Human papillomavirus and infiltration of CD8- and Foxp3-positive immune cells in sinonasal inverted papillomas

Abstract

Background: Sinonasal inverted papilloma (IP) is a benign tumor with a high risk of local recurrence and a potential to malignify and Human papillomavirus (HPV) has been suggested an etiological factor. p16INK4a (p16) overexpression is considered a surrogate marker for HPV, but whether p16 and HPV correlate to IP is uncertain. Besides, a prognostic role of tumor infiltrating lymphocytes (TILs) are observed in many tumors, however their role in IP is sparsely studied.

Aims/objectives: We hence analyzed IPs for the presence and the prognostic role of HPV and p16 overexpression together with CD8+ and FoxP3+ TILs in a population-based study.

Material and methods: 98 IP patients diagnosed 2001–2010 were identified from the Swedish Cancer Registry and analyzed for HPV by PCR and p16, CD8 and FoxP3 was by immunohistochemistry.

Results: In total, 12.2% of the IPs were HPV-positive (nine HPV-11, two HPV-6 and one HPV-45). Patients with HPV-positive lesions were younger (p?=?.003) and tended to present with more dysplasia. No correlation was observed between TILs and prognosis.

Conclusions and significance: Our data suggests that patients with HPV-positive IPs present with different clinical characteristics, suggesting possibly different disease entities. Moreover, recurrences may occur >5?years, which should be considered in the follow-up.     

Human papillomavirus (HPV) is absent in branchial cleft cysts of the neck distinguishing them from HPV positive cystic metastasis

Background: Distinguishing branchial cleft cysts (BCCs) from cystic metastases of a human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (OPSCC) is challenging. Fine needle aspirates (FNAs) from cystic metastasis may be non-representative, while reactive squamous cells from BCC can be atypic. Based on cytology and with the support of HPV DNA positivity many centers treat cystic metastasis oncological and thus patients are spared neck dissection. To do so safely, one must investigate whether HPV DNA and p16^INK4a overexpression is found exclusively in cystic metastases and not in BCC.

Patients and methods: DNA was extracted from formalin fixed paraffin embedded (FFPE) surgically resected BCCs from 112 patients diagnosed 2007–2015 at Karolinska University Hospital and amplified by PCR. A multiplex bead-based assay used to detect 27 HPV-types and p16^INK4a expression was analyzed by immunohistochemistry (IHC).

Results: All 112 BCCs were HPV DNA negative, and of 105 BCCs possible to evaluate for p16^INK4a, none overexpressed p16^INK4a.

Conclusions: HPV DNA and p16^INK4a overexpression were absent in BCCs. Lack of HPV DNA and p16 protein overexpression in BCCs is helpful to discriminate benign BCCs from HPV⁺ OPSCC metastasis. HPV testing definitely has a role in the diagnostics of cystic masses of the neck.     

Correlation between HPV status at T and N sites of oropharyngeal squamous cell carcinomas

Objectives: Human papilloma virus (HPV) is known to be associated with oropharyngeal squamous cell carcinomas (OPSCC) and may potentially play a vital role in tumor metastasis. The purpose of this study was to correlate HPV status of cervical lymph node metastases with their respective primary OPSCC tumor.

Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from 34 patients with cervical lymph node metastases were analyzed with HPV 16 DNA polymerase chain reaction (PCR), p16 immunohistochemistry and HPV typing. The results were correlated with the HPV status and type found in the primary tumors of OPSCC.

Results: Comparing HPV DNA status with p16 we found that 21 primary tumors and lymph node metastases were HPV positive (61.8%) and seven primary tumors and lymph node metastases were HPV negative (20.6%). Six patient samples differed when correlating the primary tumor and lymph node metastasis (17.6%).

Conclusions: In this study, HPV status in OPSCCs and their cervical lymph node metastases correlated in the vast majority of cases. However, HPV detection methods may have certain limitations resulting in varying degree of non-correlation. This should be taken into account when stratifying treatment in regard to HPV status.     

