A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen

Abstract

Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high-throughput screening of frozen and formalin-fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT-PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT-PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16-positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

P21Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis

“…To assess a firm association between virus and tumor cells, namely, it is recommended to carry out additional tests, such as p16 INK4A immunostaining Cell cycle protein expression and HPV in tonsillar carcinoma HC Hafkamp et al and/or FISH, which has been applied in our study. 55,56 In the univariate and multivariate statistical analyses, we found that p21 Cip1/WAF1 overexpression was a highly significant indicator of favorable prognosis in tonsillar squamous cell carcinomas independent of HPV status. Expression of p21 Cip1/WAF1 has also been associated with a favorable survival in patients with tongue squamous cell carcinomas, and in patients with ovarian, superficial bladder, gastric, colorectal, and esophageal cancers.…”

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…This high clinical sensitivity most probably is due to targeting ultra-short RNA sequences of only 65-75 base pairs specifically amplified after random cDNA generation as first used for HPV16 by Smeets and coworkers. 29 This high sensitivity of HPV RNA detection in FFPE tissues raises the possibility to explore the value of these RNA assays even for primary HPV infection diagnosis in FFPE tissues with poor nucleic acid conservation. Assay specificity for both HPV type and RNA is enhanced by the combination of type-specific and RNA-specific oligonucleotides, the two PCR primers and the splice-site specific probe, the latter excluding false-positive detection of amplified genomic viral DNA sequences.…”

“…28 Recently, an ultra-short amplicon, E6*I mRNA RT-PCR assay has been described for HPV16. 29 Advantages of such an assay are applicability to FFPE material and absolute RNA specificity by using a splice-site as identification target.…”

Time trends in the prevalence of HPV in oropharyngeal squamous cell carcinomas in northern Spain (1990–2009)

“…No single detection method seems to give reliable results on FFPE. Smeets et al 16 proposed a combination of p16 IHC followed by HPV-DNA GP51= 61 PCR on the p16-positive cases, which has recently been validated by comparing E6=E7 RT-PCR and p16 IHC=HPV-DNA PCR on both frozen and FFPE tissue samples of 82 patients, showing a concordance of 98%. 11 Other studies advocated a triple method using p16 IHC, HPV-DNA PCR and HPV ISH 17 or p16 IHC and histological evaluation of basaloid differentiation.…”