Alpha-thalassemia/mental Retardation Syndrome X-linked Loss Research Articles

Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. GSE197988.

Abstract 458: Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma

Abstract Introduction: Alpha thalassemia/mental retardation syndrome X-linked (ATRX) plays a role in chromatin regulation and maintenance of telomeres. It regulates incorporation of histone H3.3 into telomeric chromatin. ATRX is also a major component of various essential cellular pathways such as DNA replication and repair, chromatin higher-order structure regulation, and gene transcriptional regulation. ATRX loss was observed in grades II/III astrocytomas, oligoastrocytomas, oligodendrogliomas, and glioblastomas. In grades II/III gliomas, most ATRX-loss cases had IDH1/2 mutations. Assessment of ATRX loss by IHC staining captures the majority of mutations, giving a reasonable sensitivity in neuropathology diagnostics. In this study, the specificity and sensitivity of ATRX were analyzed on multiple tissues focusing on Astrocytoma and Glioblastoma tissues. Materials and Methods: IHC staining has been performed by using formalin-fixed paraffin-embedded tissue with standard deparaffinization steps and a heat-induced retrieval method using a citrate buffer solution in a pressure cooker (Decloaking chamber). Antibody was incubated for 30 minutes, followed by HRP detection and DAB solution for visualization. Slides were stained using semi-automated staining system (intelliPATH). ATRX polyclonal antibody [Biocare Medical, California, USA] diluted in a carrier at an optimal concentration with standard protocol. Results: ATRX stained 44% of diffuse and anaplastic astrocytoma and 57% of glioblastoma. Also, cross-reactivity testing determined ATRX polyclonal may stain normal brain in some cases. A more comprehensive data evaluation shown below: Conclusions: ATRX polyclonal shows a potential advantage when diagnosing brain tumors (especially astrocytoma and glioblastomas) even if some cross reactivity can be observed. ATRX in conjunction with other brain marker antibodies can offer a better tool in diagnosing brain tumors. Diseased Tissue Specificity and SensitivityTissuePositive CasesTotal Cases% +Astrocytoma163644Glioblastoma4757Breast Cancer132748Colon Cancer84020Lung Cancer245246Prostate Cancer164238Normal Tissue Cross-ReactivityCerebrum51145Cerebellum2367 Citation Format: Jose Jonathan Diaz, Tatiana Scoggin, Jason Ramos. Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 458.

Correlation between IDH, ATRX, and TERT promoter mutations in glioma.

According to the 2016 World Health Organization (WHO) classification of central nervous system tumors, diffuse astrocytic and oligodendroglial tumors are differentiated by the presence of isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation and the combined loss of the short arm of chromosome 1 and the long arm of chromosome 19 (1p/19q co-deletion). IDH-mutant astrocytoma often has p53 and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutation, showing the alternative lengthening of telomeres (ALT) phenotype, while IDH-mutant and 1p/19q-co-deleted oligodendroglioma often have wild-type p53 and telomerase reverse transcriptase (TERT) promoter mutation, showing telomerase activation. This study analyzed IDH, ATRX, and TERT promoter mutations, and the correlation between them. Immortalized cells overcome the telomere-related crisis by activating telomerase or ALT. In glioma, telomerase is mainly activated by TERT promoter mutation, while ALT is usually associated with ATRX mutation. Although the mechanism of how ATRX mutation induces ALT remains unclear, ATRX loss alone is believed to be insufficient to induce ALT. Treatments targeting telomere maintenance are promising.

Incidence of biomarkers in high-grade gliomas and their impact on survival in a diverse SouthEast Asian cohort - a population-based study

BackgroundGliomas consist of a heterogeneous group of tumors. This study aimed to report the incidences of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, 1p19q co-deletion, isocitrate dehydrogenase (IDH) gene mutations, and inactivating mutations of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) in high-grade gliomas in an ethnically diverse population.MethodsRecords of patients who underwent surgery for high-grade gliomas from January 2013 to March 2017 at our institution were obtained. The patients’ age, gender, ethnicity, Karnofsky Performance Scale (KPS) score, ability to perform activities of daily living (ADLs), tumor location and biomarkers status were recorded. Data were analyzed using chi-square and Mann-Whitney U tests, Kaplan-Meier estimates and log-rank test.Results181 patients were selected (56 with grade III gliomas, 125 with grade IV gliomas). In the grade III group, 55% had MGMT promoter methylation, 41% had 1p19q co-deletion, 35% had IDH1 mutation and none had ATRX loss. In the grade IV group, 30% had MGMT promoter methylation, 2% had 1p19q co-deletion, 15% had IDH1 mutation and 8% had ATRX loss. After adjusting for effects of age, surgery and pre-operative ADL statuses, only MGMT promoter methylation was found to be significantly associated with longer overall survival time in grade III (p = 0.024) and IV patients (p = 0.006).ConclusionsThe incidences of MGMT promoter methylation and IDH1 mutation were found to be comparable to globally reported rates, but those of 1p19q co-deletion and ATRX loss seemed to be lower in our cohort. MGMT promoter methylation was associated with increased overall survival in our cohort and might serve as favorable prognostic factor.

Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation

PurposeIsocitrate dehydrogenase 1 (IDH1) mutations are associated with improved survival in gliomas. Depending on the IDH1 status, TERT promoter mutations affect prognosis. IDH1 mutations are associated with alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations and alternative lengthening of telomeres (ALT), suggesting an interaction between IDH1 and telomeres. However, little is known how IDH1 mutations affect telomere maintenance.MethodsWe analyzed cell-specific telomere length (CS-TL) on a single cell level in 46 astrocytoma samples (WHO II-IV) by modified immune-quantitative fluorescence in situ hybridization, using endothelial cells as internal reference. In the same samples, we determined IDH1/TERT promoter mutation status and ATRX expression. The interaction of IDH1R132H mutation and CS-TL was studied in vitro using an IDH1R132H doxycycline-inducible glioma cell line system.ResultsVirtually all ALTpositive astrocytomas had normal TERT promoter and lacked ATRX expression. Further, all ALTpositive samples had IDH1R132H mutations, resulting in a significantly longer CS-TL of IDH1R132H gliomas, when compared to their wildtype counterparts. Conversely, TERT promotor mutations were associated with IDHwildtype, ATRX expression, lack of ALT and short CS-TL. ALT, TERT promoter mutations, and CS-TL remained without prognostic significance, when correcting for IDH1 status. In vitro, overexpression of IDHR132H in the glioma cell line LN319 resulted in downregulation of ATRX and rapid TERT-independent telomere lengthening consistent with ALT.ConclusionALT is the major telomere maintenance mechanism in IDHR132H mutated astrocytomas, while TERT promoter mutations were associated with IDHwildtype glioma. IDH1R132H downregulates ATRX expression in vitro resulting in ALT, which may contribute to the strong association of IDH1R132H mutations, ATRX loss, and ALT.

Loss of ATRX/DAXX expression and alternative lengthening of telomeres in uterine leiomyomas.

Uterine leiomyomas (ULs) are the most common gynecologic tumors and affect 3 of every 4 women by the age of 50 years. The majority of ULs are classified as conventional tumors, whereas 10% represent various histopathological subtypes with features that mimic malignancy. These subtypes include cellular and mitotically active ULs and ULs with bizarre nuclei. Uterine leiomyosarcoma (ULMS), the malignant counterpart of UL, is an aggressive cancer with poor overall survival. The early diagnosis and preoperative differentiation of ULMS from UL are often challenging because their symptoms and morphology resemble one another. Recent studies have shown frequent loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX) expression in ULMS, and this is often associated with an alternative lengthening of telomeres (ALT) phenotype. To investigate ATRX and DAXX expression and the presence of ALT in UL subtypes, immunohistochemical and telomere-specific fluorescence in situ hybridization analyses were performed. The study material consisted of 142 formalin-fixed, paraffin-embedded tissue samples representing various UL subtypes and 64 conventional ULs. A loss of ATRX or DAXX and/or ALT was detected in 6.3% of the histopathological UL subtype samples (9 of 142). Two patients whose ULs showed either ATRX loss or ALT were later diagnosed with a pulmonary smooth muscle tumor. Pulmonary tumors displayed molecular alterations found in the corresponding uterine tumors, which indicated metastasis to the lungs. All conventional ULs displayed normal ATRX, DAXX, and telomeres. These results highlight the differences between conventional and histopathologically atypical ULs and indicate that some UL subtype tumors may harbor long-term malignant potential.

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.

MGMT gene promoter methylation and its correlation with clinicopathological parameters in glioblastomas.

