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GenoScape - example of HPV genotype profiling report and visualization. Number, characteristics and percentage of reads that map to thirteen (13) HPV types, human, bacteria and unknown genomes after profiling reads of the clinical sample TrDNA-456 by tiered read mapping.

Scatter plots of HPV 16-L1 methylation in cervical brush (S7a) and urine ccfDNA (S7b). HPV16-L1 qMSP methylation can discriminate bisulfite treated cervical epithelium DNA and urine ccfDNA from patients with normal cytology, from women with dysplastic cytology, and premalignant cervical lesions with high Sensitivity and Specificity.

GenoScape - HPV genotype profiling from high-throughput sequencing samples consists of four analytic steps: (1) High-throughput sequencing (454, MiSeq) of HPV TrDNA; (2) Profiling by comparing sequences against several databases such as Human genome (bowtie), HPV databases -GenBank, PaVE, Enterix-HPV Hi-lo risk (sim4db) and RefSeq bacterial genomes (blast); (3) Assembly into contigs, for AlignScape visualization (newbler,minimus, trinity); and SNP discovery (samtools + filter).

Circulating cell free DNA isolation results.

Primer and probe list of genes used for quantitative Methylation Specific PCR in urine ccfDNA.

Mapping efficiency of cervical cancer cell lines, head and neck squamous cell carcinoma cell lines and clinical samples to HPV16-L1.

Dual Sequence capture of high risk HPV in TrDNA aligned to all high risk HPV types and low risk HPV types from PaVE database.

AlignScape - Pairwise alignment of the HPV16 genome with reads obtained from urine ccfDNA from seven clinical samples: TrDNA-445, TrDNA-455, TrDNA-456, TrDNA-481, TrDNA-504, TrDNA-513, and TrDNA-571. The large-scale HPV DNA server can produce a graphical 'large-scale' view of the pairwise alignments of up to twenty HPV genomes against a reference genome (HPV16 in this demo), together with annotations of genome rearrangement events.

Patient characteristics.

Reads of the HPV positive cervical cancer, head and neck cancer squamous cell carcinoma (HNSCC) cell lines and clinical samples that mapped uniquely to the reference genomes in the PAVE database.

Supplementary Methods.

Phase 1 Biomarker Development Trial to identify genes or clusters of genes that appear to be differentially methylated in cervical cancer biopsy tissues relative to control cervical brush epithelium tissue taken from women seen in the same hospital. The search for cervical cancer screening methylation biomarkers began with preclinical exploratory studies to identify characteristics unique to tumor tissue that might lead to methylated biomarkers for clinical tests that can discriminate cancer from pre-malignant and normal lesions. We used Methylated DNA Immunoprecipitation (MeDIP) and oligonucleotide sequencing arrays (Nimblegen 385K CpG Island Plus Promoter Arrays) to unveil the cervical cancer promoter region methylome (MeDIP-chip). We then used bisulfite sequencing and quantitative Methylation Specific Polymerase Chain Reaction (qMSP) to identify potential biomarkers of progression from normal cervical mucosa epithelium in cervical brush samples from women with abnormal cytology. MeDIP-chip Discovery identified 2,044 differentially methylated probes associated to total of 444 gene loci differentially methylated when comparing tumor and normal samples. Gene loci were subdivided according to two cutoffs defined for the maximal distance between a methylation peak and the TSS: -1000 to +1000, called the standard cut-off; -500 to +500, called the narrow cut-off. We found 255 unique gene loci that are cancer specific methylated according to the standard cutoff and 189 according to the narrow cutoff. More than half of the methylated gene promoters identified with MeDIP-chip (60%) were hypermethylated in all cancer samples and not in normal samples. One hundred and sixty two (162) genes were jointly identified by the intersection between both lists. These stringent criteria ensured that all 10 genes selected for biomarker validation had methylated peaks within a CPG island located in the promoter region, 500 base pairs upstream from the TSS in all the hybridized tumor samples and none in the normal samples hybridized to the arrays. The top three genes in the list that contained CpG islands in their promoter regions FKBP6, ZNF516 and INTS1) were selected for further analysis with MSP and qMSP in Validation and Prevalence cohorts. Samples were tsted for HPV presence using the reverse line blot assay.