The clinical impact of p16 status in fine-needle aspirates of cervical lymph node metastasis of head and neck squamous cell carcinomas

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.     

Clinical and molecular characteristics of HNSCC patients with brain metastases: a retrospective study

Among the metastasis patterns of head and neck squamous cell carcinoma (HNSCC), intracranial spread is a rare but dreaded event. To date only very few cases have been reported and clinical and molecular data are sparse. We screened our archives for HNSCC patients from 1992 to 2005 who were diagnosed with brain metastases (BM). For retrospective analysis, all clinico-pathological data including disease-free survival (DFS), local progression-free survival (LPFS), and overall survival (OS) were compiled. Additionally, we assessed the mutational status of the TP53 gene and the prevalence of HPV serotypes by PCR and Sanger sequencing. Immunohistochemistry was applied to detect p16INK4A expression levels as surrogate marker for HPV infection. The prevalence rate of BM in our cohort comprising 193 patients with advanced HNSCC was 5.7 %. Of 11 patients with BM, 3 were female and 9 were male. Seven of the primary tumors were of oropharyngeal origin (OPSCC). LPFS of the cohort was 11.8 months, DFS was 12.1 months and OS was 36.0 months. After the diagnosis of BM, survival was 10.5 months. Five tumors showed a mutation in the TP53 gene, while five of the seven OPSCC tumors had a positive HPV status displaying infection with serotype 16 in all cases. Compared with patients who harbored TP53wt/HPV-positive tumors, patients with TP53 mutations showed a poor prognosis. Compared with the whole cohort, the interval between diagnosis of the primary and the detection of BM was prolonged in the HPV-infected OPSCC subgroup (26.4 vs. 45.6 months). The prognosis of HNSCC patients with BM is poor. In our cohort, most tumors were OPSCC with the majority being HPV positive. Our study points toward a putatively unusual metastatic behavior of HPV-positive OPSCC.     

Tumor stromal desmoplasia and inflammatory response uniquely predict survival with and without stratification for HPV tumor infection in OPSCC patients

Abstract

Background: Oropharyngeal squamous cell carcinoma (OPSCC) positive for human papillomavirus (HPV) increases wolrd wide.

Aims/objectives: The objective for this study has been to evaluate tumor phenotypes and tumor host responses with respect to five-year disease-specific survival (DSS) in HPV(+) and HPV(?) patients.

Material and methods: Two hundred patients with OPSCC have been treated between 1992 and 2010. Histopathology slides from these patients have been morphologically evaluated in formalin-fixed, paraffin-embedded (FFPE) stained with hematoxylin–eosin (HE). From HE-stained sections tumor phenotype (keratinization, fraction of mature cancer cells and pattern of invasion) and tumor host responses (inflammation and stromal desmoplasia) were evaluated with respect to five years DSS.

Results: High tumor inflammatory response and low stromal desmoplasia had an independent effect predicting better five-year DSS among all patients and when analyzed separately in the HPV(?) and HPV(+) cohort of patients using a Cox regression survival analysis that also included standard clinical prognostic variables among OPSCC patients.

Conclusion: Tumor host responses, inflammation and stromal desmoplasia may become part of routine work-up in OPSCC patients due to prognostic value.

Significance: We present a method, accessible in most clinical locations and would give important additional information about prognosis in OPSCC patients.     

Visual gravitational vertical perception in peripheral vestibular hypofunction

Conclusion The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs).

Objective HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR).

Material and methods DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products.

Results Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyer's tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n=6; HPV33, n=1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).     

Usefulness of human papillomavirus detection in oral rinse as a biomarker of oropharyngeal cancer

Conclusion: The detection of human papillomavirus (HPV)-DNA in oral rinse with auto-nested GP5+/GP6?+?PCR is useful as a biomarker of oropharyngeal cancer.

