In the present study, we sought to explore the epigenetic alterations associated to grade II PXA and those occurring associated with the acquisition of histological malignant features, such as mitoses (as above mentioned) and/or necrosis. PXA WHO grade II is a slow-growing astrocytic tumor, which is considered benign and presents a 10-years survival rate of 70% and a recurrence-free lapse of 61% [
8,
33]. However, up to 20% PXA will develop anaplastic features and may further progress to secondary glioblastoma, exhibiting much more aggressive phenotype and dropping significantly survival rates with a median survival time of 15 months [
8].
DNA methylation changes, and moreover those associated to malignant transformation of grade II PXA, had not been investigated previously. In the present study, we analyzed DNA hypermethylation on the promoter sequences of a panel of cancer-related genes in order to investigate epigenetic alterations associated to this process. To our knowledge, only MGMT methylation status has been previously studied in PXA [
28], while the data on the epigenetic regulation of
CD81,
HCK,
TES,
HOXA5 and
ASCL2 in PXA patient have not been documented before. Despite the low cohort size, we observed comparable increases on the DNA methylation levels in independent samples used for validation. Interestingly, when analyzing differences in DNA methylation affecting each sample type, we found a much higher number of changes occurring in anaplastic PXA and the grade II precursor tumor (116 genes) as compared with fewer occurring in all other grade II PXA (19 genes) samples (Additional file
4: Table S2 and Additional file
5: Table S3). Accumulation of genetic alterations has been found associated to the pathogenesis and progression of astrocytic tumours [
34], and concomitantly, accumulation of epigenetic lesions is present as well. Among the changes identified, we observed a set of DNA hypermethylation events in anaplastic PXA, its corresponding precursor grade II tumor, overlapping with DNA methylation alterations also found in GBM (Figure
3). Several studies have previously reported frequent epigenetic disruption of
CD81 in glioblastoma [
29,
35], supporting its tumor-suppressor roles in this cancer type. Moreover, promoter hypermethylation of the gene coding for Testin (
TES) was also reported by us [
29] and others [
36,
37]. Its role acting as a negative regulator of cell growth supports a role for tumor-suppression, as has been proposed in diverse cancer types including ovarian cancer [
38] and acute lymphocytic leukemia [
39]. DNA hypermethylation of the transcription factor
ASCL2 has been identified in other cancers [
40] and is involved in the regulation of gene expression in the central and peripheral nervous system [
41]. Additionally, the pathways deregulated by DNA methylation changes (Figure
4) showed great consistency with those targeted in GBM, and are concordant with previous observations reported by us and others [
29,
37]. This data suggest that malignant progression of grade II PXA towards anaplastic relapses could be triggered by molecular mechanisms also involved in GBM. This change of methylation status during malignant progression could be also confirmed at the end-point of anaplastic PXA cases (Additional file
4: Table S2). The subset of genes validated in our study (
CD81, TES, HOXA5, ASCL2, and
HCK) were not affected by DNA hypermethylation in the grade II patients analyzed, and therefore could be specifically associated to the progression of the disease in this patient (Figure
2 and
3). On the other hand, we cannot rule out that the epigenetic alterations observed could be attributable to individual-specific DNA methylation phenomena; however, methylation of these genes has also been found in GBM in larger population studies [
29,
35‐
37,
42], thus indicating that they are most probably involved in the pathogenesis and malignant progression of astrocytic tumors.
Of note, considerable promoter hypermethylation was found in
MGMT promoter region, both in the precursor and the anaplastic tumors (Additional file
3: Figure S2). A recent report analyzing the methylation status of
MGMT in 11 grade II PXA concluded that this event was infrequent in PXA [
28]. Albeit this finding needs to be assessed in larger population studies, the extensive promoter hypermethylation we found in the anaplastic PXA patient supported the indication of chemotherapy with temozolomide in a similar pattern as it is in other malignant tumors of astrocytic lineage [
40].
The pathogenesis of PXA is largely unknown. Nevertheless, a series of molecular studies have described genomic alterations in PXA patients [
28,
42‐
48] Chromosomal gains and losses have been associated with PXA pathogenesis, although prevalent losses -frequently involving chromosomes 7 and 9- were observed in grade II astrocytomas of poor prognosis [
28,
42‐
44], accounting for a potential inactivating mechanism of tumor suppressor genes. Further genetic studies have also unveiled the high frequency of BRAF V600E mutations in WHO grade II PXAs and PXAs with anaplastic features (65 and 66% of cases, respectively) [
48], as well as homozygous deletion of CDKN2A/p14(ARF)/CDKN2B in six out of ten tumors analyzed in a different cohort [
46]. DNA methylation alterations have been scarcely analyzed in PXA. Marucci and colleagues examined
MGMT promoter methylation in 11 grade II PXA [
28], but, to our knowledge, no additional studies have been done to examine epigenetic alterations in this setting. Methylation markers in a variety of human cancers have proved trustworthy in clinical trials for diagnostic and prognostic purposes. In other solid tumors, DNA methylation markers have shown their relevance in early diagnosis, prognosis of tumor progression, or response to therapy and chemo-resistance [
49,
50]. For instance, DNA methylation profiles were shown to correlate with clinical parameters; specifically hypermethylation of
GATA6 transcription factor was found associated with poor survival in GBM patients [
35], or hypermethylation of the pro-apoptotic CASP8 is a differential feature of GBM relapses [
51]. Though limited by the size of the populations analyzed, our study suggests that DNA hypermethylation mediated silencing of tumor suppressor genes in PXA could be a relevant event contributing to malignant progression, as also defined by other diffuse astrocytic tumors. The diagnosis of grade II PXA at a high risk to recur as a malignant tumor widens the therapeutic window for intervention, in the form of early onset of adjuvant chemotherapy, even at a grade II tumor stage. Thus, in order to depict biomarkers with prognostic value on PXA patients, broader studies should be undertaken in this and further low-grade astrocytic tumors with risk to undergo malignant transformation.