Human Papillomavirus DNA In Tissue Research Articles

Detection of high-risk human papillomavirus DNA in tissue from primary cervical cancer tumor, pelvic lymph nodes and recurrent disease

ObjectivesThe present study investigated Human Papillomavirus (HPV) DNA genotyping in primary tumor, pelvic lymph nodes (PLN) and recurrence in early-stage cervical cancer patients. MethodsWe conducted a hospital-based case-control study. From 2003 to 2015, 282 patients underwent surgery for cervical cancer in the Department of Gynecology, Aarhus University Hospital, Denmark. Twenty-nine recurrent cases were identified. HPV DNA genotyping was performed on formalin-fixed, paraffin-embedded tissue specimens from the primary tumor, PLN, and recurrent disease. ResultsIn the primary tumor, HPV DNA was detectable in 18(72%) of 25 tissue specimens from recurrent cases and in 15(83%) of 18 controls. HPV DNA-positive PLN was significantly associated with recurrence, 83%(95%CI: 52–98%), compared to patients with HPV-negative PLN, 38%(95%CI: 18–62%)(p < 0.05). HPV DNA genotyping was positive in eight of 12(67%) patients with recurrent disease. The genotype was identical in all three tissues types.The positive predictive value for recurrence was the same for detection of HPV-DNA and metastases in the PLN, with reasonable sensitivity. The negative predictive value for recurrence, however, was best for HPV-DNA, 62%(95%CI: 38–98%). ConclusionsIn conclusion, our data suggest that the presence of HPV in pelvic lymph nodes is associated with an increased risk of recurrence.

Abstract LB-133: 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 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.

High-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma—Letter

See the Reponse, p. 409We read with interest the recent article excluding human papillomavirus (HPV) as a significant contributor in esophageal squamous cell cancers (ESCC), at least in Australia (1). This is in contradistinction to oropharyngeal cancers (OPC) where there is compelling epidemiologic and molecular evidence indicating HPV as the etiology of a subset of cases. Within Australia, there is a changing epidemiology of OPCs. In a recent study of 302 cases, the incidence of HPV-related OPCs increased from 19% in 1987 to 1990, 47% in 2001 to 2005, to 60% in 2005 to 2006 (2). Reports worldwide vary considerably in finding HPV in ESCC: with rates up to 81% (3). The clinical implications of excluding HPV as a major contributor to ESCC are important, particularly as HPV-related cancers have better prognosis and chemoradiation responses than their non-HPV counterparts.We question whether the HPV rates have been fully determined in this ESCC series, due to potential methodologic problems. The authors used internal control primers which amplify a 110-bp region of the β-globin gene (1). This contrasted with the 150-bp HPV L1 amplicon generated with GP5+/6+ primers. It would be standard practice to use an internal control of longer or equal length to the HPV amplicon, to ensure DNA below 150 bp has not degraded (4). Also, there are several aspects of the described PCR method which could contribute to lower assay sensitivity. Firstly, the authors modified the MgCl2 concentration to 2 mmol/L from the original assay description of 3.5 mmol/L. This would increase PCR stringency, but reduce sensitivity. The assay sensitivity in detecting HPV in tissue should be stated.Secondly, the amplicons were analyzed by gel electrophoresis, a less sensitive method than using HPV-specific probes. If original paraffin-embedded tissue blocks were small, faint amplicon bands could be missed: although would not explain so many negatives. (This would be further exacerbated if there were DNA degradation). In addition, if there were HPV integration there could be interruption of the site being amplified.It is standard when looking for HPV DNA in tissue that the sample used for PCR also has samples collected either side, to be rechecked histologically for tumor. This ensures that the sample is adequate for HPV PCR (5). We note that 22 (10%) of the total samples analyzed were from slide sections: the remainder from tissue blocks. Amplification from slide sections is less sensitive than from tissue. This is of relevance given that none of the 22 slides examined were HPV positive.S. M. Garland, advisory board for cervical cancer vaccines, honoraria for speakers bureau, and through her institution received monies for research on cervical cancer vaccine phase 3 trials.No other potential conflicts of interest were disclosed.

