Human papillomavirus in carcinomas of the tongue: clinical and prognostic implications

Abstract

It is not clear whether the presence of human papillomavirus (HPV) in squamous cell carcinomas of the tongue (SCCT) is of etiopathogenic and clinical significance. This study was designed to establish the incidence of HPV in SCCT and to determine the influence of HPV detection on clinical parameters and the prognosis. Clinical and histopathological data of 64 patients with SCCT were collected. Thirty benign lesions of the tongue were analyzed in parallel, in order to compare the HPV incidence and genotypes in these lesions with those of SCCT. Paraffin blocks of all cases were collected and PCR was carried out using SPF10 primers and the INNO-LiPA genotyping methodology. HPV was detected in 26.2% of the patients. Hybridization results showed that all patients except one had high-risk (HR)-HPV. HPV56 was the most common (42.1%), followed by HPV18 (26.3%), HPV16 (10.5%), HPV66 (10.5%), HPV39 (5.3%), and HPV51 (5.3%). The odds ratio of HR-HPV infection in cases vs. controls was statistically significant (9.45, 95% confidence interval 1.18–75.46). Among the results of the univariate analysis correlating the presence of HR-HPV with different clinical parameters, only mortality showed a statistically significant correlation, being higher in HR-HPV patients (odds ratio 3.97, 95% confidence interval 1.07–14.7).

Squamous cell carcinoma of the tongue (SCCT) is the most frequent oral cancer. In approximately two-thirds of cases, the lesion is located in the oral tongue, ahead of the circumvallate papillae, and in the other third the lesion is located at the base of the tongue (pharyngeal tongue). Most cases of SCCT, around 55–70% of cases, occur at the lateral border of the tongue. In addition, SCCT have the highest incidence of metastatic dissemination at the time of diagnosis amongst all oral squamous cell carcinomas (OSCC); approximately half of the cases have lymph node metastasis at the time of diagnosis. Surgical excision of the tumour with wide margins, often combined with ipsilateral or bilateral regional lymphadenectomy, is the elective therapy for SCCT. This therapy is sometimes combined with adjuvant radiotherapy or adjuvant chemotherapy, or a combination of both. The 5-year survival of patients with SCCT varies between 15% and 70%, depending mainly on the size of the tumour and on the presence of metastasis.

Smoking and alcohol intake are known risk factors that influence the development of oral cancer. During the last few years, human papillomavirus (HPV) has been detected in a variable proportion of OSCC, and HPV has been postulated to have an important etiopathogenic influence on carcinogenesis in some head and neck squamous cell carcinomas, especially in oropharyngeal carcinomas.

HPVs are viruses belonging to the Papillomaviridae family, and have the capacity to infect basal cells of excoriated epidermal tissues. Once these cells have phagocytosed the viruses, they are transported to the nucleus, where they are multiplied by episomal replication. In some cases the viral genome is integrated into the host genome, producing a cell-cycle deregulation that has carcinogenic potential.

The HPV oncogenic potential has been demonstrated in anogenital carcinomas, especially in carcinomas of the uterine cervix, where the presence of HPV is considered to be necessary for malignant transformation. The detection of HPV in OSCC observed in different meta-analyses varies between 23.5% and 46.5%. The implication of HPV in the development of oropharyngeal carcinomas is compelling, particularly in carcinomas of the tonsil. However, evidence of the influence of HPV in the development of tongue carcinomas is far less conclusive, and it is not clear whether the presence of HPV in these tumours is of etiopathogenic and prognostic significance.

The aggressive therapy that is required for the treatment of carcinomas of the tongue, and the poor prognosis for patients in whom these types of tumour present, have prompted the search for possible etiologic factors that will assist in the development of effective prevention strategies, or in the use of less aggressive and more effective therapies. The present study was designed to establish the incidence of HPV in SCCT using a sensitive method, and to determine the clinical influence that this may represent.

