Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Abstract

Keywords: biomarker, DAPK1, expression, head and neck cancer, lymph node metastasis, methylation, MGMT, oral cancer Abbreviations: FFPE, formalin-fixed paraffin-embedded; OOSCC, oral and oropharyngeal squamous cell carcinoma; pN status, pathologically determined nodal status; (Q)MSP, (quantitative) methylation-specific PCR.Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pNC status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.

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

“…Many laboratories have discovered oncogenes and tumor suppressors for OSCC. However, cumulative evidence revealed more complex mechanisms underlying the development and progression of this disease, including, among others, interactions between genomic and epigenetic alterations [28][29][30][31] Epigenetic alterations are responsible for the regulation of ontologically-related gene expression networks, at an appropriate level of environmental conditions and time, leading to a rise of both normal and diseased phenotype development.…”

Beyond gene dictation in oral squamous cell carcinoma progression and its therapeutic implications

“…[11][12][13][14][15][16]18,19,29 Among several epigenetic modification processes, DNA methylation is considered as central to achieve the state of gene promoter methylation and also global methylation status. [11][12][13][14][15][16]18,19,29 To achieve DNA methylation of target sequence, dedicated enzymes have been reported to carry out such regulatory process such as DNA methyltransferase (DNMTase), a family of enzymes with an ability to transfer methyl group to target DNA. Among the family of DNMTase, DNMT1 is reported to be highly active in cancer to silence tumor suppressor genes via hypermethylation of gene promoter.…”

Protein expression and molecular profiling to predict lymph node status and prognosis in breast cancer

“…MGMT methylation was associated with a negative lymph node status and tumours also showed reduced protein expression with immunohistochemistry. DAPK1 methylation was associated with a positive lymph node status, but hypermethylation did not associate with protein expression on immunohistochemistry (Melchers et al, 2015).…”

“…MGMT methylation was associated with a negative lymph node status and tumours also showed reduced protein expression with immunohistochemistry. DAPK1 methylation was associated with a positive lymph node status, but hypermethylation did not associate with protein expression on immunohistochemistry (Melchers et al, 2015).In colorectal cancer, DNA hypermethylation of the promoter of GCNT2 is associated with aberrant expression of GCNT2 and hypomethylation of GCNT2 variant 2 has been shown to be associated with lymph node metastasis in primary colorectal carcinoma (Nakamura et al, 2015). A CpG island methylator phenotype (CIMP) has also been identified in colorectal carcinoma where hypermethylation of a number of genes is associated with malignancy (Tahara et al, 2014, Toyota et al, 1999.…”

“…Melchers et al (Melchers et al, 2015) looked at 28 methylation markers in oral and oropharyngeal squamous cell carcinoma and the association with lymph node metastases.…”