HPV 28 anyplex II seegene Real Time PCR on samples collected using cytobrush and microbiopsy for the detection of HPV infection in patients affected by oral lichen planus and leukoplakia
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1
Università degli Studi di Bari, Interdisciplinary Department of Medicine, Italy
Aim. Human Papillomaviruses (HPVs) have been implicated in the pathogenesis of different skin and mucosal epithelial lesions. These viruses can cause a variety of benign lesions of the genital mucosa and they are etiologically associated with cervical and anogenital cancers. HPV infection also appears to be associated with a range of benign papillomatous oral diseases such as oral squamous papilloma, oral condyloma acuminatum, oral verruca vulgaris and focal epithelial hyperplasia, while the role of these viruses in the etiopathogenesis of oral premalignant disorders and tumors is controversial. Debated is the correlation between HPV infection and oral leukoplakia (OL) and oral lichen planus (OLP). The aim of this study was to evaluate the prevalence of HPV infection in OLP and OL lesions and identify the genotypes involved. Association between the HPV presence and the demographical, behavioural and clinical variables was calculated. We also compared two different specimen collection methods: microbrush and microbiopsy.
Materials and Methods. This study involved consecutive patients with a clinical and confirmed histopathological diagnosis of OLP and OL referred to the Oral Medicine Section of Policlinico of Bari. Demographic details such as age, gender and personal habits (tobacco and alcohol consumption) and clinical history were obtained. Each patient was undergone to lesional cytological scraping using a cytobrush, then the samples were sent to the Department of Microbiology for HPV detection. On the same lesion, a surgical biopsy was performed and a part of the bioptic specimen was addressed to the PCR procedures. The histopathological examination was performed for the definitive diagnosis.
HPV DNA presence was assessed by HPV 28 Anyplex II Seegene Real Time Polymerase Chain Reaction (RT-PCR), that allows to identify 28 different HPV genotypes (19 HR and 9 LR). According to manufacturer indications, we used 2 TOMs (TOCE Oligo Mixes): the TOM A set contains primer of DNA for 14 genotypes of HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and the TOM B set for the amplification of 5 moderate risk genotypes (26, 53, 69, 73, 8) and of 9 LR-HPV DNA (11, 34, 40, 42, 43, 44, 54, 61, 70). The compatible system CFX96 Real Time PCR (Bio-Rad) and the Seegene Viewer software were used for the data analysis and detection.
Cohen’s test was employed to calculate the agreement between cytobrush and microbiopsy considering the agreement result as: poor, fair, moderate, good and very good. The association between HPV infection and age, sex, smoking habit, use of alcohol, type of mucosal epithelium (keratinized/not keratinized) and oral lesion localization was calculated using Chi-square test (p significant value ≤ 0.05).
Results. Eighty-five patients (38 men and 47 women, mean age 61±14 years) were enrolled. Forty-five patients were affected by OL, while 40 received a diagnosis of OLP. Twenty-five patients (29%) were tobacco users and 27 (32%) drunk alcohol. The 55% of oral lesions was localized in non-keratinized sites, while the remaining was localized in keratinized mucosa. Most of the lesions (62%) was localized in the anterior region of the mouth.
HPV DNA was detected in 16,5% of total lesions (17,8% of OL and 15% of OLP). Considering each method separately, the frequency rate was higher for biopsy then cytobrush (15,3% vs. 5,3%). The HPV prevalence in OL was 17,8% in microbiopsy and 5,1% in cytobrush specimens. In OLP, HPV infection rate was 12,5% and 5,6% in microbiopsy and cytobrush specimens respectively. Ten samples collected by cytobrush were not considered because of poor cellularity. Both methods identified the same HPV status in 65/75 patients of which 64 were negative and 3 positive. Eight patients showed HPV discordant results (7 cases with HPV-positive biopsy and HPV-negative cytobrush and 1 case with HPV-negative biopsy and HPV-positive cytobrush). Cohen’s K index revealed a fair concordance between the two techniques in OLP patients (K=0.357) and OL patients (K=0.395).
HPV infection in OLP and OL patients did not show a statistical significance. There was no significant difference between HPV-negative or HPV-positive lesions in term of age, gender, tobacco and alcohol consumption, mucosal site (p>0.05). HPV 6, 11 and 42 were the most frequently detected viruses (29%, 25% and 21% respectively), while HPV 16 (8%), 53 (8%), 35 (4%) and 43 (4%) were found less frequently. None case of HPV 18 infection was recorded.
Discussion. Based on the present study, we can assume that the presence of HPV DNA detected by HPV 28 Anyplex II Seegene RT-PCR is not related to OL and OLP lesions. Previous studies showed discordant results about the relationship between HPV, OL and OLP. In fact, the reported rates of HPV DNA detection in OPMDs and OSCC range from 0% to 100%. This extreme variation is owing to difference in ethnicity, geographic locations and different methods used for HPV detection. Our study demonstrated a lack of concordance between the two specimen collection methods and microbiopsy results more accurate for HPV detection than brushing, as previously reported in part of literature. A plausible explanation is that microbiopsy provides cells from the basal layer where the virus could be present in a latent form. Despite that, this method is more invasive and generally performed only on lesions that will be subjected to histopathological examination. On the other hand, cytological scraping is an economical, easy to perform and more readily accepted by patients than microbiopsy and could be used for screening.
Similarly to other studies, we found no statistically significant relation between HPV. infection and clinical variables (mean age, gender, alcohol and tobacco use, keratinization and localization of lesions) even if the low number of patients makes the result unreliable. In our opinion it is important to improve the studies about the role of HPV in OPMDs and their neoplastic counterpart. Further studies and future researches are needed to better understand the pathogenic mechanisms related to HPV infection and related preventive strategies.
References
1. Campisi G, Panzarella V, Giuliani M, Lajolo C, Di Fede O, Falaschini S, et al. Human papillomavirus: Its identity and controversial role in oral oncogenesis, premalignant and malignant lesions (review). Int J Oncol 2007;30:813-23.
2. Syrjanen S, Lodi G, von Bultzingslowen I, Aliko A, Arduino P, Campisi G, et al. Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review. Oral Dis 2011; 17(Suppl.):158-72.
3. Ferreira L, Biasoli E, Bernabè D, Nunes C, Miyahara G. Plasma HPV DNA is detectable in oral leukoplakia patients. Pathology-Research and Practice 213(2017)759-765.
Keywords:
HPV,
Leukoplakia,
Oral lichen planus,
Cytobrush,
Microbiopsy
Conference:
5th National and 1st International Symposium of Italian Society of Oral Pathology and Medicine., Ancona, Italy, 19 Oct - 20 Oct, 2018.
Presentation Type:
Poster Presentation
Topic:
Oral Diseases
Citation:
Patano
A,
Federico
A,
Benizio
G,
Del Prete
R,
Ninivaggi
R and
Petruzzi
M
(2019). HPV 28 anyplex II seegene Real Time PCR on samples collected using cytobrush and microbiopsy for the detection of HPV infection in patients affected by oral lichen planus and leukoplakia.
Front. Physiol.
Conference Abstract:
5th National and 1st International Symposium of Italian Society of Oral Pathology and Medicine..
doi: 10.3389/conf.fphys.2019.27.00079
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Received:
05 Nov 2018;
Published Online:
09 Dec 2019.
*
Correspondence:
Dr. Assunta Patano, Università degli Studi di Bari, Interdisciplinary Department of Medicine, Bari, 70121, Italy, fedemurkez@gmail.com