Materials and methods

Patients, tissue samples, and diagnosis

A retrospective search of all cases of SCCT diagnosed or treated in the oral and maxillofacial surgery department of the hospital from 2002 to 2010, identified 64 cases. Clinical data were obtained from the clinical histories of the patients held in the city tumour registry and, when necessary, by means of telephone or personal interview with the patient. Clinical data obtained were: age, gender, size and location of the lesions, alcohol intake and tobacco smoking habits, regional lymph node involvement, treatment procedures, recurrence and second primary tumour appearance, and mortality. Cancer staging was performed in accordance with the 2002 American Joint Committee on Cancer sixth edition staging criteria.

To compare the incidence of HPV in carcinomas versus benign lesions of the tongue, samples of 30 benign lesions of the tongue diagnosed from 2002 to 2010 were selected. Glass slides, paraffin blocks, and histopathological reports were obtained for all the cases from the files of the hospital pathology department. In addition, 30 biopsy samples of the benign tongue lesions were selected. This retrospective study was approved by the hospital ethics committee.

Tissue preparation and nucleic acid isolation

Three 5-μm-thick sections from each paraffin block were placed in sterile Eppendorf tubes. To prevent possible cross-contamination between samples during the polymerase chain reaction (PCR) procedure, each microtome was cleaned with 70% ethanol before cutting the blocks, and each block was cut using a new disposable microtome blade. Tissue sections were deparaffinized using xylene, and washed with ethanol. The tissue sections were digested overnight with proteinase K in a volume of 250 μl at 56 °C. Proteinase K was heat-inactivated at 95 °C for 10 min. A 1/10 dilution of the sample was used for the PCR (10 μl).

HPV DNA PCR detection and genotyping

Broad-spectrum HPV DNA amplification was performed using the short PCR fragment (SPF10) primer set with the INNO-LiPA HPV genotyping technology of Innogenetics NV (Gent, Belgium). The SPF10 primers amplify a 65-bp fragment of the L1 region of the HPV genome. For HPV amplification, a 9-min denaturation step at 94 °C was followed by 40 cycles of amplification using 1.5 IU DNA polymerase (AmpliTaq Gold DNA Polymerase; Applied Biosystems, Foster City, CA, USA) in a thermocycler (GeneAmp 9700; Applied Biosystems). These cycles included denaturation at 94 °C for 30 s, primer annealing at 52 °C for 45 s, and chain elongation at 72 °C for 45 s. The final elongation step was prolonged to 7 min. To confirm DNA amplification, a second PCR using primers for the human β-globin gene was conducted (primers Hg063/064). Amplification products were first tested for the presence of HPV DNA by DNA enzyme-immunoassay (DEIA), which consists of hybridization with conserved probes in a microtiter-plate assay format (Universal DNA ELISA Kit; Labo Bio-medical Products BV, Rijswijk, Netherlands). SPF10 amplimers from DEIA HPV-positive samples were subsequently analyzed by reverse hybridization in an HPV line-probe assay, LiPA25 system version 1 (Labo Bio-medical Products), at high stringency, generating a type-specific hybridization pattern. In this assay, 10 μl of denatured HPV PCR product was hybridized to the genotype-specific probes immobilized as parallel lines on a nitrocellulose strip. After the washing step, the products of hybridization were detected by a colour reaction with alkaline phosphatase–streptavidin conjugate and substrate (5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium), which results in a purple precipitate. The results of the hybridization were assessed visually by comparison to the standard grid. The HPV LiPA25 version 1 permits specific detection of 25 HPV types: HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74. These assays were performed automatically using the Auto-LiPA system (Innogenetics Diagnostica Iberia, Barcelona, Spain) for 48 strips.

