Dana Holzinger

Viral RNA patterns and high viral load reliably define oropharynx carcinomas with active HPV16 involvement.

Oropharyngeal squamous cell carcinomas (OPSCC) that are associated with human papilloma virus (HPV) infection carry a more favorable prognosis than those that are HPV-negative. However, it remains unclear which biomarker(s) can reliably determine which OPSCC specimens are truly driven by HPV infection. In this study, we analyzed 199 fresh-frozen OPSCC specimens for HPV DNA, viral load, RNA expression patterns typical for cervical carcinomas (CxCaRNA(+)), and the HPV-targeted tumor suppressor protein p16(INK4a) as markers for HPV infection. In this set of specimens, there was a 49% prevalence of DNA for the cancer-associated HPV type 16 (HPV(+)). However, there was only a 16% prevalence of high viral load and only a 20% prevalence of CxCaRNA(+), a marker of HPV16 carcinogenic activity. Among the CxCaRNA(+) tumors, 78% of the specimens exhibited overexpression of p16(INK4a), which also occurred in 14% of the HPV-negative tumors. Using a multivariate survival analysis with HPV negativity as the reference group, CxCaRNA(+) as a single marker conferred the lowest risk of death [HR = 0.28, 95% confidence interval (CI), 0.13-0.61] from oropharyngeal cancer, closely followed by high viral load (HR = 0.32, 95% CI, 0.14-0.73). In contrast, a weaker inverse association was found for OPSCC that were HPV(+) and p16(INK4a) high (HR = 0.55, 95% CI, 0.29-1.08). In summary, our findings argued that viral load or RNA pattern analysis is better suited than p16(INK4a) expression to identify HPV16-driven tumors in OPSCC patient populations.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma

Background:Human papillomavirus (HPV) is a causal factor in virtually all cervical and a subset of oropharyngeal squamous cell carcinoma (OP-SCC), whereas its role in laryngeal squamous cell carcinoma (L-SCC) is unclear.Methods:Formalin-fixed paraffin-embedded (N=154) and deep-frozen tissues (N=55) of 102 L-SCC patients were analysed for the presence of 51 mucosal HPV types. HPV DNA-positive (HPV DNA+) cases were analysed for E6*I mRNA transcripts of all high risk (HR)/probably/possibly (p)HR-HPV identified, and for HPV type 16 (HPV16) viral load. Expression of p16INK4a, pRb, cyclin D1 and p53 was analysed by immunohistochemistry.Results:Ninety-two patients were valid in DNA analysis, of which 32 (35%) had at least one HPV DNA+ sample. Among the 29 single infections, 22 (76%) were HPV16, 2 (7%) HPV56 and 1 each (4%) HPV45, HPV53, HPV70, HPV11 and HPV42. Three cases harboured HPV16 with HPV33 (twice) or HPV45. Only 32% of HPV DNA+ findings were reproducible. Among HPV16 DNA+ L-SCC, 2 out of 23 (9%) had high viral loads, 5 out of 25 (21%) expressed E6*I mRNA and 3 out of 21 (14%) showed high p16INK4a and low pRb expression (all three HPV16 RNA-positive), immunohistochemical marker combination not identified in any other HPV DNA+ or HPV DNA-negative (HPV DNA−) L-SCC, respectively.Conclusion:HPV type 16 has a causative role in a small subgroup of L-SCC (<5% in this German hospital series).

Identification of oropharyngeal squamous cell carcinomas with active HPV16 involvement by immunohistochemical analysis of the retinoblastoma protein pathway.

Viral oncogene RNA expression is regarded as reliable biomarker to identify oropharyngeal squamous cell carcinomas (OPSCC) with active HPV16 involvement. This study addressed whether the expression profile of the cellular proteins p16INK4a, pRb, Cyclin D1 and p53 provide surrogate marker combinations that identify OPSCC with active HPV16 in situations where only formalin‐fixed biopsies are available. Protein expression was analyzed by immunohistochemistry on tissue microarrays created from 188 OPSCC of which the HPV16 DNA and RNA status had been established previously from snap‐frozen biopsies. Associations of single markers and of marker combinations with HPV16 DNA, viral RNA expression patterns and overall survival as primary end point were evaluated by statistical analysis. Most tumors with active HPV16 involvement (RNA+ group; n = 40) showed a specific protein pattern, that is, high p16INK4a (80%), low pRb (85%), low Cyclin D1 (95%) and normal p53 (73%). This pattern was significantly different from the pattern observed in HPV DNA‐negative tumors (HPV– group) and in HPV16 DNA‐positive tumors lacking viral RNA expression patterns (RNA– group). The combination of high p16INK4a and low pRb levels was distinctly superior to p16INK4a alone; it was strongly associated with RNA+ tumors (OR 41.4, 95%CI 10.7–162.5), with improved survival (HR 0.37, 95%CI 0.2–0.8) and had best predictive values (78% sensitivity, 93% specificity, 78% PPV, 93% NPV). In conclusion, if only formalin‐fixed biopsy material is available, the marker combination high p16INK4a/low pRb is well suited to identify OPSCC with biologically active HPV16 which represent a distinct OPSCC entity with improved prognosis.

