Eva Hamsikova

Human papillomavirus seropositivity and risks of head and neck cancer

We examined antibody response to VLP HPV‐16, HPV‐16 E6 and E7 antibodies as potential seromarkers of HPV‐related head and neck cancer (HNC). The study included 204 HNC cases and 326 controls evaluated for HPV presence in sera using ELISAs for anti‐HPV VLP antibodies and HPV‐16 E6 and/or E7 antibodies, and in tumor tissue using PCR and DNA sequencing. Anti‐HPV‐16 VLP was detected in 33.8% of cases and 22.4% of controls, anti‐E6 in 20.6% of cases and 0.9% of controls and anti‐E7 in 18.6% of cases and 0.6% of controls. HPV‐16 DNA was detected in 26.1% of tumors. The adjusted risk of HNC was elevated among those seropositive for HPV‐16 VLP (odds ratio (OR) = 1.7, 1.1–2.5), E6 (OR = 32.8, 9.7–110.8) or E7 (OR = 37.5, 8.7–161.2). Compared to HPV DNA‐negative/seronegative cases, tumor HPV‐16 cases had increased risk of detection with anti‐VLP antibodies (OR = 6.8, 3.1–14.9). The odds were more pronounced among cases seropositive for E6 (OR = 69.0, 19.3–247) or E7 (OR = 50.1, 14.7–171). Antibodies against E6 or E7 were associated with risk of cancer in the oral cavity (OR = 5.1, 1.2–22.4) and oropharynx (OR = 72.8, 16.0–330), and with disease characteristics: stage, grade and nodal status. Anti‐E6 and/or E7 antibodies were found in 74% of tumor HPV‐16 positive cases but in only 5% of tumor HPV‐negative cases (K =0.7, 0.6–0.8) suggesting good correlation between the serologic marker and HPV tumor status. Antibodies to HPV‐16 E6 and/or E7 represent a more specific biomarker than anti‐HPV‐16 VLP of an HPV‐related HNC. Because of the survival advantage of HPV‐related HNC, HPV‐16 E6/E7 detection may be useful in therapy targeted for HPV‐related tumors.

Demographic and risk factors in patients with head and neck tumors

The association between human papillomavirus (HPV) infection and the development of head and neck cancer has been documented recently. In this study on 86 head and neck cancer patients and 124 controls, data regarding demographics, behavioral risk factors, and risks related to HPV exposure were collected. HPV detection was carried out using polymerase chain reaction in the tumors and in oral exfoliated cells, and HPV typing by a reverse line blot assay specific for 37 HPV types. Sera were tested by an enzyme‐linked immunosorbent assay specific for HPV proteins. Head and neck cancer cases report significantly more oral‐anal contact (P = 0.02) and tobacco and alcohol use than controls (P = 0.001; P = 0.02, respectively). High‐risk HPV DNA was detected in 43% of oral washings of cases and 4% of controls (P < 0.0001). The association between the presence of high‐risk HPV DNA in oral exfoliated cells and in tumor tissues was statistically significant (adjusted P < 0.0001). The prevalence of HPV‐specific antibodies was significantly higher in cases than in controls (adjusted P < 0.0001). These results provide epidemiological and immunological evidence for HR HPV as a strong risk factor (OR = 44.3, P < 0.0001) for head and neck cancer, even after controlling for age, tobacco and alcohol use. The detection of high‐risk HPV DNA in oral exfoliated cells and HPV‐specific antibodies in serum can be considered as clinically relevant surrogate markers for the presence of a HPV‐associated head and neck cancer, with a high sensitivity (83%) and specificity (88%). J. Med. Virol. 81:878–887, 2009.

