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.