Goals and Primary Aims of the five phases of the National Cancer Institue Early Detection Research Network biomarker development trials.

Amplification curves for the HPV TrDNA-qPCR assay on TrDNA from women with Cervical Intraepithelial Neoplasia 1 (CIN1), Cervical Intraepithelial Neoplasia 2-3 (CIN2-3) and women with no Intraepithelial Lesions or Neoplasia (NILM). TrDNA samples were analyzed for high risk HPV DNA using our HPV TrDNA SYBR green qPCR assay. HeLa genomic DNA was included as a positive control. Ct ranges are variable, but are in a similar range as the genomic control. NILM samples did not amplify.

Clinically useful molecular tools to triage women for a biopsy upon referral to colposcopy are not available. We aimed to develop a molecular panel to detect cervical intraepithelial neoplasia (CIN) grade 2 or higher lesions (CIN2+) in women with abnormal cervical cytology and high-risk HPV (HPV+). We tested a biomarker panel in cervical epithelium DNA obtained from 211 women evaluated in a cervical cancer clinic in Chile from 2006 to 2008. Results were verified in a prospective cohort of 107 women evaluated in a high-risk clinic in Puerto Rico from 2013 to 2015. Promoter methylation of ZNF516, FKBP6, and INTS1 discriminated cervical brush samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM) with 90% sensitivity, 88.9% specificity, 0.94 area under the curve (AUC), 93.1% positive predictive value (PPV), and 84.2% negative predictive value (NPV). The panel results were verified in liquid-based cervical cytology samples from an independent cohort with 90.9% sensitivity, 60.9% specificity, 0.90 AUC, 52.6% PPV, and 93.3% NPV, after adding HPV16-L1 methylation to the panel. Next-generation sequencing results in HPV+ cultured cells, and urine circulating cell-free DNA (ccfDNA) were used to design assays that show clinical feasibility in a subset (n = 40) of paired plasma (AUC = 0.81) and urine (AUC = 0.86) ccfDNA samples obtained from the prospective cohort. Viral and host DNA methylation panels can be tested in liquid cytology and urine ccfDNA from women referred to colposcopy, to triage CIN2+ lesions for biopsy and inform personalized screening algorithms. Cancer Prev Res; 9(12); 915–24. ©2016 AACR.

Primer and probe list of genes used for quantitative Methylation Specific PCR.

Trak-Fellermeier MA, Campos M, Meléndez M, et al.Diabetes Metab Syndr Obes. 2019;12:225–238. On page 225, Abstract, Results subheading, “Althoughnot statistically significant, women in the interventiongroup (6/15) were 1.7 times more likely to achieveappropriate weekly GWG until delivery when comparedwith controls (4/16 women) (adjusted incidence rateratio = 1.67; 95% CI: 0.40, 6.94).” should read“Although not statistically significant, women in theintervention group (6/15) were 1.7 times more likely toachieve appropriate total GWG until delivery whencompared with controls (4/16 women) (adjusted incidencerate ratio = 1.67; 95% CI: 0.40, 6.94).” Appropriate GWG (baseline to delivery) section, 2nd to 4th lineon page 230, “Forty percent (6/15) of women in the interventionachieved appropriate GWG/week compared with 25%(4/16) in the control group.” should read “Forty percent (6/15) ofwomen in the intervention achieved appropriate total GWGcompared with 25% (4/16) in the control group.” The authors apologize for not noticing the inconsistenciesbetween the text and Table 3 earlier. Read the original article

This is the case of a 38 year-old female patient with an intrauterine pregnancy, in which a previous incidentally identified adrenal mass proved to be a pheochromocytoma during the antenatal period. The patient was started on α-and β-adrenergic blockade to maintain hemodynamic stability, and surgical removal of the lesion was performed during the second trimester without major complications. In view of the rarity of this disorder in pregnancy, it is imperative to have a high index of suspicion for a prompt and dedicated management, since this tumor, if unrecognized, is associated with high fetal and maternal mortality.