Statistical analysis

Basic descriptive statistics methods were used to characterize the study patients, including the mean and standard deviation (SD) for continuous variables and the absolute frequency and percentage for discrete variables. On the basis of HPV detection, patients were divided into two groups: high-risk (HR)-HPV-positive and HR-HPV-negative; the variables observed in each group were compared. Before parametric tests were performed, the normal distribution of the continuous variables was ascertained by means of the Kolmogorov–Smirnov test. For the univariate analysis, the Mann–Whitney U -test was applied to compare continuous variables and Fisher’s exact test was used for contrasting categorical variables. A P -value of less than 0.05 was considered to indicate a statistically significant difference. Finally, a multivariate, non-conditional logistic regression model was developed, with the dependent variable being the presence or not of HR-HPV. The associated measure was the odds ratio (OR), with calculation of the corresponding 95% confidence interval (CI).

Overall survival was defined as the time from the date of surgery (operated patients), or the date of diagnosis (non-operated patients) to the date of death, or to the last date of follow-up. Survival data were analyzed using the Kaplan–Meier method. Survival curves were compared by log-rank test in univariate analysis. All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Statistical analysis

Basic descriptive statistics methods were used to characterize the study patients, including the mean and standard deviation (SD) for continuous variables and the absolute frequency and percentage for discrete variables. On the basis of HPV detection, patients were divided into two groups: high-risk (HR)-HPV-positive and HR-HPV-negative; the variables observed in each group were compared. Before parametric tests were performed, the normal distribution of the continuous variables was ascertained by means of the Kolmogorov–Smirnov test. For the univariate analysis, the Mann–Whitney U -test was applied to compare continuous variables and Fisher’s exact test was used for contrasting categorical variables. A P -value of less than 0.05 was considered to indicate a statistically significant difference. Finally, a multivariate, non-conditional logistic regression model was developed, with the dependent variable being the presence or not of HR-HPV. The associated measure was the odds ratio (OR), with calculation of the corresponding 95% confidence interval (CI).

Overall survival was defined as the time from the date of surgery (operated patients), or the date of diagnosis (non-operated patients) to the date of death, or to the last date of follow-up. Survival data were analyzed using the Kaplan–Meier method. Survival curves were compared by log-rank test in univariate analysis. All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Results

Clinical characteristics of the patients at the time of diagnosis

Of the 64 patients with SCCT who were included in the study, 18 were women (28.1%) and 46 were men (71.9%). The mean age of the patients was 61.7 years (range 16–86 years). Information on tobacco smoking habits was available for 57 patients, and 71.9% of these patients were smokers or ex-smokers. Information on alcohol intake was available for 55 patients, and 47.3% of these patients had a history of heavy alcohol intake; 20% of the patients had a low or only occasional alcohol intake. The mean diameter of the lesions at diagnosis was 3.26 cm. The predominant location of the lesions was the lateral border of the tongue (62.5%), followed by the ventral area of the tongue (28.1%), the base of the tongue (7.8%), and the dorsum of the tongue (1.6%). A surgical procedure was performed in 58 patients (90.6%), radiotherapy was performed in one patient (1.6%), and five patients (7.8%) received no treatment. Of the surgically treated patients, 8.6% received neoadjuvant chemotherapy treatment, 10.5% adjuvant chemotherapy and radiotherapy, and 40.4% only adjuvant radiotherapy.

The clinical stage (cTNM) was determined in non-operated patients, while the pathological stage (pTNM) was determined in operated patients. Most patients ( n = 27) had a tumoral stage of IVA at the time of diagnosis. Seventeen patients were stage II, 10 were stage I, nine were stage III, and one was stage IVB.

HPV DNA detection

Insufficient DNA was obtained for three of the 64 SCCT, and these were excluded from HPV detection. Sixteen of the remaining 61 cases were HPV-positive (26.2%); 45 cases were HPV-negative (73.8%). A single HPV genotype was identified in 12 of the positive cases (75%), whereas a combination of two different HPV genotypes was detected in each of the other four cases (25%). The results of hybridization showed that all patients but one, who had an indeterminate genotype (X-HPV), had HR-HPV.

The detected HR-HPV genotypes were as follows: HPV56 (eight cases, 42.1%), HPV18 (five cases, 26.3%), HPV16 (two cases, 10.5%), HPV66 (two cases, 10.5%), HPV39 (one case, 5.3%), and HPV51 (one case, 5.3%).

