Prognostic value of the non invasive procedure based on DNA methylation analysis in patients surgically treated for Oral Cancer
-
1
University of Bologna, Department of Biomedical and Neuromotor Sciences - Section of Oral Sciences, Italy
Aim. Patients treated for Oral Squamous Cell Carcinoma (OSCC) have a risk of 30-40% to develop a second neoplastic manifestation during follow-up period. A genetic non invasive procedure for early detection of patients at risk to develop a secondary tumor is an attractive strategy to improve the prognosis of OSCC. Recently our research group developed a non-invasive method to detect high-risk OSCC lesions based on oral brushing as method for collecting samples followed by DNA methylation analysis of a set of 13 genes. In the present study this procedure was applied in a group of patients surgically treated for OSCC. Aim of the study is to assess an association between test positivity and poor prognosis in terms of locoregional control (LRC) of disease (appearance of local recurrence or second primary tumor).
Materials and Methods. Population study was composed of 42 consecutive patients who had completed OSCC treatment at least 6 months prior to brushing collection. Two oral brushing specimens were collected from each patient during routine follow-up visits after primary oral cancer surgical treatment: the first brushing specimen was collected within the regenerative area after OSCC resection (with or without presence of a skin graft used for tissue reconstruction after OSCC resection) and the second one in the clinically normal distant mucosa (opposite cheek). In all brushing specimens the DNA methylation level of ZAP70, GP1BB, KIF1A, ITGA4, LINC00599, MIR193, MIR296, TERT, LRRTM1, NTM, EPHX3, FLI1 and PARP15 was evaluated by quantitative Bisulfite-Next Generation Sequencing (NGS). Bioinformatic analysis was performed in a Galaxy Project environment. Each sample was considered either as a numeric and a dichotomic variable (pos/neg) in relationship to a pre-definite cut off value based on our developed algorithm. Follow-up examinations of OSCC treated patients were performed monthly during the first year, every 3 months during the second year, and every 6 months thereafter.
Statistical analysis: One Way ANOVA analysis was used to evaluate presence of any significant difference between group of patients who experienced a second neoplastic manifestation and group of patients who didn’t develop a second tumor during follow-up period. Presence of a positive score was analyzed for their relationship with appearance of secondary tumor. Survival rate was estimated using the Kaplan-Meier method. Statistical significance was evaluated using the log rank test. Time was defined as the period between brushing collecting sample and appearance of the second neoplastic lesion or last follow-up visit. P values < 0.05 were considered statistically significant in all analyses.
Results. 6/42 (14,3%) patients developed a second neoplastic manifestation (5 Second Primary Tumor and 1 Local Recurrence) during follow up period (mean follow up: 14.3 months). 16/42 (38,09%) brushing samples collected in surgically treated oral mucosa were detected as positive, while epithelial cells collected in clinically healthy distant mucosa showed 7/42 (16,7%) samples who exceeded the threshold value. One Way ANOVA analysis revealed that patient who experienced second tumoral event showed significant higher numeric values both in specimens collected in regenerative area after OSCC resection (p<0.01), and in specimens collected in clinically healthy distant mucosa (p<0.05).
The presence of a positive score resulted a variable significantly related with a worse LRC of disease (p<.05), both regarding brushing samples collected in regenerative area after cancer resection and regarding brushing samples collected in distant mucosa. Indeed, 5/16 patients with a positive score in OSCC resection area develop a second neoplasia with respect to 1/28 patients with negative score. Moreover 3/7 patients with a positive score in distant mucosa developed a second neoplasia with respect to 3/37 patients with a negative score.
Discussion. The poor survival rate of OSCC patients has traditionally been ascribed to the high rate of secondary tumors, and deaths due to comorbidity. Non-invasive detection techniques for the identification of patients with an high risk to develop a second neoplastic manifestation are needed.
In a recent paper, using our noninvasive method based on brush sampling and a 13-genes-panel methylation analysis, we correctly stratified OSCC from healthy donors (sensitivity 97,1%, specificity 100%, AUC 0.981). Highly sensitivity and specificity of the method stimulated us to apply the procedure in patients surgically treated for OSCC to elucidate the prognostic potential of our assay.
In the present study 16/42 (38.09%) brushing samples collected in surgically treated oral mucosa and 7/42 normal distant mucosa (16.7%) were detected as positive indicating the presence of an altered epigenetic pattern in oral brushing specimens from normal appearing buccal mucosa. This data may be probably related to the presence of a field cancerization effect detectable after surgical resection in close and distant proximity to the index tumour.
An interesting finding of the present study was the presence of a significant relationship between presence of a positive score and appearance of a secondary tumor, both considering brushing samples collected from regenerative area after OSCC resection and considering brushing samples collected from normal distant mucosa.
Conclusions: These preliminary results seem to indicate that our novel assay based on quantitative bisulfite NGS analysis could be a method to identify patients with an high risk to develop a second neoplasia starting from non-invasive, easy-to-perform brush sampling. Further studies with a larger cohort of patients with a long follow-up period are needed to elucidate the intrinsic prognostic potential of our assay.
References
1. DNA methylation analysis by bisulfite next-generation sequencing for early detection of oral squamous cell carcinoma and high-grade squamous intraepithelial lesion from oral brushing Morandi L, Gissi D, Tarsitano A, Asioli S, Monti V, Del Corso G, Marchetti C, Montebugnoli L, Foschini MP; J Craniomaxillofac Surg. 2015 Oct;43(8):1494-500
2. CpG location methylation level are crucial factors for the early detection of oral squamous cell carcinoma in brushing samples and using bisulfite sequencing of a 13-gene panel; Morandi L, Gissi D, Tarsitano A, Asioli S, Gabusi A, Marchetti C, Montebugnoli L, e Foschini MP, Clinical Epigenetics (2017) 9:85
Keywords:
oral cancer,
Brushing,
Methylation,
prevention,
OSCC
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:
Rossi
R,
Morandi
L,
Tarsitano
A,
Kavaja
S,
Gabusi
A and
Gissi
B
(2019). Prognostic value of the non invasive procedure based on DNA methylation analysis in patients surgically treated for Oral Cancer.
Front. Physiol.
Conference Abstract:
5th National and 1st International Symposium of Italian Society of Oral Pathology and Medicine..
doi: 10.3389/conf.fphys.2019.27.00078
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
05 Nov 2018;
Published Online:
09 Dec 2019.
*
Correspondence:
Dr. Roberto Rossi, University of Bologna, Department of Biomedical and Neuromotor Sciences - Section of Oral Sciences, Bologna, Emilia-Romagna, 40126, Italy, fedemurkez@gmail.com