Background Laparoscopic cholecystectomy, the standard procedure for removing the sick gallbladder of children, is generally performed leaving the child overnight in the hospital. Purpose This study aimed to determine if there is a safe advantage in performing laparoscopic cholecystectomy as an outpatient procedure while setting the clinical parameters for those who will benefit from in-hospital stay. Methods Thirty-five patients were selected for the study and were divided into group A, if the outpatient procedure was done, and group B, if the child was left overnight in the hospital. Retrospective review of medical charts was performed. Statistical significance was defined as P Results Group A consisted of 13 patients and group B of 22 patients. All patients in group A left the hospital the same day of surgery. Distribution by age and sex in the groups was not statistically different. Preoperative symptoms of vomiting were statistically significantly higher in group B. Presence of an associated medical condition was higher in the in-hospital patients. Concomitant procedures, blood loss estimates, and duration of surgery showed no statistical difference. No child was readmitted after release from the hospital. Pre-, intra-, and postoperative pain management were the same in all patients. Mean postoperative stay and medical charges were statistically significant between the groups. Conclusions Laparoscopic cholecystectomy can safely be done as an outpatient procedure. Children with a complicated gallbladder disease process or associated medical condition benefit from an overnight stay. Perioperative pain management is crucial in all cases. Reduced hospital stay and medical charges are significant advantages in performing laparoscopic cholecystectomy as an outpatient procedure.

Cervical cancer is the fifth most common cancer among women in Puerto Rico (PR). Information about which obstetricians and gynecologists in PR are following current cervical cytology guidelines is not available. Our two objectives were to determine whether current guidelines for abnormal cervical cytology management were being followed adequately by OB-GYN residents and faculty at 3 university hospitals and, in addition, to evaluate the knowledge of the current guidelines that is possessed by these individuals.A retrospective medical record review (records from January 2009 through December 2010) of patients with abnormal cervical cytology results (n=166) to evaluate the management they were given and a cross-sectional survey of OB-GYN residents (n=34) and faculty (n=46) to evaluate their knowledge of cervical cytology guidelines were both performed.One hundred and sixty-six medical records were reviewed. The level of management for 45% of the patients was found to have been optimal."Thirty-three percent were lost to follow-up. Eleven percent of the patients were managed sub optimally and the management of 12% of the patients was considered to have been poor. The survey showed that the faculty were less updated with regard to current guidelines for the management of cervical cytology than were the residents. Study limitations were the lack of electronic medical records in the hospital--which makes access to patient information more difficult--and the low response rate of the faculty.OB-GYN physicians and residents were not consistent with their management, according to current guidelines, of cervical cytological abnormalities. The high-risk nature of the patients and their poor adherence to treatment (loss to follow-up) may have been the cause of this inconsistency. Meetings aimed at addressing the lack of compliance by local OB-GYN physicians with the guidelines for cervical cancer screening have been Better screening strategies are also needed if we are to improve patient compliance in our population.

