ATRX loss promotes immunosuppressive mechanisms in IDH1 mutant glioma.

BACKGROUND: ATRX inactivation occurs with IDH1R132H and p53 mutations in over 80% of Grades II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1R132H/p53mut glioma cells and xenografts. METHODS: Isogenic astrocytoma cells (+/-IDH1R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline-inducible IDH1R132H, ATRX shRNA, or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1R132H+/-ATRXloss on cell migration, growth, DNA repair, and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays, and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization, and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1R132H/p53mut/ATRXloss cells. RESULTS: Adding ATRX loss to the IDH1R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune-suppressive transcriptome consisting of up-regulated immune checkpoints (e.g., PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g., IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune-suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition. CONCLUSIONS: ATRX loss activates a BRD-dependent immune-suppressive transcriptome and immune escape mechanism in IDH1R132H/p53mut astrocytoma cells.

Association of MGMT Gene Promoter Methylation With Clinicopathological Parameters in Patients With Wild-type IDH Glioblastoma.

BACKGROUND: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter plays a key role in response to temozolomide chemotherapy and disease prognosis in patients with wild-type isocitrate dehydrogenase (IDH) glioblastoma (GBM). PATIENTS AND METHODS: The MGMT promoter methylation status and its association with clinicopathological parameters were retrospectively analysed in a cohort of 316 patients with GBM with wild-type IDH. RESULTS: MGMT methylation was significantly associated with ATRX chromatin remodeler (ATRX) loss and completion of the standard Stupp protocol. The median durations of overall and progression-free survival for the unmethylated, low-methylated (10-39%), and hypermethylated (≥40%) groups were 15, 23, and 30 months and 11, 18, and 21 months, respectively. However, the improvement in the survival of the hypermethylated group was not statistically significant. CONCLUSION: We suggest a possible association between MGMT methylation status and ATRX mutations in GBM with wild-type IDH.

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative "C-circle" assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

Higher Plasma Fibrinogen Levels are Associated with Malignant Phenotype and Worse Survival in Patients with Glioblastomas.

Preoperative plasma fibrinogen levels were associated with poor clinical outcomes in malignancies. There were few data about the prognostic value of plasma fibrinogen in glioblastomas (GBMs). The objective of our study was to investigate the association between fibrinogen and patients' clinicopathological factors and overall survival (OS). From 2008 to 2016, 315 patients with GBMs who had a surgical treatment at our institute, were retrospectively involved in this study. IDH (isocitrate dehydrogenase) mutations and ATRX (alpha thalassemia/mental retardation syndrome X-linked) loss were detected with IHC (Immunohistochemistry). The preoperative plasma fibrinogen levels ranged from 1.00 to 5.22 g/L, with a mean of were 2.57 g/L. There were increased levels of plasma fibrinogen in patients aged ≥ 65 years, secondary GBMs, IDH mutation (p = 0.033) and ATRX loss (p = 0.040). Moreover, the plasma fibrinogen level was the highest in the subtype of IDH-1R132H wildtype - ATRX expression, which showed a shorter OS compared to the group of IDH-1R132H mut and IDH-1R132H wildtype - ATRX loss (p = 0.001, log-rank test). ROC curves for fibrinogen and IDH-1R132H wildtype - ATRX expression was also plotted, and indicated a potential diagnostic value of fibrinogen in molecular pathology. Univariate analysis found that younger age, higher KPS (Karnofsky Performance Score), gross total resection, complete chemoradiotherapy, IDH-1R132H mutations and lower levels of fibrinogen were associated with favorable outcomes. Multivariate analysis proved that chemoradiotherapy, IDH-1R132H and fibrinogen were independent prognostic factors. In conclusion, plasma fibrinogen could predict clinical outcome and molecular subtype in GBMs.

Detection of ATRX and IDH1-R132H immunohistochemistry in the progression of 211 paired gliomas.

Recurrence and progression to higher grade lesions are key biological events and characteristic behaviors in the evolution process of glioma. A small residual population of cells always escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence or progression. IDH mutation (isocitrate dehydrogenase) and ATRX (alpha-thalassemia/mental retardation, X-linked) loss/mutation occur in association and may represent early genetic alterations in the development of gliomas. However, their prognostic value in the evolution of gliomas still needs further investigation.Two hundreds and eleven serial sampling of gliomas were included in our study. We used immunohistochemistry (IHC) to detect IDH1-R132H mutation and ATRX status and showed that the IDH1-R132H and (or) ATRX status could be necessary to provide the basic molecular information for the "integrated diagnosis" of gliomas. We illustrated an evaluation formula for the evolution of gliomas by IDH1-R132H combined with ATRX immunohistochemistry and identified the association of IDH1-R132H/ATRX loss accompanied by longer progression time interval of patients with gliomas. Furthermore, we observed that most recurrences had a consistent IDH1 and ATRX status with their matched primary tumors and demonstrated the progressive pattern of grade II astrocytoma/oligodendroglial tumors and anaplastic oligoastrocytoma with or without IDH1-R132H. Identification of IDH1-R132H and ATRX loss status in the primary-recurrent gliomas may aid in treatment strategy selection, therapeutic trial design, and clinical prognosis evaluation.