Evidence for Vertical Transmission of HPV from Mothers to Infants

Few large studies have evaluated concordance based on a broad spectrum of human papillomavirus (HPV) types in oral and genital specimens of mothers and their recently born infants. This information is important in determining whether HPV vaccines administered prior to pregnancy may be useful for preventing vertical transmission. HPV DNA was positive in 30% of mothers and 1.5% of newborns. Maternal/newborn concordance (HPV+/+ or HPV-/-) was 71%. Among HPV DNA+ mothers, only 3% of their infants were DNA+ and only 1 pair had the same HPV type. Among HPV- women, 0.8% of infants were HPV+. HPV DNA detected in hospitalized newborns reflects current infection transmitted to infants during pregnancy or delivery. None of the mother/baby HPV DNA+ concordance pairs detected viral types found in HPV vaccines suggesting that vaccination prior to pregnancy is unlikely to be efficacious in preventing vertical transmission.

HPV involvement in tonsillar cancer: Prognostic significance and clinically relevant markers

The association of high‐risk human papillomaviruses (HR HPVs) with tonsillar cancer (TC) has been documented. Because patients with HPV‐associated tumors show better survival rates, modification of their treatment regimen is being considered. It is therefore crucial to find markers for the identification of patients whose tumors are linked to viral infection. A cohort of 109 patients with primary TC was screened for HPV DNA presence in the tumor tissues and HPV‐specific antibodies in sera. Data regarding risk factors and clinical parameters were collected. Forty‐five specimens were analyzed for the expression of viral E6 and E2‐region mRNA, and the p16 and p53 protein expression status was assessed by immunohistochemistry. The overall prevalence of HPV DNA in TC tissues was 65.1%. Ninety‐three percent of HR HPV DNA‐positive samples expressed E6*I mRNA. E2‐region mRNA expression was detected in 36% of positive samples, which implies that the virus is integrated in 64% of HPV DNA/RNA‐positive tumors. p16 overexpression and the presence of antibodies specific to HPV16 E6/E7 oncoproteins correlated well with HPV DNA and RNA presence. The disease‐specific survival rate of patients with HPV DNA‐positive tumors was significantly higher than that of HPV DNA‐negative patients. In addition to providing further evidence of the involvement of HPV infection in the etiopathogenesis of a proportion of TC cases, our study demonstrates that p16 immunostaining and anti‐E6/E7 antibodies as surrogate markers of HPV involvement represent specific, sensitive and clinically accessible assays for the identification of TC patients who have a considerably better prognosis.

Human papillomavirus (HPV) profiles of vulvar lesions: possible implications for the classification of vulvar squamous cell carcinoma precursors and for the efficacy of prophylactic HPV vaccination.

The term vulvar intraepithelial neoplasia (VIN) introduced in 1986 incorporates 3 grades of usual VIN (u-VIN I-III) and the differentiated VIN (d-VIN). Although u-VIN is etiologically associated with the human papillomavirus (HPV) infection, d-VIN represents an alternative HPV negative pathway of vulvar carcinogenesis. In 2004, the u-VIN I category was abandoned and u-VIN II and III were merged. Further, an alternative Bethesda-like terminology scheme presenting the term vulvar intraepithelial lesion was proposed recently. To analyze the impact of HPV profiles of vulvar precancerous lesions for their classification and to assess the presumable efficacy of the prophylactic HPV vaccination, 269 vulvar excisions representing lichen sclerosus, lichen simplex chronicus, condylomata acuminata, d-VIN, all grades of u-VIN and squamous cell carcinomas were subjected to the HPV typing by use of GP5+/6+ polymerase chain reaction and reverse line blot hybridization. The results showed different HPV profiles, and also differing frequency of multiple-type HPV infection and the age structure in patients with u-VIN II and III. The biologic heterogeneity within the u-VIN II category was also demonstrated. u-VIN I was distinguished as a rare disorder associated with high-risk HPV infection. We conclude that the original VIN terminology proposed in 1986 seems to be appropriate for the classification of vulvar squamous dysplastic lesions. The spectrum of HPV types found in vulvar squamous cell carcinomas indicates that the efficacy of HPV vaccination in preventing vulvar cancer might be diminished in the studied population, because the recently developed prophylactic vaccines are targeted against a limited number of HPV types.

