Viral markers in tumour samples from 53 Italian cases of epithelial upper airways cancers

Background

Several histotypes of both epithelial and non-epithelial neoplasms belong to the heterogeneous family of the primary upper airways cancers (UAC), i.e., the overall set of the malignant neoplasms respectively arising from the nasopharynx (ICD-10 C11), the nasal cavities (ICD-10 C30), and the paranasal sinuses (ICD-10 C31). 

Both nasopharyngeal cancers (NPC) and sinonasal cancers (SNC) recognize complex and, at present, only partially defined webs of causation. 

Previous research suggests that a wide range of airborne viruses and chemicals is therein (with varying degrees of evidence) involved. 

A list of the agents for which sufficient or limited evidence of carcinogenicity in humans emerges is made available and constantly updated by IARC; the version based on the IARC Monographs from 1 through 140 shows that:

sufficient evidence sustains a causal relationship of Epstein-Barr Virus (EBV), formaldehyde, Chinese-style salted fish, and wood dust to the NPC;

limited evidence sustains a causal relationship of traditional Asian pickled vegetables to the NPC;   

sufficient evidence sustains a causal relationship of isopropyl alcohol manufacture using strong acids, leather dust, Nickel compounds, Radium-226 and its decay products, Radium-228 and its decay products, tobacco smoking, and wood dust to the SNC

limited evidence sustains a causal relationship of work in carpentry and joinery, hexavalent Chromium compounds, formaldehyde, and work in textile manufacturing industry to the SNC.¹

While NPC are very uncommon in most countries, including Italy, they are relatively common in some specific areas, such as Southern China and Southeastern Asia, relating to both alimentary factors and EBV infection.^2,3 

Several volatile N-nitrosamines are emitted by both Chinese-style salted fish and traditional Asian pickled vegetables.⁴ Thus, one or more volatile N-nitrosamines could be involved in the aetiology of the NPC in respect of some Asian foods.

In 2007, IARC dedicated the whole Monograph 90 to the Human Papilloma Virus (HPV), stating that multiple serotypes of the agent resulted as established or probable carcinogens to humans at multiple sites; however, the UAC were not evaluated in that context.⁵ Even later, no IARC evaluation of a possible specific relationship between the HPV and the UAC have been published.  

A IARC Monographs Meeting dedicated to the Hepatitis D Virus, the Human Cytomegalovirus (HCMV), and the Merkel cell polyomavirus took place in June 2025; HCMV was evaluated for the first time. The IARC study group, considering the figure of a positive association between HCMV and childhood acute lymphoblastic leukaemia, classified HCMV as possibly carcinogenic to humans (Group 2B); the hypothesis of a specific relationship between the virus and the UAC was not evaluated.⁶

In Italy, two specialized Cancer Registries, respectively dedicated to the SNC (a relevant fraction of which is due to occupational exposures) and to all malignancies recognising a so-called ‘lower occupational aetiological fraction’ (which include NPC) have been established by law in 2008. Both of them consider and analyse individual anamnestic data, collected by means of standardised questionnaires. At present, the Italian Cancer Registry dedicated to the SNC includes only the neoplasms of epithelial origin. 

In the perspective of launching future, registry-based studies on the role of airborne viruses and chemicals (particles and vapours) in the causation of the UAC, we present here the results from a preliminary case-series study, exploring the presence of EBV, HCMV, HPV16, and HPV18 markers in tumour tissues collected from patients with primary epithelial UAC diagnosed in an Italian hospital.

Methods

All the UAC diagnosed from 01.01.1991 through 31.12.2012 at the Histopathology and Cytopathology Unit of the local hospital in Macerata (Italy) were considered, in the first instance, as potentially eligible for the study; the non-epithelial primary UAC and the secondary UAC were subsequently excluded. 

The reason why only cases which had been diagnosed years earlier were considered was that we wanted to be sure to avoid consuming, in this merely exploring phase, any sample still suitable for clinical investigation in the interest of the individual patients. 

With this in mind, two possible limitations were therefore, consciously taken in account. 

First, the long time elapsed from the tissue collection to the analysis might have entailed some degradation of the viral genetic material, thus lowering the sensibility of the tests and raising the probability of false negative results. 

Second, most cases were diagnosed before the institution of the above-mentioned Cancer Registries, thus we could not use any anamnestic information in the present analysis.

Each sample was tested for EBV, HCMV, HPV16, and HPV18, performing two multiplex real-time PCR and two simplex real-time PCR.

The PCR tests are known for their high sensitivity and specificity, i.e., their capability to both accurately detect the presence of a specific target, namely a pathogen’s DNA or RNA, and to avoid a false identification of similar, but different, sequences; the latter is achieved by means of highly specific primers binding only to the target sequence, allowing the amplification of only that specific region of DNA or RNA.^7,8   

Results

The study included samples from 53 patients (44 males and 9 females) diagnosed with the following cancers:

• 11 squamous or undifferentiated carcinomas arising in the nasopharynx (10 male patients, 1 female patient);
• 1 poorly differentiated adenocarcinoma arising in the nasopharynx (1 male patient);
• 15 intestinal-type adenocarcinomas (ITAC) arising in the sinonasal cavities (14 male patients, 1 female patient);
• 15 adenocarcinomas of non-intestinal-type or not otherwise specified adenocarcinomas arising in the sinonasal cavities (13 male patients, 2 female patients), including 3 cystic adenoidal carcinomas (2 male patients, 1 female patient);
• 11 squamous or undifferentiated carcinomas arising in the sinonasal cavities (6 male patients, 5 female patients).

