Paired Box 5 Methylation Detection by Droplet Digital PCR for Ultra-Sensitive Deep Surgical Margins Analysis of Head and Neck Squamous Cell Carcinoma

Abstract

Molecular deep surgical margin analysis has been shown to predict locoregional recurrences of head and neck squamous cell carcinoma (HNSCC). In order to improve the accuracy and versatility of the analysis, we used a highly tumor-specific methylation marker and highly sensitive detection technology to test DNA from surgical margins. Histologically cancer-negative deep surgical margin samples were prospectively collected from 82 eligible HNSCC surgeries by an imprinting procedure (n=75) and primary tissue collection (n=70). Bisulfite treated DNA from each sample was analyzed by both conventional quantitative methylation-specific polymerase chain reaction (QMSP) and QMSP by droplet digital PCR (ddQMSP) targeting PAX5 gene promoter methylation. The association between the presence of PAX5 methylation and locoregional recurrence free survival (LRFS) was evaluated. PAX5 methylation was found in 68.0% (51/75) of tumors in the imprint samples and 71.4% (50/70) in the primary tissue samples. Among cases which did not have postoperative radiation, (n=31 in imprint samples, n=29 in tissue samples), both conventional QMSP and ddQMSP revealed that PAX5 methylation positive margins was significantly associated with poor LRFS by univariate analysis. In particular, ddQMSP increased detection of the PAX5 marker from 29% to 71% in the non-radiated imprint cases. Also, PAX5 methylated imprint margins were an excellent predictor of poor LRFS (HR=3.89, 95%CI:1.19-17.52, P=0.023) by multivariate analysis. PAX5 methylation appears to be an excellent tumor-specific marker for molecular deep surgical margin analysis of HNSCC. Moreover, the ddQMSP assay displays increased sensitivity for methylation marker detection.

Epigenetic Alterations Associated with the Overall Survival and Recurrence Free Survival among Oral Squamous Cell Carcinoma Patients

“…The survival difference between the two identified clusters, cluster 4 vs. cluster 6, represented a clear difference in the lifetime of these patients. These results fall in agreement with previous reports that showed a significant association between promoter methylation status and worse OSCC patients' survival [31][32][33][34]. These results demonstrated the utility of epigenetic alterations detection for potential clinical application in OSCC patients.…”

JAK3 Variant, Immune Signatures, DNA Methylation, and Social Determinants Linked to Survival Racial Disparities in Head and Neck Cancer Patients

“…Dysregulated expression of PAX5 is involved in differentiation block (55), somatic hypermutation and immunoglobulin heavy chain class switch recombination (56), leukemogenesis (57), and positive regulation of c-Met transcription (58). PAX5 association to different tumor types suggests it has diagnostic or prognostic utility in lung cancer (59), Hodgkin lymphoma (60), acute lymphocytic leukemia (61), breast cancer (61), oral cancer (62,63), gastric cancer (64), head and neck cancer (65,66), and esophageal cancer (66). A recent study demonstrated that the lack of expression of PAX5 in lymphoid neoplasms is associated with promoter hypermethylation, leading to PAX5 silencing in cases characterized by poor clinical outcome (67).…”

PAX5 gene as a novel methylation marker that predicts both clinical outcome and cisplatin sensitivity in esophageal squamous cell carcinoma

“…The bisulfite-modified DNA was used as a template for fluorescence-based QMSP as described. 5,6,47 The primer and probe sequences are shown in Supplementary Table S2. QMSP assays were performed in triplicate using StepOnePlus (Thermo Fisher Scientific).…”