HPV infection has been shown to cause oropharyngeal cancer. However, the clinical significance of HPV infection in head and neck squamous cell cancer other than oropharygeal cancer is yet to be determined. The prevalence of laryngeal and hypopharyngeal squamous cell carcinomas that are HPV positive varies from 0% [2, 15, 16] to 75% . This difference in the prevalence of HPV in laryngeal cancer has been explained by the sensitivity of diagnostic techniques, ethnic and geographical differences in patients, small study samples, low quality of the specimens, and differences in methods of sample storage and lesion localization [7, 11].
PCR and ISH are widely used HPV detection systems. ISH is commonly used to detect HPV in clinical biopsy specimens. Some studies report that PCR-based methods are more sensitive than ISH. Besides, PCR-based methods can yield false positive results, because they may not differentiate biologically irrelevant HPV from clinically significant HPV, whereas ISH correlates with biologically and transcriptionally active HPV, differentiating between clinically significant and non-tumorigenic HPV DNA [18,19,20,21]. HPV DNA PCR amplification only shows the existence, whereas ISH can show the integration of the viral DNA into the host genome [12, 22].
In the current study we used HR-HPV chromogenic ISH for detection of HR-HPV; however, none of the 82 laryngeal specimens of squamous cell carcinoma and none of 11 laryngeal specimens of normal laryngeal mucosa exhibited a positive cISH staining pattern. Although the reported prevalence of HPV positive squamous cell cancer of the larynx and hypopharynx varies widely, the literature generally indicates that the prevalence is higher than that observed in the present study, but some studies have also reported no HPV positivity or a very low prevalence of HPV positivity [2, 15, 23,24,25]. The study by Castellsague et al. including 1042 laryngeal cancer patients from 29 countries tested the specimens with PCR and a DEIA for the presence of HPV-DNA and samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16INK4a, pRb, p53, and Cyclin D1 immunohistochemistry . This study, the largest exploring HPV attribution in head and neck cancers also found a small percentage of HPV positive laryngeal cancer cases. Some studies report that PCR-based methods are more sensitive than ISH and that PCR-based methods can yield false positive results [15, 18,19,20,21]. Such false positivity was avoided in the present study via use of cISH, which could be one of the reasons why HPV positivity was not observed in any of the present study’s laryngeal specimens. Gallo et al.  examined the role of HPV virus in laryngeal cancer using PCR and taking all necessary precautions to avoid false positive, as well as false negative findings. They reported that none of the 40 cases of squamous cell carcinoma showed presence of HPV genome, which supports the notion that earlier reports of high prevalence of HPV positivity might have been due to false positive results, and that many the majority of the laryngeal cancers are not related to HPV infection, as the current findings indicate.
The effect of tobacco use on laryngeal cancer is well-known, and we also reviewed the data and/or asked the patients about their tobacco use. Although the data were not very reliable because they were collected via self-report and the patients did not remember precisely how long they smoked or how much they smoked, all our patients had a history of tobacco use. Gheit et al.  reported that 75% of their patients harboring viral DNA had a history of tobacco use, and suggested that tobacco use could act together with HPV induced cancer formation. In the present study all patients had a history of tobacco use, but no HPV DNA was detected in any of the laryngeal specimens.
Some studies regarding head and neck cancer found out that HPV positivity was observed in younger patients compared to patients with HPV negative cancer [3, 4]. The current study’s patients were aged 40–75 years; none were considered young. Based on those earlier reports, it is possible that had the present study included younger patients, some with HPV positivity would have been identified [3, 4]. The transmission of HPV has been widely studied, and orogenital sexual contact and multiple sex partners were shown to increase the rate of transmission of the virus . Roshan et al.  conducted a study in Iran and reported that HPV was not encountered in any of the specimens; neither in biopsies of patients with laryngeal cancer nor in biopsies obtained from healthy people, as in the present study. They attributed their findings to the rarity of high-risk sexual behavior in Iran. Their study differs from the present study as they only investigated HPV 16 and 18, whereas the present study investigated all HR-HPV types; in addition, the present study’s patient group was larger.
Earlier studies on HPV positivity in laryngeal cancer in Turkey reported that 7.4%  and 10.6%  of patients had HPV DNA. The difference in those reported percentages and that found in the current study might be secondary to differences is diagnostic techniques; Guvenc et al.  used hybrid capture to detect HPV and Gungor et al.  used PCR genotyping. These studies also included LR-HPV infections—another possible cause for the differences in findings, as when only HR-HPV infection was considered, the HPV positivity rate in Gungor et al.’s study was only 1% . Other studies reported low rates of HPV positivity in normal laryngeal mucosa samples, of which many cases showed presence of low-risk HPV [12, 15, 18]. Similarly, none of the present study’s normal laryngeal mucosa specimens were HPV positive.
The present study is among the few from Turkey to investigate HPV infection in patients with laryngeal cancer, and compare it to HPV positivity in normal laryngeal tissue. The cISH technique used in the present study is not widely used for studying laryngeal cancer. A strength of the present study is that HPV negativity based on cISH was confirmed via genotyping. As some earlier studies reported, HPV positivity was not observed in any of the present study’s laryngeal specimens (both cancerous and normal) [2, 15, 16] indicating that HPV does not play a major role in the etiology of malignant laryngeal lesions. Nonetheless, the present study included only patients over 40 years old and a small overall population, which might be considered limitations. Lastly, as none of the present study’s laryngeal specimens were HPV positive, it was not possible to obtain any data on the effect of HPV positivity on recurrence, survival, or chemotherapy sensitivity.