Positive results emerged from samples relating to 20 patients, as reported in table 1. 

Overall, the prevalence of positive tests for EBV, HCMV, and HPV18 was 24.5%, 5.5%, and 5.5%, respectively. 

EBV tested positive in 10 squamous or undifferentiated carcinomas (9 out of 11 arisen in the nasopharynx and 1 out of 11 arisen in the sinonasal tract) and in 4 adenocarcinomas (the only one arisen in the nasopharynx and 3 out 31 arisen in the sinonasal tract). 

HCMV tested positive in 1 out of 11 squamous or undifferentiated carcinomas arisen in the sinonasal tract and in 2 out of 31 adenocarcinomas arisen in the sinonasal tract.

HPV18 tested positive in 3 out of 31 adenocarcinomas arisen in the sinonasal tract.

All of the samples tested negative for HPV16.

Discussion

Caution is certainly warranted when interpreting these results, as the study design – a case-series – and the limited number of the studied patients affect the possibility to interpret them from an etiopathogenetic perspective.

It should be noted that viruses can infect the upper airways without exerting a carcinogenic action and that a virus may persist inside a transformed cell just as an associated ‘passenger’; the local persistence and long-lasting replication of a potentially carcinogenic virus is, moreover, a relevant condition, increasing the odds of an agent’s contribution to the transformative processes.⁹ 

On the other hand, positivity for the tested viruses could have been lost through a degradation of the viral DNA over the years. 

The lack of any positivity for HPV16 may be explained by an actual non-involvement of the agent in the pathogenesis of the UAC, but also hypothesizing that the agent enters at some early phase of the carcinogenetic processes, then fades during the following stages; the multifaceted contribution of HPV16 to the carcinogenesis has been discussed some years ago.¹⁰

With all these caveats, we think that some of the results are intriguing and could foster further aetiological research. 

The observed positivity for EBV in 4 adenocarcinomas, 1 arising in the nasopharynx (ICD10 C11), and 3 arising in the sinonasal cavities (ICD10 C30 + C31) could be of interest, as the association of EBV to both nasopharyngeal and sinonasal adenocarcinomas has been only rarely reported so far.^11,12

Moreover, it is noteworthy that 2 out of 3 samples which tested positive for HMCV referred to sinonasal adenocarcinomas; no previous reports have been found about an association between the agent and this kind of histotype, referred to the sinonasal tract as well in other sites. 

All the 3 samples which tested positive for HPV18 referred to sinonasal adenocarcinomas; an association between ‘High-Risk HPV’ and sinonasal adenocarcinoma was just recently reported.¹³

Future studies based on the two specialized Italian Cancer Registries respectively dedicated to the SNC and to all malignancies recognising a so-called ‘lower occupational aetiological fraction’ could be able to shed light on the complex aetiology of UAC, by integrating the best pathological evidence and the best systematic acquisition of standardized anamnestic data, meanwhile taking in consideration both the viral presence in the cancer tissues and the patients’ self-referred exposures to particles and vapours.

Conflicts of interest: none declared.

Financing: the purchase of all the necessary primers and probes used in the Real-Time PCR tests was financed by the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano (Italy), in the context of the research sub-project “Big data and deep learning in the surveillance of the occupational tumors (BEST)”, in turn supported by grants from the Italian National Insurance Institute (INAIL), Rome (Italy) inside its BRiC Project ID-56 2023-25. No other source of funding was used.  

Author contributions: conceptualization and supervision, RC and MP; methodology, validation, formal analysis, writing – original draft preparation, writing – review and editing, visualization, RC, MP, and SM; investigation, MP, SB, LC, and FC; resources and data curation, RC, MP, and SM; project administration, RC. All authors have read and agreed to the published version of the manuscript.

Ethical considerations: this study, as it used anonymized secondary data without any direct interaction with human subjects, has been classified at minimal risk and not requiring approval from an ethics review board.  

Informed consent statement: patient consent was waived due the considerations that the analysed samples referred to old and already completely diagnosed cases, and that the study results could in no way affect either the therapeutic choices or the prognosis, so that no patients interest subsists regarding either the treatment of the samples or the study results. 

Data availability statement: a dataset containing copies of all the original histological reports is available at AST Macerata, Occupational Safety and Health, Occupational Epidemiology Unit, Civitanova Marche (Italy); a dataset containing the original reports of all the analytic results is available at IZSUM - Istituto Zooprofilattico Sperimentale Umbria e Marche “Togo Rosati” in Fermo (Italy).

Acknowledgments: we are very grateful to Dr. Gerardo Ferrara and Mrs. Roberta Alidori, respectively, director and supervisor of the Unit of Pathological Histology and Cytology of the Macerata General Hospital (Italy), for their kindness and precious collaboration in providing all the cancer tissues samples. 

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