MGMT (O6-methyl guanine DNA methyl transferase) promoter hypermethylation is a prognostic and predictive biomarker for glioblastomas (GBM). To evaluate the frequency of MGMT methylation status in a single institute series of 134 GBMs and correlate it with clinical (age, sex, location, survival) and other molecular parameters [such as p53 expression, alpha thalassemia/mental retardation syndrome X-linked (ATRX) expression, isocitrate dehydrogenase (IDH) 1R132H mutation, and epidermal growth factor receptor (EGFR) gene amplification]. One hundred and thirty-four GBMs were evaluated by methylation-specific polymerase chain reaction (MSP) for MGMT promoter methylation status. The results were correlated with the above mentioned clinicopathological parameters. MGMT gene promoter methylation was identified in 49.2% (66/134) GBMs, and was significantly associated with IDH1R132H mutation (14/66; 21%; P - value, 0.01) and ATRX loss (15/66; 23%; P - value, 0.01). Confluent necrosis was found to be significantly associated with MGMT unmethylation status (P - value: 0.002). Multivariable logistic regression analysis showed confluent necrosis as a single independent predictor (odds ratio [OR], 2.5; confidence interval [CI], 1.0-5.8; P - value, 0.04) of MGMT unmethylation status among all the parameters studied. The frequency of MGMT promoter methylation in GBMs was 49.2%, which was significantly associated with IDHR132H mutation and ATRX loss. In addition, the presence of confluent necrosis was significantly associated with MGMT unmethylation and was found to be an independent predictor of the same.

In vivo molecular profiling of human glioma using diffusion kurtosis imaging.

The purpose of this study is to assess the diagnostic performance of diffusion kurtosis imaging (DKI) for in vivo molecular profiling of human glioma. Normalized mean kurtosis (MKn) and mean diffusivity (MDn) metrics from DKI were assessed in 50 patients with histopathologically confirmed glioma. The results were compared in regard to the WHO-based histological findings and molecular characteristics leading to integrated diagnosis (Haarlem Consensus): isocitrate-dehydrogenase (IDH1/2) mutation status, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression, chromosome 1p/19q loss of heterozygosity (LOH), and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status. MKn was significantly lower in tumors with IDH1/2 mutation (0.43 ± 0.09) and ATRX loss of expression (0.41 ± 0.11) than in those with IDH1/2 wild type (0.57 ± 0.09, p < 0.001) and ATRX maintained expression (0.51 ± 0.10, p = 0.004), respectively. Regarding the integrated molecular diagnosis, MKn was significantly higher in primary glioblastoma (0.57 ± 0.10) than in astrocytoma (0.39 ± 0.11, p < 0.001) and oligodendroglioma (0.47 ± 0.05, p = 0.003). MK may be used to provide insight into the human glioma molecular profile regarding IDH1/2 mutation status and ATRX expression. Considering the diagnostic and prognostic significance of these molecular markers, MK appears to be a promising in vivo biomarker for glioma. The diagnostic performance of MK seems to fit more with the integrated molecular approach than the conventional histological findings of the current WHO 2007 classification.

MPTH-30ATRX CAN OCCUR IN IDH WILD-TYPE GBM

BACKGROUND: ATRX (alpha thalassemia/ mental retardation syndrome X-linked) is a transcriptional regulatory protein which contains an ATPase/helicase domain. In gliomas loss of ATRX expression has been recently described and is mostly restricted to IDH mutant tumors who seem to have a significantly better prognosis. ATRX loss is thought to be extremely rare in IDH wild type (WT) tumors and in that setting one group reported H3F3A mutation. METHODS: Tissue samples of 23 patients with IDH wild-type malignant gliomas were analyzed for ATRX expression using immunohistochemistry. ATRX status was associated with clinical characteristics, histology and other molecular markers (MGMT, BRAF, EGFR). RESULTS: Median age in all patients was 49,3 years. Loss of ATRX expression was detected in 4 out of 23 patient tumors. All ATRX loss patients were primary glioblastomas. Median survival in ATRX mutated gliomas is 237 weeks which was longer than overall survival in gliomas with retained ATRX (88 weeks). All of our patients underwent concurrent chemoradiotherapy with temozolomide. One out of four ATRX loss tumors was BRAF mutated, three out of four ATRX loss patients tested for EGFR were found to be wild type and the two ATRX mutated patients tested for MGMT were methylated. CONCLUSION: ATRX mutation can occur in IDH wild-type patients. In that setting it appears to be associated with a better outcome. Our data is hypothesis generating. Tumors with IDH WT/ ATRX loss should be evaluated prospectively in a larger cohort and possibly checked for H3F3A mutation.

