometrium. On completion of imaging, the remaining intracavitary saline and endometrial tissue were aspirated through the saline-infusion sonohysterography catheter and submitted for pathologic evaluation. Based on the clinical, pathologic, and ultrasonographic results, the patients underwent surgical treatment with hysteroscopy, hysterectomy, or clinical observation. Follow-up results and outcomes were collected using electronic medical records. Sensitivity, specificity, and predictive values of saline-infusion sonohysterography, endometrial aspiration, and combined approaches for endometrial aspiration and sonohysterography were assessed. RESULTS: Six hundred three patients underwent endometrial aspiration at the time of sonohysterography. Endometrial tissue was present in 567 (94.0%) and outcome data were available for 540 (89.5%). In 194 (35.9%) patients, final pathology was obtained by surgical intervention. The remaining 346 (64.1%) patients were monitored for at least 6 months. Thirty patients (5.6%) had cancer or endometrial hyperplasia. A sequential model, in which endometrial aspiration was done only for positive saline-infusion sonohysterography findings, yielded sensitivity of 86.7% (95% confidence interval [CI] 69–96%) and specificity of 100% (95% CI 99–100%) for detecting endometrial hyperplasia or cancer (area under the curve 0.93). Considering proliferative endometrium as abnormal endometrial aspiration reduced specificity to 88.3% (95% CI 85–91%, P<.01) without significant increase in sensitivity (100%, 95% CI 88–100%, P=.13). CONCLUSION: The high sensitivity and specificity of the sequential endometrial aspiration at the time of sonohysterography make this approach a useful and reliable screening algorithm for detecting endometrial cancer or hyperplasia in postmenopausal women at risk. Endometrial aspiration at the time of sonohysterography should be considered as an initial one-stop endometrial evaluation in this population. LEVEL OF EVIDENCE: II

Oral lopinavir use and human papillomavirus infection in HIV-positive women

Invasive cervical cancer (ICC) is the third most common female malignancy and fourth most common cause of cancer death in women globally1. The risk of ICC is several-fold higher in HIV-positive women than in HIV-negative women, as are the prevalence2–5, incidence, and persistence of oncogenic human papillomavirus (oncHPV)6–14, the infectious cause of most ICC. Although use of effective highly-active antiretroviral therapy (HAART) has been associated with reduced oncHPV prevalence and incidence15 and increased regression of cervical lesions16–19, the overall incidence of ICC has not decreased in HIV-positive women during the HAART era. By increasing survival, HAART may increase lifetime oncHPV infections, allowing accumulation of somatic mutations and epigenetic changes necessary for oncogenesis. Currently, no anti-viral medications are clinically approved to treat cervical HPV infections. In vitro studies have shown that lopinavir (LPV), an HIV-1 protease inhibitor (PI) used in some HAART regimens, may have activity against oncHPV through inhibition of viral oncogene E620, 21. Most recently, an early phase clinical trial conducted in Nairobi, Kenya studied topical application of LPV to the cervix; preliminary results showed that 21 of 23 women initially diagnosed with high-grade disease returned to normal on subsequent Papanicolaou (Pap) smears and showed visible regression of cervical lesions22. We therefore assessed the hypothesis that oral LPV use may be associated with decreased prevalence and increased clearance of oncHPV compared to other antiretroviral (ARV) regimens.

A SWIFT Method for Handing Off Obstetrical Patients on the Labor Floor

OBJECTIVE The aim of this study was to improve patient handoffs on the labor floor. METHODS A prospective cohort study of obstetrics residents at Montefiore Medical Center was performed between 2012 and 2014. Labor-floor handoffs were recorded before and after didactic sessions as well as after installation of whiteboards formatted with the mnemonic SWIFT (Subject, Why?, Issues, Fetus, Tasks). Handoff transcripts were evaluated by obstetricians blinded to timing and speaker identity. An intraclass correlation coefficient accounted for evaluator differences. Data analysis was by ordinal logistic regression, the generalized estimating equations method (correlated data), and Bonferroni adjustment (multiple comparisons). RESULTS Forty-five handoffs were evaluated (15 each predidactics, postdidactics, and postwhiteboard revision). Higher completeness scores over time were noted for admission reason, labor concerns, and task list (not statistically significant). Comprehensive score increases prelecture to postwhiteboard were seen in handoff clarity (2.81 versus 2.91) and overall quality (2.77 versus 2.81) (not statistically significant). A subanalysis of four residents who gave multiple handoffs over different periods revealed few significant changes over time. Greater interevaluator consistency was noted with more objective elements. CONCLUSIONS The mnemonic SWIFT, with formalized curricula for obstetrical resident training focusing on new learners and increased faculty involvement and reinforcement, may result in improvement of handoffs on the labor floor.