Diagnosing HPV-Related Oropharyngeal Cancers: The Need to Speak a Common Language

Oral cavity squamous cell cancer (OSCC) and Oropharynx squamous cell carcinoma (OPSCC) are the most frequent forms of Head and Neck Cancers (HNCs) [1]. About 300,000 new cases of oral cancers are being counted yearly worldwide, having registered an increase of incidence of 225% in U.S.A. in the last 20 years, with about 50% of related deaths [2,3]. The relevant advances in treatments of OSCC during the last decades, allowed updated surgery techniques, robotic surgery, intensive induction chemotherapy and hyperfractionated radiotherapy to be currently applied to the advanced cases. Unfortunately, most of these therapies often carry severe acute and chronic side effects, heavily impacting on patients’ quality of life.


Short Communication
Oral cavity squamous cell cancer (OSCC) and Oropharynx squamous cell carcinoma (OPSCC) are the most frequent forms of Head and Neck Cancers (HNCs) [1]. About 300,000 new cases of oral cancers are being counted yearly worldwide, having registered an increase of incidence of 225% in U.S.A. in the last 20 years, with about 50% of related deaths [2,3]. The relevant advances in treatments of OSCC during the last decades, allowed updated surgery techniques, robotic surgery, intensive induction chemotherapy and hyperfractionated radiotherapy to be currently applied to the advanced cases. Unfortunately, most of these therapies often carry severe acute and chronic side effects, heavily impacting on patients' quality of life.
Very interestingly, OSCC is generally associated with a history of alcohol and tobacco abuse, whereas OPSCC is frequently related to a persistent infection by high-risk Human Papilloma Viruses (HR-HPV), mainly HPV16, and frequently arises in non-smokers, younger patients than OSCC.
The striking expansion of the incidence of the OPSCC HPV-related subgroup, in the last 30 years, has led to a unprecedented change in the epidemiology of HNCs, to the point that HPV-positive OPSCCs are now regarded as "the New Face of HNCs" [4], and are considered responsible of the peak of HNCs incidence currently observed in Western Countries, where tobacco abuse started to decline years ago (i.e., U.S.A.), due to the success of decades of anti-smoking campaigns [5].
The difference between HPV-positive and HPV-negative cancers bears a signifi cance that goes beyond the epidemiology.
An increasing amount of data indicates, in fact, that the HPVrelated OPSCC subgroup shows a lesser aggressive behavior and a more radio-and chemo-responsivity than alcohol and/ or smoking related OSCCs [6].
Several reasons may underlie the important differences in biological aggressiveness among HPV-positive and HPVnegative cancers.
Changes in the tumor microenvironment and immune surveillance are emerging as one of the major determinants of clinical behavior of these tumors, through the crosstalk of cancer-directed immune cells, hypoxia regulating genes, and chemokines production [7].
Hypoxia has been reported as a common event in OSCC, that experience important adaptive changes (i.e.anaerobic glycolysis, pH stabilization) mainly directed by the hypoxia inducible factor (HIF)-1, which acts early in tumor progression, contributing to the generation of an immune escape phenotype and poor clinical outcome of patients with advanced disease [8,9]. As anticipated by zur Hausen in at the beginning of this century [11], HPV leads to the evasion from host-cell control in early events in carcinogenesis, and actually we know that it actively contributes to the impairment of the immune response, negatively modulating the IFNgamma secretion and the HLA-mediated antigen presentation, allowing early immune escaping, during the fi rst steps of infection [12].
As well, many other factors and molecular pathway need to be fully investigated, before we could exactly know how and why HPV-positive OPSCC behaves in a more favorable way, and how we can maximize our efforts to completely eradicate these tumors, even when in advanced stage, without signifi cant side-effects for patients. In other terms, although most HPV-OPSCC show an innate better outcome and a good response to current treatment strategies, sometimes they can be associated with other major risk factors, this HPV-positive OPSCC with distinct, individual risk profi les [13].
More data are needed, on even greater series of cases, worldwide, before we reach defi nitive results.
In the meantime, we still lack a defi nite panel of prognostic markers able to unequivocally detect patients with poorer outcome to be treated with intensive therapeutic regimens [26], and to date, the only discriminant parameter available in the clinical-pathological practice, is the active presence of an HR-HPV (HPV16, in the major part of cases) on formalinfi xed and paraffi n-embedded tissue (FFPE) OPSCC tissue, which is actually emerging as a real prognostic and predictive biomarker.
