Practice of the New Integrated Molecular Diagnostics in Gliomas: Experiences and New Findings in a Single Chinese Center

Abstract

Background: The latest WHO classification of CNS tumors using the integrated phenotypic and molecular parameters (IDH, ATRX, 1p19q, TERT etc.) have reestablished the CNS tumors classification in addition to traditional histology. The establishment of glioma molecular typing can more accurately predict prognosis, better guide individualized treatment to improve survival. Methods: The expression of IDH1, ATRX, PHH3, P53 and Ki67 was detected by IHC. Molecular status of IDH1/2 and TERT were analyzed using Sanger sequencing. MGMT was explored using methylation-specific PCR. 1p/19q codeletion status was firstly detected by FISH, then further confirmed by multiplex PCR-based next generation sequencing. Results: The mutation frequency of IDH1 was 68.7% (79/115) in WHO II astrocytoma, and 82 cases (82/344, 23.8%) were "triple-negative glioma" in our cohort. Multivariate COX analysis revealed that only IDH, 1p/19q, TERT and MGMT were independent prognostic factors. Noteworthily, we found 7 cases of the new molecular phenotype presented as "IDH wildtype and 1p/19q codeletion", not mentioned in the latest WHO guideline. Conclusion: We detected the newly recommended markers in a large cohort of Chinese glioma patients. Our data demonstrated a relatively lower frequency of IDH mutations and a higher prevalence of triple-negative glioma in Chinese compared with American and European, indicating ethnic and geographical difference in some markers. In addition, the new molecular phenotype "IDH wildtype and 1p/19q codeletion" glioma deserved special focus. These findings suggest that further stratification of infiltrating gliomas is needed for different treatment strategy and precision medicine.

Keywords: 1p/19q; ATRX; FISH; IDH; MGMT; Sanger sequencing; TERT; glioblastoma; glioma.

Figure 1

Representative IHC staining cases of (A) ATRX (nuclear loss expression while nuclei of non-neoplastic cells such as endothelia, microglia, lymphocytes and reactive astrocytes were strongly positive), (B) IDH1(strong diffuse cytoplasmic staining), (C) Ki67(Nuclear positive), (D) P53 (Nuclear positive), (E) PHH3 (Nuclear positive). (40X and 200X).

Figure 2

Representative Sanger sequencing images of IDH mutation, TERT promoter mutation and FISH image of 1p/19q deletion. (A) IDH1 R132H mutation (red circle), (B) TERT promoter C250T mutation (red circle), (C) 1p deletion, (D) 19q deletion, with 1 red and 2 green signals in scattered nuclei. Some signals are missing due to nuclear truncation and overlap.

Figure 3

Kaplan-Meier survival analysis of (A) IDH1 mutation status, (B)1p/19q codeletion status, (C) ATRX immunoreactivity (negative=ATRX mutation), (D)ATRX status in WHO II astrocytoma patients, (E) P53 immunoreactivity, (F) Ki-67 labelling index (cut-off value=10%), (G) Patients' age at diagnosis (cut-off value=45 years), (H) PHH3(mitosis number/10HPF, cut-off value=5 ), (I) TERT promoter status in WHO IV glioblastoma patients and (J) MGMT promoter methylation status in all the infiltrating glioma patients. Log rank test p values are also showed for each parameter. IDH1 mutation, 1p/19q codeletion and MGMT promoter methylation were good prognostic indicators in all the infiltrating gliomas. Transitional markers such as high expression of P53/Ki67/PHH3 and old age exerted unfavorable prognosis. ATRX only have prognostic value in WHO grade II gliomas, and TERT promoter mutation only have prognostic value in WHO grade IV glioma (GBM) in our cohort.