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P03.10 Combined immunohistochemistry for IDH1R132H, p53 and ATRX improves the molecular classification of diffuse gliomas in adults

ABSTRACT: Abstract Introduction: The updated 2016 edition of the WHO Classification of Tumor of the Central Nervous System has shifted from the traditional approach primarily based on microscopic features to that on genetic hallmarks. The classification of diffuse gliomas is now defined in accordance with their IDH and 1p/19q status. Although that shift has resulted in a more objective definition of diffuse gliomas, genetic testings, which are not always available worldwide, has become mandatory for WHO diagnosis. Immunohistochemistry (IHC) for mutant IDH1R132H and fluorescence in situ hybridization (FISH) for 1p/19q are the most popular methods, but IDH1R132H does not detect other loci and currently available probes for 1p36 and 19q13 are known to yield false positive results (about 15%). To overcome such disadvantages, we verified the utility of combined IHC for p53 and ATRX in addition to IDH1R132H, since about 90% of IDH mutated astrocytomas carry TP53 mutations followed by ATRX mutations in 80% of TP53 mutants and TP53 or ATRX mutations, which are detectable on IHC, are mutually exclusive to 1p/19q co-deletion. Materials and Methods: Archived 97 grade II-IV diffuse gliomas of adults with known 1p/19q status on FISH testing (Vysis/Abbotte DNA FISH probes, Chicago, IL) were served for study. Using Envision kit (Dako, Santa Clara, CA), IDHR132H (H09; Dianova, Hamburg, Germany), p53 (DO-7; DAKO) and ATRX (polyclonal HPA001906; Sigma-Aldrich, St. Louis, MO) were applied formalin-fixed, paraffin-embedded sections. Tumors with strongly positive nuclei > 10% of tumor cells on p53 IHC was regarded as positive (TP53 mutated) based on previous studies. Results: All ATRX-lost gliomas were 1p/19q non-deleted. Seven IDH1R132H-negative tumors were p53-positive and ATRX-lost. Six IDH1R132H-positive and ATRX-lost tumors were p53-negative. Of ATRX-intact 53 tumors, 19 tumors were p53-positive; 18 of which were 1p/19q non-deleted. Of 13 co-deleted tumors, two were IDH1R132H-negative. Conclusions: ATRX loss in gliomas was equivalent to 1p/19q non-deletion. IDH1R132H-negative, p53-positive and ATRX-lost gliomas were highly suspicious of carrying IDH mutations other than IDH1R132H. IDH1R132H-positive, ATRX-lost and p53-negative gliomas were suggested to have nonsense or frame-shift mutations of TP53. Although when IDH1R132H and p53 are negative and ATRX is intact it is difficult to predict 1p/19q status, the combined immunohistochemistry for IDH1R132H, p53 and ATRX improves the molecular classification of diffuse gliomas in daily practice.

SUBMITTER: Komori T

PROVIDER: S-EPMC5463599 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Project description:Abstract Immunotherapeutic concepts for brain tumors have been hampered by lack of selective measures to target the CNS and lack of truly tumor-specific antigens. But the concept of an immune privileged CNS has to be questioned due to survey of the brain by specific T-cells, CNS autoimmunity and presence of lymphatics. As tumor-specific antigens, mutated antigens have come into focus for all tumor entities. Mutations in the gene for isocitrate dehydrogenase 1 frequently occur in diffuse gliomas, mostly astrocytomas, resulting in a point mutation (mostly IDH1R132H). IDH1R132H is an ideal tumor-specific antigen, because it is tumor-specific, homogenously expressed and considered a driving mutation in glioma. Preclinical studies have shown that mutation-specific Th cell responses spontaneously occur in patients with IDH1-mutated gliomas and that a peptide vaccine encoding IDH1R132H is therapeutic in a humanized mouse tumor model. Based on these data we have initiated a multicenter, first-in-man, phase I clinical vaccine trial, which is planned to enroll 39 patients with newly diagnosed malignant IDH1R132H+ astrocytomas at eight German sites: NOA-16 (NCT02454634). The vaccine is made of an IDH1R132H peptide emulsified in mineral oil and administered with topical imiquimod. Vaccination is implemented into primary standard therapy. Primary end points are safety and immunogenicity as measured by IDH1R132H-specific antibodies and T cell responses. As of March 2016, 13 patients have been enrolled in the trial; no severe adverse events have been reported. We report here on 3 patients treated with the vaccine on a compassionate use basis. No patient experienced a regime-limiting toxicity as defined in the trial protocol. Two patients received all 8 doses of the vaccine; both developed IDH1-specific antibody responses, which were mutation-specific early after vaccination. Of these, one patient developed a vaccination-induced mutation-specific cellular response. The second patient had a high baseline mutation-specific T cell response, which was temporarily boosted by the vaccine. IFN-? catch assays from PBMCs of this patient revealed a Th cell-mediated response. To identify and clone IDH1R132H-specific TCR(s) for adoptive T cell therapy, single T cells from this patient specifically responding to IDH1R132H were sorted and subjected to TCR sequencing. 11 TCRs have been identified so far and will be cloned and validated. In summary, we demonstrate for the first time the induction of a mutation-specific humoral and cellular immune response to the IDH1R132H neoepitope after peptide vaccination of patients with IDH1R132H-mutated gliomas. The ongoing trial will analyze safety and immunogenicity of the vaccine in a defined patient cohort, which also allows for collecting data on therapeutic efficacy.