O8.06 * ATRX AND TERT EXPRESSION IN RELAPSING LOW GRADE GLIOMA

Mechanisms responsible for telomere maintenance are been recently elucidated: many cancers harbor mutations in alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene resulting in a phenomenon known as alternative lengthening of telomeres (ALT) or point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene leading to an increased telomerase expression. ATRX and TERT mutations have been recently identified also in brain tumors. In gliomas, a molecular stratification based on IDH and ATRX mutational status and 1p/19q codeletion has been suggested. ATRX mutations have been observed mainly in astrocytic tumors and TERT promoter mutations mainly in oligodendroglial tumors. Aim of our study was to evaluate the expression of both ATRX and TERT in WHO grade II gliomas and in their resected recurrences, related to a set of confirmed molecular markers and to clinical data. We performed an immunohistochemical analysis of ATRX and TERT expression in 30 grade II gliomas (astrocytomas, oligoastrocytomas and oligodendroglioma) and in their respective recurrences (grade III-IV). Also evaluation of IDH1 and p53 status and 1p/19q LOH were carried out. Loss of expression of ATRX protein has been associated to the mutated gene and ALT phenotype, while expression of TERT protein could be related to TERT promoter mutations. We found a loss of ATRX expression in 70% of cases; recurrences showed the same pattern of ATRX expression. In the most part of cases ATRX loss was associated to IDH1 and p53 mutation and intact 1p/19q, as previously observed. In the major part of cases with ATRX loss of expression TERT was not detected. TERT high expression was observed in about 30% of grade II gliomas at the first surgery, minor expression of TERT was found in grade III-IV recurrent tumors. In most case high expression of TERT was associated to 1p/19q deletion. We observed a statistically significant correlation between TERT high expression and a better outcome. Our study of protein expression of both ATRX and TERT in low grade gliomas and in their recurrences in relationship with established molecular markers and follow-up data, confirm the possibility of using immunohistochemistry in routine diagnostic procedures for better define this subset of neoplasm.

O10.01 * PROGNOSTIC AND PREDICTIVE BIOMARKER-BASED SUBGROUPS IN THE NOA-04 TRIAL

BACKGROUND: Molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, mutations and (consequently) loss of expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, improve prognostication and may even guide treatment decisions in patients with anaplastic gliomas. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) allow the determination of large-scale methylation profiles and genome-wide DNA copy number changes, enabling molecular subgrouping of tumors. In addition, algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation using this assay. METHODS: In the biomarker cohort of the NOA-04 trial, the diagnostic and prognostic performance of these molecular markers using single tests, HM450 data and HM450-based algorithms has been investigated to propose biological subgroups, which reflect outcomes and potentially influence treatment decisions. RESULTS: Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged progression-free survival (PFS) with chemotherapy or radiotherapy (RT), and thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, too, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. Comparisons of single assays and HM450-based algorithms revealed a high concordance for IDH and 1p/19q status. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of cases as conventional methylation-specific PCR, with 87/91 G-CIMP-positive tumors predicted as MGMT promoter-methylated. CONCLUSIONS: ATRX loss is a hallmark and favorable prognosticator of astrocytic tumors allowing a better definition of the clinically and morphologically mixed group of AOA. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wildtype, but not IDH1-mutant malignant gliomas of WHO grades III/IV. Combined IDH1/ MGMT assessment may help to individualize clinical decision making in neurooncology. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and associated with prognosis in the NOA-04 trial. HM450 arrays allowed clustering of anaplastic gliomas into relevant subgroups.