Characterization of humoral immune responses against p16, p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers

The cellular tumor suppressor p16 is strongly overexpressed in cervical cancers and precancers. We have previously demonstrated that infiltrating T lymphocytes reactive against p16 can be found in cervical cancer patients. Here, we analyzed whether p16 induces humoral immune responses. Sera of patients with cervical cancer, oropharyngeal cancer, colorectal cancer and autoimmune disease were included. A total of 919 sera were analyzed, including 486 matched sera from a cervical cancer case control study. p16 antibodies were analyzed in Western blot and a newly developed peptide ELISA covering the complete p16 protein. In addition, a Luminex‐based multiplex assay was used for simultaneous detection of antibodies directed against p16, p53, HPV16 E6 and HPV16 E7. In all entities, only low p16 antibody reactivity was observed. Epitope mapping revealed 2 predominant epitope regions of the p16 protein. No significant difference in p16 antibody frequency (OR = 0.9; 95% CI = 0.6–1.3) and p53 antibody frequency (OR = 0.6; 95% CI = 0.3–1.2) was found between patients and healthy controls in the cervical cancer case control study. Antibodies against the HPV16 oncoproteins E6 and E7 were detected more frequently in cervical cancer patients when compared with healthy controls (E6 OR = 27.8; 95% CI = 11.1–69.7, E7 OR = 5.7; 95% CI = 2.9–11.1). In conclusion, despite the strong expression of p16 and the observed induction of cellular immune responses, antibody reactivity against p16 was observed only at very low levels independent of the disease background.

Risk factors and survival by HPV-16 E6 and E7 antibody status in human papillomavirus positive head and neck cancer

High‐risk human papillomavirus types (HPV‐HR) are associated with head and neck cancer (HNC) risk and better survival. Most patients with HPV‐HR DNA‐positive tumors develop anti‐HPV E6/E7 antibodies; however, it is unclear whether those who mount an immune response have similar risk factors or clinical outcomes as those who do not. HPV‐16 DNA tumor‐positive HNC cases were evaluated for HPV‐16 E6 and E7 antibodies using a GST capture ELISA system. Among 57 HPV‐16 DNA tumor‐positive HNC cases, 67% were detected with HPV‐16 E6 and/or E7 antibodies. Male gender (76% vs. 42%, p = 0.02), younger age (63% vs. 16%, p = 0.001) but not tobacco or alcohol were associated with E6 and/or E7 seropositivity. Seropositivity was associated more often with late stage (76%), poor grade (65%), positive nodes (82%). and in the oropharynx (82%), Median disease‐specific and recurrence‐free survival were longer in E6 and/or E7 seropositive compared to E6/E7‐negative cases (2.2 years vs. 1.4 years, both outcomes), although results were not statistically significant. When examined jointly with p16 expression, E6 and/or E7‐positive/p16‐positive cases had better disease‐specific (2.1 years vs. 1.1 years, p = 0.06) and recurrence‐free (2.3 years vs. 1.1 years, p = 0.03) survival compared to E6‐/E7‐/p16‐ cases. These findings suggest there are 2 distinct HNC patient groups with HPV DNA‐positive tumors, distinguishable by E6 and/or E7 antibody status. Differences in antibody status are associated with distinct risk factors and clinical outcomes. This information can be available as a simple blood test at initial presentation, before the removal of tissue through biopsy or surgery.

Human papillomavirus serologic follow-up response and relationship to survival in head and neck cancer: a case-comparison study