The aim of this research was to study the relationship of abnormal placentation and cesarean hysterectomy risk factors at the University District Hospital.A retrospective study was conducted using 60 medical records of patients in the University District Hospital from 2001 to 2008 with a diagnosis of abnormal placentation. Descriptive statistics and chi square for associations were used for statistical analysis.The mean age in our population was 29 years and mean BMI was 29.6. 45 % of the patients had a previous history of cesarean, 1.7% of placenta previa and 26.7% of uterine curettage. We found that 33.3% had a cesarean hysterectomy and 41.7% had post-partum hemorrhage. Also 51.7% received blood transfusion. The mean hospital stay was 18.1 days. The probability of cesarean hysterectomy in patients with placenta previa was eleven times higher in patients with history of previous cesarean sections (OR 11, CI: 1.29-97.9, p value=0.01). There was no statistically significant difference in cesarean hysterectomy rate between obese (BMI30) and nonobese patients (OR 3.8 CI: 0.69-20.5, p value=0.11).The probability of cesarean hysterectomy in patients with abnormal placentation was significantly increased in patients with previous cesarean sections. By decreasing the rate of cesarean section we can decrease the incidence of cesarean hysterectomy and its comorbidities.

Cervical dysplasia alters the release of cytokines and inflammatory mediators in pregnant woman with cervical dysplasia. This study evaluates a cohort of pregnant patients screened for cervical dysplasia to determine the relationship between cervical dysplasia and preterm labor (PTL).Retrospective chart review of pregnant patients screened for cervical dysplasia at the San Juan City Hospital between October 2006 and December 2010. Patients with low or high-grade squamous intraepithelial lesions (LGSIL or HGSIL, respectively) were evaluated with colposcopy. Primary outcome was the event of PTL and the risk factor evaluated was presence of cervical dysplasia.A total of 2,059 patients were screened for cervical dysplasia and 59 were evaluated with colposcopy due to LGSIL or HGSIL. From those, 29% were negative for intraepithelial lesions and malignancy (NILM), 54% had cervical intraepithelial neoplasia (CIN)-1, 17% where diagnosed as CIN-2/3 where no invasive cervical cancer was identified. In the group of NILM, 24% had PTL compared to 18% in patients who screened negative on initial cytological evaluation. In cases where CIN-1 was confirmed by colposcopy 28% had PTL compared with 40% in patients with CIN-2/3. Other risk factors associated with PTL were not significantly different among the groups.Cervical dysplasia in pregnancy may represent an increase risk for premature labor.

Persistent infection with high-risk (HR) HPV is a necessary risk factor for the development of cervical cancer. Information on HPV infection is limited in Puerto Rico. This study determined the distribution of HPV types and the association of HR-HPV types with cervical pathology in a clinic-based sample of women in PR.Data from 92 female participants aged 18 to 34 years and recruited from the University of Puerto Rico-Gynecology Clinic, were analyzed. Cervical cytology was performed. HPV testing was performed using L1 consensus primer PCR with MY09/ MY11 primers and typed by dot-blot hybridization. Logistic regression modeling was used to determine the crude and covariate adjusted association between HR-HPV and cervical pathology.Twenty percent (n = 18) of the patients had abnormal cytology, 45.7% (n = 42) were HPV positive, and 30.4% (n = 28) were HR HPV-positive. Women infected with HR-risk HPV types were 7.9 (95% CI = 2.5-25.5) times more likely to have abnormal cytology as compared to women without HR infection when adjusted by age and age at first sexual intercourse.The burden of HPV infection was high, and, as expected, HR HPVs were strongly associated with dysplasia. A population-based study is needed to estimate HPV prevalence and its association with related malignancies in our population. This will be of great value in determining disease burden and will increase awareness of the HPV vaccination in our population.