Background: This study aimed to determine the usefulness of oral rinse to detect HPV-DNA as a biomarker of HPV-positive oropharyngeal cancer (OPC).

Patients and methods: One hundred and ten patients with various head and neck diseases, including 19 patients with OPC, were enrolled. Oral rinse and tonsillar swab were collected, and auto-nested GP5+/GP6?+?PCR for HPV-DNA was performed. For oropharyngeal cancer, p16 immunostaining was also conducted.

Results: The rate of HPV-DNA detection in both oral rinse and tonsillar swab was significantly higher in OPC compared with non-OPC upper respiratory tract cancer and non-cancer diseases. HPV-DNA was detected in oral rinse in nine out of 12 p16-positive OPC cases, while none of the p16-negative OPC cases demonstrated detectable HPV-DNA. All p16-positive cases were also positive for HPV-DNA in tumor tissue. Based on p16 immunostaining, the sensitivity and specificity of HPV-DNA detection in oral rinse were 75% and 100%, respectively. Among eight of nine evaluable OPC cases positive for HPV-DNA in oral rinse at diagnosis, HPV-DNA was undetectable in oral rinse in seven cases after treatment.     

Human papillomavirus co-infection and survival in oral and oropharyngeal squamous cell carcinoma: A study in 235 Brazilian patients

ObjectivesWhile unknown for oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinoma (OSCC), some studies assessing cervical carcinoma have shown that human papillomavirus (HPV) co-infection can be associated with its prognosis.MethodsThrough in situ hybridization (HPV and Epstein-Barr virus [EBV] probes) and immunohistochemistry (p16^INK4a, cyclin D1, p53, and Ki-67 antibodies), 126 OPSCC and 109 OSCC samples were assessed.ResultsAll patients were EBV-negative. OPSCC (25%) showed a significant association with HPV compared to OSCC (11%). Almost all HPV-associated cases were p16^INK4a-positive. Regarding OPSCC and OSCC, 23 and 7 cases were positive for high-risk HPV (HRHPV) only, 6 and 3 cases for low-risk HPV (LRHPV) only, and 3 and 2 cases for HRHPV/LRHPV, respectively. HPV-associated carcinomas showed a significantly higher proliferative index than HPV-unassociated carcinomas. Both carcinomas showed a similar overall survival rate, which was not affected by the HPV status. However, when comparing HPV-associated subgroups, patients with HRHPV/LRHPV-associated carcinomas showed worse survival.ConclusionLRHPV-associated and HRHPV/LRHPV-associated cases can also be detected when assessing OSCC and OPSCC. Further studies, especially in populations with a high prevalence of HPV-associated OPSCC, are necessary to understand the clinicopathological behavior of these neoplasm subgroups.     

Role of human papillomavirus in the development of head and neck squamous cell carcinomas

Objective To review the literature on the role of oncogenic human papillomaviruses (HPVs) in the carcinogenesis of the head and neck mucosa.

Material and Methods Molecular and epidemiological studies concerning the high-risk HPV types and their role in carcinogenesis in the head and neck region were screened.

Results Different studies revealed that: (i) 15–25% of head and neck squamous cell carcinomas (HNSCCs) are clonally associated with high risk HPV types (type 16); (ii) the oropharynx and particularly the tonsils are the most susceptible sites; (iii) patients with HPV-positive tumours present with more advanced stages of disease, are relatively younger, do not have extravagant tobacco and alcohol intake and seem to have a better survival; (iv) HPV-positive tumours are characterized by poor differentiation grade and a basaloid appearance; and (v) HPV-positive tumours exhibit integrated HPV DNA, wild-type p53, pRb downregulation and overexpression of p16^INK4A.

Conclusion Taken together, these data support the view that HPV-harbouring HNSCC can be considered a discrete tumour entity with, moreover, a favourable prognosis. Screening of patients, especially those with tonsillar cancers, for the presence of HPV may help to further optimize treatment protocols and to provide more accurate prognostic information.     

