Recently studies have shown that sometimes PXAs particularly anaplastic PXAs show considerable clinical, radiological, histological and molecularly overlap with eGBs resulting in difficulties when attempting to segregate these entities [2, 3, 7]. Though eGB occurs in de novo fashion in the majority of cases, some cases arose from lower-grade gliomas as well [7, 8, 9, 11, 22]. The fact that most of these lower-grade lesions documented so far were PXA and that both tumors commonly exhibit BRAF V600E mutation re-enforce the possibility that eGB and PXA are related [2, 11, 23]. Two independent studies in addition revealed a striking similarity of genetic alterations (BRAF V600E, TERT promoter mutations and CDKN2A/B homozygous deletions) in anaplastic PXAs and eGBs [24, 25]. Further, eGBs have shown PXA or astrocytoma like areas with presence of BRAF mutation in both components [5, 8, 26], thus suggesting that BRAF mutation might possibly be an early event to both tumors and additional genetic alterations such as TERT-p, LSAMP or CDKN2A, is essential for progression of PXA to eGB. [5, 13, 27]. Supporting this hypothesis were our two cases of PXA and A-PXA that progressed to eGB after acquiring CDKN2A homozygous deletion. Tanaka et al also recently reported a case of eGB developing within the tumor bed of a PXA, thirteen years after initial resection .
A recent study by Kurshonov et al documented considerable molecular and clinical heterogeneity within the eGBs by global DNA methylation and CNV analyses and established three distinct subsets: a PXA subset, with a high percentage of BRAF V600E mutations, but a relatively low percentage of TERT promoter mutations; an adult IDH-wild-type GB subset, with a relatively low percentage of BRAF V600E mutations, but a high percentage of TERT promoter mutations; and a pediatric RTK1 subset not harboring either mutation. They proposed that it is likely that the “epithelioid” GB phenotype represents a mere histologic pattern rather than a variant or separate entity .
In the current study PXA/A-PXA and e-GB exhibited some common findings on MRI: (i) contrast-enhanced solid and/or cystic; (ii) well circumscribed with no or little peritumoral edema; (iii) leptomeningeal involvement and dissemination. Histologically areas resembling PXA with focal lack of cytological uniformity along with spindled cells forming fascicles, few bizarre multinucleated cells, accompanied with infiltrating lymphocytes and perivascular lymphocytic cuffing were identified in three of fourteen (21.4%) eGB’s. However it was relatively inconspicuous in comparison to the adjacent, more abundant epithelioid rhabdoid looking tumor component as it constituted around 5–10% of the entire tumor raising the possibility that at least in few eGB’s, a precursor PXA component may be overgrown by the more malignant epithelioid component or was not sampled in the resection specimen. Mitotic figures, MVP and necrosis were found in both eGBs and A-PXA. Further, no reliable IHC or molecular differences were observed between the two glioma subtypes. In addition in our series A-PXA behaved clinically similar to eGB’s, with recurrences and death of a patient within 26 months of initial resection. All these findings suggest that eGB and PXA particularly A-PXA are either the same entity or highly related.
eGBs differs from conventional GB’s in that its course is often complicated by early recurrence, intratumoral hemorrhage and leptomeningeal spread and in addition it does not show EGFR amplification, IDH1 gene mutation or PTEN deletion, but instead, about 16.6–93% of these cases harbor BRAF V600E mutation [3, 8], a finding that remained true in our study as well.
In our study 100% concordance was found between Sanger sequencing and IHC results for BRAF V600E mutation, thus immunostaining could be used as a subsitute and cost-effective method for verification of the mutation. However any negative or low staining cases may be selected to undergo genetic analysis based on other clinical and histopathologic features. Other studies have also documented a high concordance between IHC and ARMs/Sanger sequencing with senstivity and specificity of IHC staining being 97–100% [28.29].