Measures of cutaneous human papillomavirus infection in normal tissues as biomarkers of HPV in corresponding nonmelanoma skin cancers

Cutaneous human papillomavirus (HPV) may be associated with the development of nonmelanoma skin cancer (NMSC), as suggested by reports of HPV DNA in NMSC tumors. HPV has also been investigated as an NMSC risk factor in epidemiologic studies, although findings vary across studies that used different biomarkers of HPV infection in normal tissues. To identify appropriate biomarkers for use in future epidemiologic studies, we conducted a sampling validation study. NMSC tumor tissue was obtained from 20 patients with pathology-confirmed basal or squamous cell carcinoma of the skin, in addition to several normal tissues, including eyebrow hairs, normal skin swabs obtained using multiple techniques, normal skin punch and shave biopsies, and serum for antibody measurement. Presence of cutaneous HPV DNA in tissues was measured with multiplex PCR using HPV type-specific primers and array primer extension (APEX) for HPV typing. Antibody detection was based on glutathione-S-transferase capture ELISA in combination with fluorescent bead technology. Using HPV DNA in tumor tissues as a gold standard, sensitivity and specificity were calculated for each measure of HPV infection in normal tissues. beta-Papillomavirus DNA was observed in tumor tissues in 60% of patients. The normal skin punch biopsy demonstrated optimal sensitivity (75%) and specificity (75%). Biomarkers obtained using less-invasive techniques demonstrated poor specificity when considered individually, although specificity improved when biomarkers were combined. Based on the current case series, the combinations of antibodies+eyebrow hairs or antibodies+eyebrow hairs+Dacron swabs are the optimal, minimally invasive markers of cutaneous HPV infection for use in epidemiologic studies.

Human papillomavirus infection in actinic keratosis and bowen’s disease: comparative study with expression of cell-cycle regulatory proteins p21 waf1/cip1, p53, pcna, ki-67, and bcl-2 in positive and negative lesions

We examined the presence of human papillomavirus (HPV) DNA in tissues of premalignant skin lesions, i.e., actinic keratosis (n = 13) and Bowen’s disease (n = 62), taken from 69 Japanese immunocompetent and renal transplant recipient patients. Detection and typing of HPV DNA were performed using polymerase chain reaction (PCR) and sequence analysis or restriction fragment length polymorphism (RFLP) analysis, respectively. The positivity rates of HPV DNA in tissues of actinic keratosis and Bowen’s disease were 77% and 65%, respectively. Twenty-seven HPV types were detected in 50 (67%) premalignant skin lesions, in which Z95963 (accession no. in the EMBL Databank), Z95968, AJ010823, and AJ000151 have been described as partial sequences of unknown HPV types. Furthermore, 2 unknown types, HPVX1 and HPVX2, were found in specimens of actinic keratosis. Sequence analysis showed that HPVX1 is related to HPV-37 (86.1% sequence homology) and that HPVX2 is related to HPV-38 (79.7%). These results indicate that various mucosal and epidermodysplasia verruciformis-related HPV types are associated with the pathogenesis of actinic keratosis and Bowen’s disease. In addition, 24 specimens of HPV-positive or HPV-negative premalignant skin lesions were examined immunohistochemically for proliferating cells to determine biological differences between HPV-positive and HPV-negative lesions. Immunohistochemistry for p21 Waf1/Cip1, p53, proliferating cell nuclear antigen (PCNA), Ki-67, and Bcl-2 revealed that there was no significant difference in the cell proliferation activity between HPV-positive and HPV-negative lesions, suggesting that HPV infection alone does not induce cell proliferation in those lesions.

Detection of viral DNA and RNA by in situ hybridization.

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.