BackgroundHuman papillomavirus high risk (HPV-HR) type 16 is a significant risk factor for head and neck cancers (HNC) independent of tobacco and alcohol. The purpose of this study was to determine whether antibody levels to the HPV-16 oncoproteins E6 and E7 measured in sera collected at baseline (BL) prior to treatment and at two post-treatment follow-up (FU) visits were associated with HNC risk factors or prognosis.MethodsPresence of antibodies to HPV-16 E6 and E7 was evaluated in 109 newly diagnosed HNC cases with BL and FU blood samples, using the enzyme-linked immunosorbent assay (ELISA).ResultsHPV-16 E6 and/or E7 seropositive HNC cases were associated with higher risk in younger patients (≤ 55 years), more sexual partners (≥ 10), oropharyngeal cancer, worse stage at diagnosis, poorer grade, and nodal involvement. Between BL and FU (median = 8.3 months), there were decreased antibody levels for seropositive E6 (73% vs. 27%, p = 0.02) and seropositive E7 patients (65% vs. 35%, p = 0.09) with 5% of BL E6 and 35% of BL E7 seropositive patients converting to negative status at FU. Overall mortality (OM) was significantly worse among BL E6 seronegative patients than among BL seropositive patients (40.2% vs.13.6%, p = 0.01). There were no disease specific (DS) deaths among BL E6 seropositive vs. 24% in BL E6 seronegative patients (p = 0.01). BL E7 seronegative patients also had higher mortality than BL seropositive patients (OM: 38.2% vs. 20.0%, p = 0.04; DS: 22.5% vs. 5.6%, p = 0.07).ConclusionThese findings are the first to follow post-treatment OD levels of HPV-16 E6 and E7 in HNC and suggest that these HPV antibodies may be potential prognostic markers of survival in HNC patients.

Human papillomavirus infection and tumours of the anal canal: correlation of histology, PCR detection in paraffin sections and serology†

Human papillomavirus infection is an important etiological factor in squamous cell carcinoma of the anus (SCCA). Different histological variants of anal carcinomas displaying squamous differentiation, previously classified as separate tumours, were recently reclassified as SCCA by the WHO. In our recent study the presence of HPV was detected by PCR in biopsy specimens of 42 different anal tumours, including SCCA and its histological variants (n=22), adenocarcinomas (n=5), tubulovillous adenomas (n=5) and anal condylomas (n=10). HR HPV16 (high risk – HR) was detected in 18 of SCCA specimens (81.8%). All histological variants, i.e. tumours with basaloid, squamous and mixed histological patterns, were represented among the HPV‐positive cancers. Four tumours (18.2%) were HPV negative. Low‐risk (LR) HPV types were not detected within the SCCA group. HPV16 was identified in one adenocarcinoma, while four cases were HPV negative. Two adenomas showed presence of HPV16; one showed simultaneous positivity for HPV33. The remaining three tumours were HPV negative. Seven anal condylomas (70%) were LR HPV 6 and/or 11 positive, while three were HPV negative. The presence of HR HPV types was not observed in anal condylomas. Our results provide further evidence in support of the etiological role of HR HPV infection in the development of SCCA regardless of its histological appearance.

Markers of HPV infection and survival in patients with head and neck tumors

The purpose of this study was to determine whether changes in human papillomavirus (HPV) DNA prevalence in oral rinses and/or HPV‐specific antibody levels in the sera of patients with oral/oropharyngeal cancer have prognostic significance. One hundred and forty‐two patients with oral/oropharyngeal tumors were enrolled. The presence of HPV DNA was assayed in tumor tissue and oral rinses and HPV‐specific antibodies were assessed in the sera. Oral rinses were collected before treatment and one year after the treatment. Sera were drawn before treatment, one month, and one year after the end of the treatment. Altogether, 59.2% of tumors were HPV positive. The presence of HPV DNA in the tumors correlated with HPV DNA positivity in oral rinses and with HPV‐specific antibodies in the sera. Out of 66 patients with HPV‐positive oral rinses at enrolment, 84.8% became negative at one‐year follow‐up, while most patients remained seropositive for HPV‐specific antigens. However, the mean titers of HPV16 E6 and/or E7 antibodies at follow‐up were significantly lower. Of 16 patients with recurrences at follow‐up (alive on second sampling), six were positive at enrolment for HPV16 E6 and/or E7 antibodies. In five of these, no decrease in antibody levels was observed. Titers of antibodies specific for HPV16 capsid antigens did not change during the follow‐up. Our data suggest that the detection of antibodies specific for the HPV 16 E6 and E7 oncoproteins may serve not only as a marker of HPV etiology, but also as a marker of recurrence and a prognostic indicator in patients with HPV‐positive tumors.