This constitutes a real conundrum, in the real setting.
In fact, differently from cervical cancer, there are currently no defi nitive reliable and cost-effective diagnostic tests approved by the FDA for the unequivocal determination of HPV in Head and Neck Cancers. The various techniques currently in use for HPV detection, range from consensus and type-specifi c PCR methods, real-time PCR assays, DNA in-situ hybridization (ISH), and immunohistochemical detection of surrogate biomarkers (e.g., P16 INK4a protein) [27]. None of these methods offers optimal sensitivity and specifi city levels.
P16 immunohistochemistry has been judged as the best surrogate test to use for risk stratifi cation of OPSCC in a routine pathology laboratory setting [27]. However, this test may produce false positive and false negative results.
The gold standard of the testing methods is qRT-PCR [28]. that requires fresh frozen tissue for optimal results and is technically complex, factors that restrict its use only to research laboratories. Therefore, stepwise algorithms that combine different HPV tests have been proposed as a strategy to compensate for the limitations of individual tests.
As confi rmatory tests after a positive IHC for P16 INK4a protein, fl uorescence in situ hybridization (FISH) on DNA and/ or PCR are used to detect HPV in OPSCC [29].
These tests certainly add signifi cant information to IHC, as viral DNA is found in squamous cell carcinomas of the head and neck region, and particularly the oropharyngeal region, in a proportion of cases in continuous and progressive growth. This is fully in agreement with the knowledge that persistent infection with high risk HPV (HR-HPV) causes cellular immortalization and tumorigenesis of epithelial cells involved. However, to have biological and clinical relevance, HPV should be transcriptionally active, and all the above mentioned tests do not assess the viral transcript (i.e. the E6/E7messenger RiboNucleic Acid, mRNA), which currently represents the best effective indicator of HPV status of OPSCC [30].
The presence of a transcriptionally active HPV in cancer cells is considered the direct evidence of HPV-related oncogenesis: for this reason, a ISH RNA based HPV E6/E7 for transcripts is defi nitely the preferred method to identify the "actual" HPVrelated OPSCC [31]. Therefore, the gold standard for detecting oncogenic HPV is the demonstration of transcriptionally active high-risk HPV in tumor tissue. Whereas quantitative reverse transcription and polymerase chain reaction (qRT-PCR) requires the extraction of RNA, which destroys the tumor, making impossible the morphological diagnostic correlation, new techniques, as the RNAscope® assay, allow the direct, in situ visualization of RNA on FFPE, with sensitivity at single molecule and single cell resolution. The essay for HPV RNAscope® is designed to detect the mRNA of E6/E7 of high-risk HPV genotypes (e.g. HPV 16,18,31,33,35,52 and 58) using a pool of probes specifi c for these genotypes [32][33][34].
RNAscope® can be applied also on tissue microarrays (TMA), and shows a highly simplifi ed workfl ow, similar to the IHC standard protocols, preserving the morphology of the tissues and allowing the pathologist to perform histopathological correlations [34].
HPV testing by RNAscope® demonstrated 97% sensitivity and specifi city 93%, taking as reference the qRT-PCR method.
The conventional method chromogenic medium for HR-HPV DNA ISH is highly specifi c, but has a sensitivity of 80%. There is a strong need for a consensus on tests to use when determining whether a case of OPSCC is a "real" HPVrelated neoplasm, in view of the new strategies for treatment deintensifi cation for these patients that are already under clinical evaluation.
We strongly outline the urgency to move jointly, worldwide, at the pathology laboratory level, to defi ne and standardize the best molecular algorithm able to accurately evaluate the HPV status of OPSCC. Only by speaking all the same language, it will be possible to give the right information to every patient, allowing also the scientifi c community to share precious information, constituting open, common databases useful to the further comprehension of the molecular differences among HPV-positive and negative OPSCC.
A great amount of results has been collected in the last decade, for that concerning our knowledge of the oral HPVrelated cancerogenesis. Further investigations are undoubtedly needed, but we could be very close to make a snapshot of the real face of HPV-positive OPSCC.