P17.71 * ROLE OF ATRX AND TERT EXPRESSION AS DIAGNOSTIC AND PROGNOSTIC FACTORS ON ANAPLASTIC GLIOMAS

Molecular and histological classification of anaplastic gliomas have been recently refined by the elucidation of the role of mutations in alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene and in telomerase reverse transcriptase (TERT) promoter. Loss of ATRX expression and the acquisition of alternative lengthening of telomeres (ALT) phenotype is peculiar of a subtype of astrocytic tumours with p53 and IDH1 mutations and better prognosis, while TERT overexpression, due to its promoter's mutations, is strongly associated to oligodendroglial tumours and 1p/19q codeletion, without a significant correlation with survival. Based on these findings, the aim of our study was to better clarify the role of ATRX and TERT expression in the histological classification of anaplastic gliomas, as diagnostic and prognostic markers of survival and relapse. We performed immunohistochemical and molecular analysis of ATRX and TERT expression on 60 grade III gliomas (astrocytomas, oligoastrocytomas and oligodendrogliomas) and the respective recurrences when they were available. Nuclear accumulation of p53, IDH1 status and LOH of chromosomes 1p, 19q, 9p, 17p and 10q were also evaluated. Our results showed a loss of ATRX expression in 60% of anaplastic oligoastrocytomas and 40% of anaplastic astrocytomas, while ATRX was wild-type in oligodendroglial tumours. We then found a strong positive correlation between loss of ATRX expression and p53 nuclear accumulation, while its strong expression was present mostly in tumours with 1p/19q codeletion. Finally, ATRX loss was correlated with a better prognosis both in anaplastic oligoastrocytomas and in anaplastic astrocytomas according to literature. Regarding TERT expression, we did not find a strictly correlation with 1p/19p codeletion and the oligodendroglial histological pattern, but, surprisingly, we found a possible role in relapse of anaplastic gliomas. In our series, tumours with high TERT expression tend to recur within 25 months, while anaplastic gliomas with low o null expression of TERT tend to recur within 60 months. In conclusion, these set of data could allow us in the future to better classify anaplastic gliomas and their tendency to recur adding immunohistochemistry for ATRX and TERT to other molecular markers in routine diagnostic procedures, and to define new molecular profiles for their therapy.

PROGNOSTIC AND PREDICTIVE BIOMARKER-BASED SUBGROUPS IN THE NOA-04 TRIAL

BACKGROUND: The WHO classification, based on morphological criteria, may be increasingly supplemented with defined molecular aberrations. These might help to resolve the discrepancy between classification and clinical outcome. Molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, mutations and (consequently) loss of expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, improve prognostication and may even guide treatment decisions in patients with anaplastic gliomas. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) allow the determination of large-scale methylation profiles and genome-wide DNA copy number changes, enabling molecular subgrouping of tumors. In addition, algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation using this assay. METHODS: In the biomarker cohort of the NOA-04 trial, the diagnostic and prognostic performance of these molecular markers using single tests, HM450 data and HM450-based algorithms has been investigated to propose biological subgroups, which reflect outcomes and potentially influence treatment decisions. RESULTS: Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged progression-free survival (PFS) with chemotherapy or radiotherapy (RT), and thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, too, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. Comparisons of single assays and HM450-based algorithms revealed a high concordance for IDH and 1p/19q status. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of cases as conventional methylation-specific PCR, with 87/91 G-CIMP-positive tumors predicted as MGMT promoter-methylated. CONCLUSIONS: ATRX loss is a hallmark and favorable prognosticator of astrocytic tumors allowing a better definition of the clinically and morphologically mixed group of AOA. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wildtype, but not IDH1-mutant malignant gliomas of WHO grades III/IV. Combined IDH1/ MGMT assessment may help to individualize clinical decision making in neurooncology. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and associated with prognosis in the NOA-04 trial. HM450 arrays allowed clustering of anaplastic gliomas into relevant subgroups. SECONDARY CATEGORY: Tumor Biology.

ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis

Mutation/loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression has been described in anaplastic gliomas. The present study explored the role of ATRX status in the molecular classification of anaplastic gliomas and its impact on survival in the biomarker cohort of the NOA-04 anaplastic glioma trial. Patients (n = 133) of the NOA-04 trial were analyzed for ATRX expression using immunohistochemistry. ATRX status was correlated with age, histology, isocitrate dehydrogenase (IDH), 1p/19q, alternative lengthening of telomeres (ALT) and O6-methylguanine-DNA methyltransferase (MGMT) status, and the trial efficacy endpoints. Loss of ATRX expression was detected in 45 % of anaplastic astrocytomas (AA), 27 % of anaplastic oligoastrocytomas (AOA) and 10 % of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. The ALT phenotype was significantly correlated with ATRX loss. ATRX and 1p/19q status were used to re-classify AOA: AOA harboring ATRX loss shared a similar clinical course with AA, whereas AOA carrying 1p/19q co-deletion shared a similar course with AO. Accordingly, in a Cox regression model including ATRX and 1p/19q status, histology was no longer significantly associated with time to treatment failure. Survival analysis showed a marked separation of IDH mutant astrocytic tumors into two groups based on ATRX status: tumors with ATRX loss had a significantly better prognosis (median time to treatment failure 55.6 vs. 31.8 months, p = 0.0168, log rank test). ATRX status helps better define the clinically and morphologically mixed group of AOA, since ATRX loss is a hallmark of astrocytic tumors. Furthermore, ATRX loss defines a subgroup of astrocytic tumors with a favorable prognosis.