Project description:BackgroundGlioma is the most common of all primary brain tumors with poor prognosis and high mortality. The 2016 World Health Organization classification of the tumors of central nervous system uses molecular parameters in addition to histology to redefine many tumor entities. The new classification scheme divides diffuse gliomas into low-grade glioma (LGG) and glioblastoma (GBM) as per histology. LGGs are further divided into isocitrate dehydrogenase (IDH) wild type or mutant, which is further classified into either oligodendroglioma that harbors 1p/19q codeletion or diffuse astrocytoma that has an intact 1p/19q loci but enriched for ATRX loss and TP53 mutation. GBMs are divided into IDH wild type that corresponds to primary or de novo GBMs and IDH mutant that corresponds to secondary or progressive GBMs. To make the 2016 WHO subtypes of diffuse gliomas more robust, we carried out Prediction Analysis of Microarrays (PAM) to develop DNA methylation signatures for these subtypes.ResultsIn this study, we applied PAM on a training set of diffuse gliomas derived from The Cancer Genome Atlas (TCGA) and identified DNA methylation signatures to classify LGG IDH wild type from LGG IDH mutant, LGG IDH mutant with 1p/19q codeletion from LGG IDH mutant with intact 1p/19q loci and GBM IDH wild type from GBM IDH mutant with an accuracy of 99-100%. The signatures were validated using the test set of diffuse glioma samples derived from TCGA with an accuracy of 96 to 99%. In addition, we also carried out additional validation of all three signatures using independent LGG and GBM cohorts. Further, the methylation signatures identified a fraction of samples as discordant, which were found to have molecular and clinical features typical of the subtype as identified by methylation signatures.ConclusionsThus, we identified methylation signatures that classified different subtypes of diffuse glioma accurately and propose that these signatures could complement 2016 WHO classification scheme of diffuse glioma.

Project description:IntroductionDiffuse glioma constitutes 28% of primary brain tumors. Until recently morphologic appearance was the only criterion for classifying these tumors. However, WHO 2016 incorporates molecular information in the primary diagnosis of gliomas such as Isocitrate dehydrogenase 1 (IDH1), Alpha thalassemia/mental retardation syndrome X inked (ATRX) as well as 1p/19q codeletion on FISH. In a resource-limited setup where FISH is not available, Alpha internexin (INA) has been suggested as a surrogate IHC marker.Material and methodsCross-sectional study conducted in the Department of Pathology for two years. Tissue blocks and clinical as well as radiological details were obtained from departmental archives. After assessing the morphologic details, routine IHC markers such as GFAP, Ki67 and P53 along with molecular markers like IDH-1, ATRX, and lNA were applied.ResultsOut of 55 cases of diffuse glioma, 23 cases of astrocytoma and 32 cases of oligodendroglioma with an overall mean age of presentation of 41.49 ± 12.47 years. IDH-1 expression among diffuse glioma was 89.1% in our study. Alteration in the ATRX gene expression was observed in 95.7% of astrocytomas. 75% of oligodendrogliomas expressed INA with no significant difference in expression between the two grades. Based on the algorithmic approach using molecular surrogate markers, diffuse gliomas were categorized into six distinct groups. IDH-mutant, ATRX loss of expression astrocytoma and IDH-mutant, INA positive oligodendroglioma are two categories that do not require further molecular testing. This comprises 72.7% of the cases and these do not warrant further workup.ConclusionImplementation of combined phenotypic-genotypic diagnosis with the use of histomorphology and immunohistochemical surrogates for molecular genetic alterations will yield more homogeneous and narrowly defined diagnostic entities which will provide better prognostication and definitive treatment. It also is cost-effective in a resource-limited setup.

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P03.10 Combined immunohistochemistry for IDH1R132H, p53 and ATRX improves the molecular classification of diffuse gliomas in adults

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