Whole exome sequencing and system biology analysis support the "two-hit" mechanism in the onset of Ameloblastoma

Abstract

Background Ameloblastoma is the most frequent odontogenic tumor. Various evidence has highlighted the role of somatic mutations, including recurrent mutation BRAF V600E, in the tumorigenesis of Ameloblastoma, but the intact genetic pathology remains unknown. Material and Methods We sequenced the whole exome of both tumor tissue and healthy bone tissue from four mandibular ameloblastoma patients. The identified somatic mutations were integrated into Weighted Gene Co-expression Network Analysis on publicly available expression data of odontoblast, ameloblast, and Ameloblastoma. Results We identified a total of 70 rare and severe somatic mutations. We found BRAF V600E on all four patients, supporting previous discovery. HSAP4 was also hit by two missense mutations on two different patients. By applying Weighted Gene Co-expression Network Analysis on expression data of odontoblast, ameloblast, and Ameloblastoma, we found a proliferation-associated gene module that was significantly disrupted in tumor tissues. Each patient carried at least two rare, severe somatic mutations affecting genes within this module, including HSPA4, GNAS, CLTC, NES, and KMT2D. All these mutations had a ratio of variant-support reads lower than BRAF V600E, indicating that they occurred later than BRAF V600E. Conclusions We suggest that a severe somatic mutation on the gene network of cell proliferation other than BRAF V600E, namely second hit, may contribute to the tumorigenesis of Ameloblastoma. Key words: Ameloblastoma, whole exome sequencing, somatic mutation, BRAF, HSPA4, two-hit theory.

The Molecular Pathology of Odontogenic Tumors: Expanding the Spectrum of MAPK Pathway Driven Tumors

“…In 2014, the pioneer study by the group of Heikinheimo reported for the first time recurrent activating BRAF p.V600E mutations in ameloblastomas [9]. Considering that MAPK/ERK signaling can be activated by stimulation of transmembrane receptors, including EGFR, and that overexpression of EGFR had previously been reported in ameloblastomas, when [9], [10], [51], [52], [53] e , [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [ V600E mutation in all five ameloblastic carcinoma samples evaluated and this frequency was much higher than reported by previous studies, and therefore we did not add the results to the other ones when calculating mutation frequency.…”

“…Following these pieces of research, several studies have also assessed the presence of BRAF p.V600E in conventional ameloblastoma, either by molecular techniques or a combination of mutation screening and immunohistochemistry, with mutation frequencies varying from 55 to ∼90% [10,[53][54][55][56][57][58][59][60][61][62][63]. In two studies with limited sample numbers, the mutation was detected in all samples [2/2 [64] and 4/4 [65]]. The frequency of BRAF p.V600E mutation in studies that have assessed the mutation only using immunohistochemistry varied from 33 to 79% [66][67][68][69][70][71].…”

“… a Individual frequency per study ranged from 30 to 90%, except for two studies that have reported mutations in all samples probably due to limited sample size [2/2 [ 64 ] and 4/4 [ 65 ]] . b In eight out of 13 (61.5%) cases the mutation was KRAS p.G12R and in one sample KRAS p.L56_G60dup.…”

Genetic Profile of Adenomatoid Odontogenic Tumor and Ameloblastoma. A Systematic Review

“…Two articles that performed WES reported the presence of mutations affecting KMT2D occurring in the background of BRAF mutations [13,15]. Guan et al [13], reported 2/10 ameloblastomas to harbor non-sense mutations in KMT2D, whereas Shi et al [15], identified 1/4 ameloblastomas with a frameshift deletion in the same gene. Odontogenesis-related genes have been widely associated with the etiopathogenesis of ameloblastoma.…”

“…The prevalence of BRAF V600E in ameloblastoma ranges from 46 [6] to 90% [11], with a mean value of 68%. Other somatic mutations have been reported, either in MAPK or non-MAPK pathways [6,8,10,[12][13][14][15][16]. Some of these, such as mutations in PTEN, SMARCB1, EGFR, TP53, CTNNB1, and PIK3CA [6,[8][9][10], can occur in the background of the classical BRAF V600E mutation.…”