Soenke J. Weissenborn

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Genetic Signatures of HPV-related and Unrelated Oropharyngeal Carcinoma and Their Prognostic Implications

Purpose: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. Experimental Design: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16INK4A immunostaining. The results were correlated with HPV status and clinical data from patients. Results: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P = 0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P = 0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P = 0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P = 0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P = 0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P = 0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P = 0.008; disease-free survival, P = 0.01) and none of these patients had a tumor recurrence. Conclusions: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Abstract. Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyers tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site (P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per β-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading (P=0.008) and alcohol consumption (P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.

p16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Klingenberg B, Hafkamp H C, Haesevoets A, Manni J J, Slootweg P J, Weissenborn S J, Klussmann J P & Speel E‐J M
(2010) Histopathology 56, 957–967
p16INK4Aoverexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Penile Intraepithelial Neoplasia Is Frequent in HIV-Positive Men with Anal Dysplasia

Anogenital human papillomavirus (HPV)-infection is common in HIV-infected men who have sex with men (HIV+MSM). These patients have a strongly increased risk of HPV-induced anal cancer and its precursor lesion, anal intraepithelial neoplasia (AIN), and a moderately increased risk for penile cancer. Only limited data exist on penile intraepithelial neoplasia (PIN) in HIV+MSM. We determined the prevalence and evaluated the virologic characteristics of PIN and AIN in 263 HIV+MSM. In case of histologically confirmed PIN (and AIN), HPV-typing, HPV-DNA load determination, and immunohistochemical staining for p16(INK4a) were performed. PIN was detected in 11 (4.2%) and AIN in 156 (59.3%) patients. Ten PIN patients also had AIN within the observation period. Four clinical types of PINs could be distinguished. High-risk-alpha-HPV-DNA was found in 10 PIN lesions, with HPV16 being the most frequent type. Infections with multiple HPV-types were common. All high-grade lesions had high-risk-HPV-DNA-loads > or = 1 HPV-copy/beta-globin-gene-copy. Cutaneous beta-HPVs were found in PIN and AIN, but beta-HPV-DNA loads were very low, irrespective of the histological grade. p16(INK4a) Expression was detectable in all PIN lesions and correlated both with the histological grade and with high-risk HPV-DNA loads. In view of the PIN prevalence found in our study, all HIV+MSM should be screened for PIN in addition to AIN screening.

Expression of p16 protein is associated with human papillomavirus status in tonsillar carcinomas and has implications on survival.

Our recent analysis of papillomavirus (HPV) DNA in different malignant head and neck tumors revealed that HPV infections occurred most frequently in tonsillar carcinomas (58%) and that 84% of positive cases contained the highly oncogenic HPV type 16. We could also present data in favor of the hypothesis that in view of their clinical behavior and the involved risk factors HPV-positive and HPV-negative tonsillar carcinomas may represent two separate tumor entities. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection/typing we analyzed p16 protein expression in 34 tonsillar carcinomas for their correlation with HPV status. p16 is an inhibitor of cyclin-dependent kinases 4 and 6 which activate the negative cell cycle regulator protein pRB which in turn downregulates p16 expression. It could be shown that in neoplastic cells of the cervix uteri E7 protein of the high-risk HPVs can interfere with this regulatory circuit by its virtue to inactivate pRB and thus lead to the overexpession of p16. We found 53% of the tested tonsillar carcinomas to be HPV positive. 56% of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only 1 of the HPV-negative carcinomas showed focal p16 staining (p < 0.001). Clinical outcome analysis revealed a significant correlation of p16 expression with increased disease-free survival (p = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV DNA detection.

Human Papillomavirus Load in Eyebrow Hair Follicles and Risk of Cutaneous Squamous Cell Carcinoma

Background: Beta-human papillomavirus (betaPV) may play a role in the development of cutaneous squamous cell carcinoma (SCC). However betaPV is highly prevalent, and it may only be people with a higher viral load who have increased risk of SCCs. We therefore examined the association between betaPV load and SCCs. Methods: We recruited 448 immunocompetent cases with SCCs and 464 controls from Italy and Australia and 497 immunosuppressed organ transplant recipients (OTR; 179 cases and 318 controls) from Europe. We used reverse hybridization to genotype 25 betaPV types in eyebrow hair follicles and determined the viral load for eight selected types using quantitative PCR. We used logistic regression to assess associations between type-specific and cumulative viral load and SCCs. Results: Australian and OTR participants in the highest cumulative load tertile were at significantly higher risk of SCCs than those in the lowest tertile. Those with more than four betaPV types in the high load tertile were at approximately three-fold increased risk of SCCs. In Australia, HPV23 and 36 loads were significantly associated with SCCs, with borderline associations for HPV5 and 38. In OTR, HPV8 and 38 loads were significantly associated and HPV20 and 36 were borderline. We found little evidence for an association between load and SCCs in Italy. Conclusions: High viral load may be associated with risk of cutaneous SCCs, with total load seemingly more important than the load of any specific type. Impact: Our findings lend weight to the hypothesis that HPV plays a role in skin carcinogenesis. Cancer Epidemiol Biomarkers Prev; 22(4); 719–27. ©2013 AACR.

No indications for HPV involvement in the hypertrophic skin lesions of a Darier disease case without ATP2A2 gene mutations

Darier disease (DD) is a relatively common genodermatosis characterized by impaired differentiation and abnormal cell‐to‐cell adhesion. Haploinsufficiency of the ATP2A2 gene product, sarco/endoplasmic reticulum Ca2+ ATPase isoform 2 (SERCA2), is the underlying cause of most cases. Although DD may have a papillomatous appearance, few and controversial results have been reported about the role of human papillomavirus (HPV) in this disease. The aim of this study was to determine a possible correlation between development of hypertrophic lesions in DD and infection by HPV. We report the case of an 84‐year‐old woman with a hypertrophic DD variant that has been successfully treated with oral retinoids. HPV analysis for a broad spectrum of cutaneous and mucocutaneous genotypes was performed on surgical specimens obtained from the cutaneous lesions and snap‐frozen plucked eyebrows. Genetic analysis of the ATP2A2 gene did not detect any mutations. Epidermal expression of SERCA2b was shown by immunohistochemistry. We describe a patient with DD lacking mutations of the ATP2A2 gene, but with reduced SERCA2b expression in the epidermal keratinocytes. The results obtained by polymerase chain reaction (PCR) genotyping, quantitative real‐time PCR, and in situ hybridization indicate that HPV replication was very low and suggest no direct role of the virus in the development of the disease.

Detection of a Novel 1905C→T Mutation within the Dihydropyrimidine Dehydrogenase Gene and Potential for Misclassification with the Exon 14-skipping Mutation

Dihydropyrimidine dehydrogenase (DPYD) is the initial and rate-limiting enzyme in the metabolism of the chemotherapeutic drug 5-fluorouracil (5-FU), thus affecting its pharmacokinetics, efficacy, and toxicity (1). Application of 5-FU is restricted by a narrow therapeutic index because of severe toxicity of WHO grades III–IV (2). Polymorphisms within the DPYD gene have been reported, with deficiency in enzyme activity leading to severe 5-FU-related toxicity in cancer patients (3). The so-called exon 14-skipping mutation at the 5′-splice donor site of exon 14 (1905 + 1G→A) has been detected in ∼25% of affected patients (4). To identify patients at increased risk for severe 5-FU-induced toxicity, many medical centers routinely screen for the exon 14-skipping mutation before starting chemotherapy. We directly compared DNA sequencing with …