Multifocal human papillomavirus detection in palatine and pharyngeal tonsils

Background: Multifocal human papillomavirus (HPV) infection into the palatine and pharyngeal tonsils, which might be linked to a second primary tumor of HPV-positive oropharyngeal cancer (OPC), was investigated.

Patients and methods: One hundred and five patients with various head and neck diseases including 14 patients with OPC were enrolled in this study. Swabs from the palatine and pharyngeal tonsils were collected in each individual, and auto-nested GP5+/GP6+ PCR for HPV DNA was performed.

Results: HPV DNA was detected in the palatine tonsil or the pharyngeal tonsil in a small subset of upper respiratory tract cancer other than OPC (URTC) and non-cancer diseases. Furthermore, HPV DNA was detected in both the palatine and pharyngeal tonsils in the same individual in 2 of 48 (4%) URTC cases, and 1 of 43 (2%) non-cancer cases. On the other hand, p16-positive OPC cases demonstrated a higher HPV DNA detection rate (4 of 9, 44.4%) compared with other disease groups.

Conclusion: HPV DNA detection in both the palatine and pharyngeal tonsils in the same individual, especially in HPV-OPC, suggested the ability of HPV to infect tonsillar tissues of Waldeyer’s ring multifocally.     

Association of BMI-1 and p16 as prognostic factors for head and neck carcinomas

Conclusions BMI-1 is an upstream repressor of tumor suppressor p16 and their inverse expression patterns have been linked with patient survival in OPSCC. In this material only p16 remained a relevant prognostic marker in OPSCC.

Objectives HNSCC tumors carry variable phenotypes and clinical outcomes depending on their anatomical location. In OPSCC, expression of tumor suppressor p16 is used as a surrogate marker of HPV infection and has prognostic value. There are no good prognostic biomarkers for HNSCC tumors of other anatomical locations.

Aim To study the expression patterns of p16 and BMI-1 in not only oropharyngeal but also oral, hypopharyngeal, and laryngeal squamous cell carcinomas and to clarify their putative connections with clinical parameters, survival, and each other.

Method Hospital records on 130 patients (59 OPSCC, 18 OSCC, 20 HPSCC, and 33 LSCC) diagnosed between 1997–2008 at the Helsinki University Hospital, Finland, were reviewed. BMI-1 and p16 expressions were studied by immunohistochemistry.

Results Sixty-eight per cent of OPSCC expressed p16 and expression correlated with lower age, lower T- and higher N-category, and with improved OS and DFS. BMI-1 expression was most prevalent in OPSCC and LSCC, but had no clinical correlations. No correlation between p16 and BMI-1 expression was found.     

Humane Papillomavirusinfektionen bei Plattenepithelkarzinomen des Kopf- und Halsbereichs

There is increasing evidence worldwide that human papillomavirus is a major risk factor for head and neck cancer. Only few studies on this association have been performed in Germany to date. For the purposes of the present study, tumor specimens from 223 patients with squamous cell cancer of the oral cavity, oropharynx, hypopharynx and larynx were analyzed for HPV DNA and p16INK4a expression. The prevalence of HPV genotype 16 (HPV16) DNA in the study population was 17.5?%. Further high-risk HPV types were not detected. All HPV16-positive tumors showed intense p16INK4a expression. HPV16 prevalence was highest in tonsillar carcinoma (37.5?%) and lowest in laryngeal cancer (2.8?%). We observed a significantly higher incidence of cervical lymph node metastases in patients with HPV16-positive tonsillar carcinoma in comparison to HPV-negative tumors (p?<?0.016). Tobacco and/or alcohol consumption was significantly lower in patients with HPV-positive tumors (p?<?0.0001).     