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer

Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA‐) are similar to HPV‐negative (DNA‐) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response‐related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA‐ tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53‐mutation status for patient stratification and identify associations of an immune response‐related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

New concepts for translational head and neck oncology: lessons from HPV-related oropharyngeal squamous cell carcinomas

Human papillomavirus (HPV) infection is well established as an etiological agent responsible for a number of pathologies affecting the stratified epithelia of skin and anogenital sites. More recently, the infection by (mucosal) high-risk HPV types has also been found to be causally associated with squamous cell carcinoma in the head and neck region (HNSCC), especially in the oropharynx. Intriguingly, HPV-related oropharyngeal squamous cell carcinomas (OPSCC) represent a distinct clinical entity compared to HPV-negative tumors with particular regard to treatment–response and survival outcome. The association between HPV infection and OPSCC may therefore have important implications for the prevention and/or treatment of OPSCC. The improved survival of patients with HPV-related tumors also raises the question, as to whether a better understanding of the underlying differences may help to identify new therapeutic concepts that could be used in targeted therapy for HPV-negative and improved therapy for HPV-positive cancers. This review summarizes the most recent advances in our understanding of the molecular principles of HPV-related OPSCC, mainly based on functional genomic approaches, but also emphasizes the significant role played by the tumor microenvironment, especially the immune system, for improved clinical outcome and differential sensitivity of HPV-related tumors to current treatment options.

Proteomic analysis of field cancerization in pharynx and oesophagus: a prospective pilot study

‘Field cancerization’ in head and neck squamous cell carcinoma (HNSCC) is poorly understood and it may extend from the pharynx into the oesophagus. Both local recurrences and second primary carcinomas/second field tumours may originate from field cancerization. Our prospective pilot study aimed at the identification of patients suffering from field cancerization on the basis of mucosal protein profiles. Five mucosal biopsies from the oropharynx, hypopharynx and from three regions of the oesophagus were taken from 24 patients. Protein profiles were generated from the mucosal biopsies. After classifier learning, using the profiles of the patients without tumour diagnosis (n = 9), we were able to discriminate between the different mucosal sites and between healthy mucosa and HNSCC using tumour and healthy tissue samples. Mucosal biopsies of tumour patients (n = 15) revealed changes in the protein profiles similar to those in the tumours. During 42 months median follow‐up, six tumour patients experienced local recurrences and second field tumours, of which three occurred in the oesophagus. In all six cases, tumour relapse was correctly predicted by altered mucosal protein profiles (p = 0.007, Fishers exact test, two‐tailed). Consequently, molecular field cancerization had a strong impact on progression‐free survival (p = 0.007, log‐rank test). Protein profiles of small diagnostic biopsies hold great promise to improve personalized risk assessment in HNSCC. Larger studies are needed to further substantiate these findings. Copyright

From HPV-positive towards HPV-driven oropharyngeal squamous cell carcinomas

The incidence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), which is both biologically and clinically distinct from tobacco- and alcohol-related OPSCC, is dramatically increasing. The finding that individuals with HPV-positive local/regionally advanced OPSCC have a significantly better prognosis than their negative counterparts have led to efforts to de-escalate treatment in those patients to avoid serious side effects and to improve their long-term quality of life, while maintaining treatment efficacy. Identifying diagnostic tests that are able to distinguish cancers etiologically associated with HPV is thus becoming a pressing challenge for researchers. The purpose of this review is to provide an overview of the diagnostic tools presently available to evaluate HPV status in patients with OPSCC and, in particular, to discuss their strengths and weaknesses in identifying those infections that are the real driving force in the oropharyngeal carcinogenesis process.

Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma

To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16‐driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh‐frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV‐driven in the presence of HPV16 DNA and HPV16 transformation‐specific RNA transcript patterns (E6*I, E1∧E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16‐driven. HPV16 E6 seropositivity was the best predictor of HPV16‐driven OPSCC (diagnostic accuracy 97% [95%CI 92–99%], Cohens kappa 0.93 [95%CI 0.8–1.0]). Of the 66 HPV‐driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non‐HPV‐driven OPSCC, resulting in a sensitivity of 96% (95%CI 88–98) and a specificity of 98% (95%CI 90–100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16‐driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19–81), but was highly specific (100%, 95%CI 96–100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16‐driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16‐driven HNSCC outside the oropharynx.

Mucosal Alpha-Papillomaviruses are not associated with Esophageal Squamous Cell Carcinomas: Lack of Mechanistic Evidence from South Africa, China and Iran and from a World-Wide Meta-Analysis.

Epidemiological and mechanistic evidence on the causative role of human papillomaviruses (HPV) in esophageal squamous cell carcinoma (ESCC) is unclear. We retrieved alcohol‐ and formalin‐fixed paraffin‐embedded ESCC tissues from 133 patients seropositive for antibodies against HPV early proteins, from high‐incidence ESCC regions: South Africa, China and Iran. With rigorous care to prevent nucleic acid contamination, we analyzed these tissues for the presence of 51 mucosotropic human alpha‐papillomaviruses by two sensitive, broad‐spectrum genotyping methods, and for the markers of HPV‐transformed phenotype: (i) HPV16/18 viral loads by quantitative real‐time PCR, (ii) type‐specific viral mRNA by E6*I/E6 full‐length RT‐PCR assays and (iii) expression of cellular protein p16INK4a. Of 118 analyzable ESCC tissues, 10 (8%) were positive for DNA of HPV types: 16 (4 tumors); 33, 35, 45 (1 tumor each); 11 (2 tumors) and 16, 70 double infection (1 tumor). Inconsistent HPV DNA+ findings by two genotyping methods and negativity in qPCR indicated very low viral loads. A single HPV16 DNA+ tumor additionally harbored HPV16 E6*I mRNA but was p16INK4a negative (HPV16 E1 seropositive patient). Another HPV16 DNA+ tumor from an HPV16 E6 seropositive patient showed p16INK4a upregulation but no HPV16 mRNA. In the tumor tissues of these serologically preselected ESCC patients, we did not find consistent presence of HPV DNA, HPV mRNA or p16INK4a upregulation. These results were supported by a meta‐analysis of 14 other similar studies regarding HPV‐transformation of ESCC. Our study does not support the etiological role of the 51 analyzed mucosotropic HPV types in the ESCC carcinogenesis.