With the success of Immune checkpoint blockade (ICB) in various solid organ malignancies, the potential candidature of brain tumors for ICB is now being actively explored. Recent studies have reported PDL-1 and CTLA4 expression in GBs, which are targetable by prospective immunotherapies. Quantification of PDL-1 expression on tumor cells and CTLA-4 on T lymphocytes by IHC is widely being used as a predictive biomarker for ICB response [17, 30]. Adaptive upregulation of PDL-1 as an immune escape mechanism is usually seen in tumors with increased CTL infiltrates . A recent clinical trial reported lack of response to ICB in a small cohort of recurrent GBs despite PDL-1 expression and attributed the failure to the scarcity of intra-tumoral T-lymphocytes . Hereby we used a simplistic approach of IHC for PDL-1, CTLA-4, CD3 and CD8 to identify whether eGB’s and PXA are potential candidates for ICB.
In our small cohort the frequency and TPS of PDL-1 expression in PXA and eGB’s was found to be higher than 1°GB though the difference could not meet statistical significance p-value = 0.097 and 0.342). The frequency of PDL-1 expression was 71.4% in eGB, 66.67% in APXA and 60% in PXA as compared to 1°GB (44.4%). Although overall positivity rate reported in adult gliomas is 44.72%, a wide range of PDL-1 expression (6.1 to 100%) has been noted across different studies . Contrary to the studies on gliomas by Garber et al and Bergoff et al who documented high frequency of PDL-1 expression in only grade IV tumors, we observed its higher expression in PXA (grade II) and A-PXA (Grade III) cases [34, 35]. Although limited, the available studies present considerable differences in correlating PDL-1 expression with the patient outcome. In meta-analysis of 1052 patients, Xue et al. demonstrated high/positive PDL-1 expression to be associated with poor OS, which was also documented by Nduom et al [36, 37]. However Berghoff et al and Heiland et al observed no association of PDL-1 with OS [35, 38]. In present study also we could not elucidate any association between PDL-1 positivity and OS or PFS in tumor subtypes. Further, in all tumor subtypes, there was no association of PDL-1 with any specific genetic alteration (Fig. 2).
Both frequency and intensity of CTLA-4 expression was found to be significantly higher in eGB and PXA cases as compared to 1°GB respectively (P-value = 0.001 and 0.0008). Though CTLA-4 is a well studied immune checkpoint protein, its expression in glioma and its effects on prognosis was yet not examined until recently Liu et al in his study documented that CTLA-4 have a positive correlation with PD-1 and TIL’s and its higher expression are observed in higher grades of glioma and resulted in poor OS . Though no association of CTLA-4 with overall survival was documented in our study (P-value = 0.1286), a variety of clinical trials targeting CTLA-4 have demonstrated promising benefits in patients with glioma therefore comprehensive analysis of CTLA-4 expression will still be required to identify its enrichment criteria in gliomas .
Frequency and density of TIL’s expression was significantly higher in PXA (100% for both CD3 and CD8) and eGB (CD3-100%, CD8-92.9%) cases as compared to 1°GB (CD3-66.7%, CD8-55.6%) (P-value = 0.001 each, P-value = 0.0004 and 0.0001). Our findings were concordant with previous study by Berghoff et al and Kim et al who reported CD3 expression in 66.7 to 89.6% and CD8 expression in 44.4 to 77.6% GB’s [35, 41]. Though there was statistically no significant difference between low and high-density groups of TIL with respect to overall survival in our study (p-value = 0.21), few studies have observed prolonged survival with increased TIL [41, 42]. However Hans documented that CD8 + TILs alone cannot effectively predict patient outcome in GB whereas high CD4 + TIL levels in combination with low CD8 + TIL levels were associated with unfavourable prognosis .
We acknowledge that the power of survival analyses in our series is limited by the sample size and the retrospective study design, and our results surely need confirmation in larger and prospectively collected cohorts. These observations together offer some circumstantial evidence that PXA and eGB’s are more immunogenic thus reinforcing exploitation of PD-1/PDL-1 and CTLA-4 immune checkpoints for immune evasion.