Humane Papillomviren bei Plattenepithelkarzinomen der Kopf- und Halsregion

Human papilloma viruses (HPV) are responsible for approximately half of all oropharyngeal squamous cell carcinomas (OPSCC) and incidence rates of HPV-associated OPSCC continue to increase substantially. The defined viral carcinogenesis permits development of specific diagnostic, therapeutic, and prophylactic approaches. Laboratory identification of HPV-associated OPSCC may be achieved by p16^INK4a immunohistochemistry combined with HPV DNA detection by polymerase chain reaction (PCR) using tumor tissue. Patients with HPV-associated OPSCC have a relatively good prognosis; therefore, the HPV status plays an important role in patient guidance. Due to the relatively favorable prognosis, ongoing studies are evaluating whether less rigorous therapy for HPV-positive patients results in equally good cure rates. The criteria for patient selection are, however, still uncertain. Particularly markers for detection of HPV-positive patients with a high risk of treatment failure are lacking. Besides tumor stage and comorbidities, distinct genomic, epigenetic, and immunologic alterations are prognostically relevant for HPV-associated OPSCC, and might be of predictive value. Furthermore, the characteristic molecular alterations suggest the possibility of novel vigilant and specific therapy approaches. These may be inhibitors of the phosphatidylinositol 3?kinase (PI3K) pathway, which is frequently activated in HPV-associated OPSCC, and immunotherapeutic methods, e.?g., therapeutic vaccination. Although prophylactic HPV vaccinations may also prevent development of HPV-associated OPSCC, foreseeable effects on OPSCC incidence will be low, given the low vaccination rates in Germany. This highlights the fact that interdisciplinary research networks should enhance the necessary activities related to HPV-associated OPSCC.     

The role of human papillomavirus in head and neck cancer and the impact on radiotherapy outcome

Conclusion: HPV?+?HNSCC patients have improved Overall Survival (OS), Disease Specific Survival (DSS), Disease Free Survival (DFS), and Progression Free Survival (PFS). The radiotherapy treatment can’t improve the Survival of the HPV-negative HNSCC patients.

Objective: To investigate the role of Human papillomavirus in head and neck cancer and the impact on radiotherapy outcome.

Methods: A search in PubMed and Chinese CNKI (2000–2015) was performed. This meta-analysis was done using RevMan 5.1 software. Outcomes included OS, DSS, DFS, PFS, and Treatment responses rates (RR).

Results: A total of 2620 patients in 10 studies were included. The Positive detective rates of HPV and P16 are 32.5% (425/1309) and 42.5% (526/1239). OS and PFS were improved in HPV?+?patients compared to HPV???patients (HR?=?0.48; 95% CI?=?0.37–0.62, p?p?p?p?p?= 0.05).     

Prognostic significance of HPV status in the re-irradiation of recurrent and second primary cancers of the head and neck

Purpose

To evaluate the prognostic significance of human papillomavirus (HPV) status among patients treated by salvage radiation therapy for local-regional recurrences and second primary cancers of the head and neck arising in a previously irradiated field.

Comparison of psychosocial factors over time among HPV+ oropharyngeal cancer and tobacco-related oral cavity cancer patients

Introduction

The role of human papilloma virus (HPV) in the pathogenesis of oropharyngeal squamous cell carcinoma (OPSCC) is well documented, as is the excellent prognosis of patients with HPV-associated disease; in contrast, oral cavity squamous cell carcinoma (OCSCC) is associated with tobacco and alcohol use and has a worse prognosis. While causative factors, staging, and treatment guidelines differ between these cancer subsets, few studies have compared psychosocial factors in these groups.

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction

Background: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial.

Objectives: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16^INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated.

Materials and Methods: Tissue samples from 31 submandibular gland lesions were evaluated. p16^INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed.

Results: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16^INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16^INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers.

Conclusions: HPV-DNA positivity and p16^INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.     

Human papillomaviruses in lymph node neck metastases of head and neck cancers

CONCLUSION: The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs). OBJECTIVE: HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR). MATERIAL AND METHODS: DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products. RESULTS: Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyer's tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n = 6; HPV33, n = 1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).