Abstract
ObjectiveTo compare six HPV detection methods in pre-treatment FFPE tumour samples from patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who received postoperative (N = 175) or primary (N = 90) radiochemotherapy. Materials and methodsHPV analyses included detection of (i) HPV16 E6/E7 RNA, (ii) HPV16 DNA (PCR-based arrays, A-PCR), (iii) HPV DNA (GP5+/GP6+ qPCR, (GP-PCR)), (iv) p16 (immunohistochemistry, p16 IHC), (v) combining p16 IHC and the A-PCR result and (vi) combining p16 IHC and the GP-PCR result. Differences between HPV positive and negative subgroups were evaluated for the primary endpoint loco-regional control (LRC) using Cox regression. ResultsCorrelation between the HPV detection methods was high (chi-squared test, p < 0.001). While p16 IHC analysis resulted in several false positive classifications, A-PCR, GP-PCR and the combination of p16 IHC and A-PCR or GP-PCR led to results comparable to RNA analysis. In both cohorts, Cox regression analyses revealed significantly prolonged LRC for patients with HPV positive tumours irrespective of the detection method. ConclusionsThe most stringent classification was obtained by detection of HPV16 RNA, or combining p16 IHC with A-PCR or GP-PCR. This approach revealed the lowest rate of recurrence in patients with tumours classified as HPV positive and therefore appears most suited for patient stratification in HPV-based clinical studies.
Highlights
By using the human papilloma virus (HPV) DNA polymerase-chain reaction (PCR)-based array method (A-PCR), HPV DNA was detected in 59 cases including 57 HPV16 DNA (32.6%) cases as well as 1 HPV18 DNA and 1 HPV33 DNA positive case, whereas the HPV DNA GP5+/GP6+ qPCR method (GP-PCR) revealed HPV positivity in 51 cases (29.1%)
Multivariable regression revealed that HPV status is an important prognostic factor independent of tumour localization and ECE status on the postoperative cohort and of N stage and tumour volume on the primary cohort. This is the first study investigating the relative power of different HPV biomarkers for the outcome of postoperative or primary radio(chemo)therapy using patient cohorts that followed narrow inclusion criteria and were treated with state-of-the-art contemporary treatment schedules. This kind of methodological comparison is urgently needed, because there are several ongoing prospective trials which aim at personalizing radio(chemo)therapy in patients with oropharyngeal squamous cell carcinomas (OPSCC) based on the HPV status and employ different means of detection
In the subgroup of patients with OPSCC, 80% of p16 positive tumours were positive for HPV16 DNA, whereas other tumour locations revealed HPV16 DNA positivity in only 50% of the p16 positive cases, showing that the technology may not be suited for all tumour locations
Summary
Since fresh or frozen tumour material is not routinely available, and mRNA isolation from formalin-fixed and paraffin-embedded (FFPE) tissues and the subsequent PCR are technically challenging [23], detection of p16 over-expression by IHC is being used as a surrogate marker for HPV infection alone or in combination with assessment of HPV DNA using different PCR-based approaches in a number of clinical studies and in clinical routine (reviewed in [23,24]) It has not been systematically analysed which of the methods is the most reliable one for patient stratification with particular focus on the response to radio(chemo)therapy, i.e. in a field where an increasing number of trials are currently undertaken and critically require precise means of HPV status detection. The overall aim of this study was to compare six methods, which are available for HPV status analysis in pretreatment FFPE samples of HNSCC and to evaluate the results in connection with the clinical outcome of radio(chemo)therapy
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