ObjectiveThis study aimed to describe the prevalence and concordance between cervical and anal human papillomavirus (HPV) infection and compare cervicovaginal and anal self-collection methods for HPV testing between physician and self-collected specimens in women in Puerto Rico.Materials and methodsSpecimens for HPV-DNA testing were obtained from 100 women aged 18 to 34 years attending a general gynecology clinic for a routine Pap smear. Human papillomavirus testing was performed using polymerase chain reaction MY09/MY11 primers. Positive samples were typed for 39 genotypes. Agreement between sampling methods was determined by percent agreement and the κ statistic.ResultsFor the 39 genotypes evaluated, 38.4% (38/99) of cervicovaginal and 33.7% (30/89) of anal physician-collected samples were HPV+, whereas 35.1% (34/97) of cervicovaginal and 32.0% (31/97) of anal self-collected samples were positive. Human papillomavirus type 16 was the most common type identified in the cervix (8.3%, 8/97) and the anus (5.6%, 5/89) of physician-collected samples, with similar prevalence in self-collected samples. Concordance between cervical and anal HPV infection was high (>90%) for all types evaluated. There was a strong percent agreement between physician- and self-collected cervicovaginal and anal samples (>95% for all HPV types) and good to excellent agreement (κ > 0.60) for most HPV types.ConclusionsThe clinic-based prevalence of anal and cervicovaginal HPV infection was high, with a strong concordance between cervical and anal infection and good to excellent agreement between physician- and self-collected samples. This study supports the feasibility of using cervical and anal self-sampling methods in future population-based studies of HPV infection in Puerto Rico and as an HPV screening method in women.

We set out to develp novel workflows to identify panels of methylated human papilloma virus (HPV) and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. Using Human DNA methylation arrays and massively parallel Next Generation Sequencing (NGS) for Discovery and quantitative Methylation Specific PCR (qMSP) for validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from no intraepithelial lesions or malignancy (NILM) in cervical brush samples: 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for massively parallel Next Generation Sequencing (NGS) in 454 and MiSeq instruments, respectively to identify high risk HPV and the HPV methylome. Using our NGS results, we designed Syber Greeen qPCR assays to detect high risk HPV DNA in tissue and in Trans Renal DNA (TrDNA). We also designed qMSP primers-probe sets to detect methylated DNA in the HPV16-L1 gene region and ZNF516, FKBP6, and INST1 in TrDNA. TrDNA is circulating DNA that gets filtered by the kidneys and we isolate from urine as fragmented DNA ranging is size from 50 to 250 bp. We replicated the cervical brush epithelium results in liquid prep samples from another cohort, by adding HPV16-L1 methylation to the panel of methylated host genes (ZNF516, FKBP6, and INST1): 90.9% sensitivity, 60.9% specificity, AUC = 90.1, 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC = 80.7) and TrDNA (AUC = 86.1) isolated from a subset of patients who provided liquid prep samples. Our results suggest that a biomarker panel that quantifies DNA methylation in the HPV-LI gene, together with promoter methylation of three host genes, ZNF516, FKBP6, and INST1, may be used as a reflex test in liquid prep to triage high-risk HPV positive women into colposcopy in countries with established cervical cancer screening programs. Our results also suggest that a panel of human and viral DNA methylation markers can be combined with a high-risk HPV DNA assay, to develop urine screening and triage tests in countries where cervical cancer screening programs are not effective due to cultural or infrastructure factors. Country specific DNA methylomes of normal and premalignant cervical epithelium for different age groups can be created as reference methylomes, to better understand the cultural-, socio-economic- and age-specific variations in the cervical epithelium and their correlation with HPV DNA methylomes. Citation Format: Rafael E. Guerrero-Preston, Blanca L. Valle, Anne Jedlicka, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Edgardo De Jesus-Rodríguez, Keimari Méndez-Martínez, José Rodríguez-Orengo, Teresa Díaz-Montes, David Sidransky, Josefina Romaguera. Triage of high risk HPV positive women before colposcopy with reflex tests in pap smears and screening of high risk HPV in Transrenal DNA isolated from urine, using novel workflows to identify panels of methylated viral and human DNA. [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 LB-133.

Gestational trophoblastic disease has been reported to be responsive to chemotherapy, with a 90% cure rate. Several factors place patients at high risk of experiencing treatment failure with single agent chemotherapy. Choriocarcinoma following term pregnancy is very rare and associated with a poor prognosis and a mortality rate of 33-40%. We present a rare case of cutaneous metastasis of choriocarcinoma to the left third digit.