Prognostic stratification for IDH-wild-type lower-grade astrocytoma by Sanger sequencing and copy-number alteration analysis with MLPA

Abstract

The characteristics of IDH-wild-type lower-grade astrocytoma remain unclear. According to cIMPACT-NOW update 3, IDH-wild-type astrocytomas with any of the following factors show poor prognosis: combination of chromosome 7 gain and 10 loss (+ 7/- 10), and/or EGFR amplification, and/or TERT promoter (TERTp) mutation. Multiplex ligation-dependent probe amplification (MLPA) can detect copy number alterations at reasonable cost. The purpose of this study was to identify a precise, cost-effective method for stratifying the prognosis of IDH-wild-type astrocytoma. Sanger sequencing, MLPA, and quantitative methylation-specific PCR were performed for 42 IDH-wild-type lower-grade astrocytomas surgically treated at Kyoto University Hospital, and overall survival was analysed for 40 patients who underwent first surgery. Of the 42 IDH-wild-type astrocytomas, 21 were classified as grade 4 using cIMPACT-NOW update 3 criteria and all had either TERTp mutation or EGFR amplification. Kaplan-Meier analysis confirmed the prognostic significance of cIMPACT-NOW criteria, and World Health Organization grade was also prognostic. Cox regression hazard model identified independent significant prognostic indicators of PTEN loss (risk ratio, 9.75; p < 0.001) and PDGFRA amplification (risk ratio, 13.9; p = 0.002). The classification recommended by cIMPACT-NOW update 3 could be completed using Sanger sequencing and MLPA. Survival analysis revealed PTEN and PDGFRA were significant prognostic factors for IDH-wild-type lower-grade astrocytoma.

Figure 1

(A) The cell plot shows characteristics of all 42 IDH-wild-type astrocytomas. (B) The schema of three step classification of IDH-wild-type astrocytomas. TERTp mutation was examined by Sanger sequencing, and EGFR amplification and combination of EGFR gain and PTEN loss were examined by multiplex ligation-dependent probe amplification (MLPA). The case with red square was diagnosed as “astrocytoma. grade 4”.

Figure 2

Survival analysis of 40 patients whose tumours were removed at their first surgery, were shown. The overall survival was analysed stratified by the following factors associated with the cIMPACT-NOW update 3 criteria; (A) the diagnosis of “astrocytoma, grade 4”, (B) TERTp mutation, or (C) EGFR amplification, in the groups of all IDH-wild type astrocytomas, IDH-wild type diffuse astrocytomas, and IDH-wild type anaplastic astrocytomas. The p values were calculated log-rank test and Wilcoxon test, and p < 0.05 was shown with red letters.

Figure 3

Survival analysis of 40 patients whose tumours were removed at their first surgery, were shown. (A) The overall survival of IDH-wild type diffuse astrocytomas (grade II) and anaplastic astrocytomas (grade III) were compared in the groups of all IDH-wild type astrocytomas, “astrocytoma, grade 4”, and not-“astrocytoma, grade 4”. The overall survival was also analysed stratified by the following factors associated with factors not included in cIMPACT-NOW update 3 criteria; (B) PDGFR amplification, (C) PTEN status (intact, hemizygous loss, or homozygous loss), and (D) the combination status of PTEN loss and EGFR gain. The p values were calculated log-rank test and Wilcoxon test, and p < 0.05 was shown with red letters.