All the control cases had sufficient and well preserved DNA; HPV was detected in five cases (16.7%). Each of these positive cases showed a single HPV genotype: two low-risk (LR)-HPV genotypes (HPV6 and HPV11) and three indeterminate genotype (X-HPV).

After inserting a fictitious HR-HPV-positive case into the control group, the OR of infection with HR-HPV was 9.45 (95% CI 1.18–75.46) ( P = 0.012). When adjusted for age and sex, the OR was 10.75 (95% CI 1.28–90.35) ( P = 0.03). Likewise, even on introducing a fictitious case of LR-HPV into the case group, we could not find any statistically significant differences between cases and controls in LR-HPV and total HPV.

Evolution and survival analysis of patients with carcinoma of the tongue

The mean follow-up of the patients was 30.2 months. Of the treated patients, ‘35.1’ showed tumour recurrences (50% were local recurrences, 40% regional recurrences, 5% local and regional, and 5% distant metastasis). The percentage of recurrences was directly related to the tumoral stage. The global mortality rate of the patients was 48.4%, with 67.9% of these deaths directly related to the SCCT, 21.4% due to postoperative complications, and 10.7% due to other causes. Mortality showed a direct correlation with the tumoral stage.

Mean survival was 5.34 years (95% CI 4.01–6.67), with a median of 3.18 years (95% CI 1.48–4.88) ( Fig. 1 ). Seventy-three percent of the patients were alive at the first year follow-up, 57% at 2 years, 49% at 3 years, and 41% at 4 years.

Fig. 1
Global survival of patients with squamous cell carcinoma of the tongue.

Tables 1 and 2 show the results of the univariate analysis to correlate the presence of HR-HPV with the different clinical parameters analyzed. Only mortality showed a statistically significant correlation with the presence of HPV, being higher in HR-HPV-positive patients. The OR for the correlation of mortality and HR-HPV was 3.97 (95% CI 1.07–14.70; P = 0.032). In a search of the literature we found that tumoral stage, age, alcohol intake, and tobacco smoking may act as possible confounding factors. When the OR was adjusted to take these parameters into account, there was a considerable increase in the observed mortality (OR 13.4, 95% CI 1.5–117.6), being 84.3% the predictive capacity of the model.

Table 1
Univariate results of qualitative variables.
HR-HPV-negative ( n = 45) HR-HPV-positive ( n = 15) OR (95% CI) P -value
Gender
Female 12 (75%) 4 (25%) 1.0 (0.27–3.75) 1.00
Male 33 (75%) 11 (25%)
Tobacco smoking habits
Non-smoker 10 (66.7%) 5 (33.3%) 0.58 (0.16–2.14) 0.493
Smoker or former smoker 31 (77.5%) 9 (22.5%)
Alcohol intake
None 14 (82.4%) 3 (17.6%) 1 (reference)
Occasional drinker 7 (63.6%) 4 (36.4%) 2.67 (0.46–15.35) 0.272
Heavy drinker or former heavy drinker 20 (80%) 5 (20%) 1.17 (0.24–5.67) 0.849
Surgical or radiation treatment
No 3 (75%) 1 (25%) 1.02 (0.98–10.67) 0.984
Yes 41 (74.5%) 14 (25.5%)
Regional lymph node involvement
No 28 (71.8%) 11 (28.2%) 1.47 (0.46–6.08) 0.43
Yes 17 (81.0%) 4 (19.0%)
Recurrence or second primary
No 24 (75%) 8 (25%) 0.88 (0.25–3.17) 0.848
Yes 17 (77.3%) 5 (22.7%)
Mortality
No 27 (87.1%) 4 (12.9%) 3.97 (1.07–14.70) 0.032
Yes 17 (63.0%) 10 (37.0%)
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Jan 21, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Human papillomavirus in carcinomas of the tongue: clinical and prognostic implications
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