Assessment of CDKN2A homozygous and hemizygous deletion status in gliomas.

Abstract

e14021 Background: Homozygous deletion of CDKN2A (Homdel) has been included in the WHO CNS5 classification as a grade 4 definition in IDH mutated gliomas without the 1p-/19q-codeletion ( IDH mu-noncodel). Hemizygous CDKN2A deletion (Hemdel) has also been reported to be associated with poor prognosis. The identification methods for CDKN2A deletion include next generation sequencing (NGS), methylation array, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). However, at present, there is no gold standard threshold for FISH to judge the homozygous and hemizygous deletion. Methods: We collected formalin fixed paraffin-embedded (FFPE) tissue samples from 99 patients with adult-type diffuse glioma and all samples were tested using DNA-based NGS profiling (Simceredx). In addition, CDKN2A status was also detected by FISH and IHC (P16 and MTAP). Based on the CDKN2A status determined by NGS, the FISH were evaluated using ROC curves, including Area Under Curve (AUC), sensitivity, specificity, and cut-off values of Homdel and Non-Homdel, and deletion (Homdel and Hemdel) and non-deletion. The consistency of CDKN2A status identified by NGS, FISH IHC-P16 and IHC-MTAP was compared by Kappa test and Cramer V coefficient. Results: In 99 enrolled patients, NGS results showed 49 patients with CDKN2A Homdel, 18 patients with CDKN2A Hemdel, and 32 patients with CDKN2A Normal. There were no significant differences in age, gender, location of occurrence, IDH mutation status, or receiving chemoradiotherapy among the three groups. There were statistically significant differences in cancer subtype (p value=0.015) and grade (p value=0.010) among the three groups: oligodendroglioma and 2-3 grade was more likely to be normal in CDKN2A and grade 4 significantly predisposed to be Homdel. The ROC curve determines that cutoff of Homdel and Non- Homdel by FISH is 23%, and greater than 23% is Homdel. The ROC curve showed that the AUC of FISH method using 23% cutoff is 0.958, and the sensitivity and specificity were 85.11% and 100%, respectively. The consistency between FISH and NGS was 0.854. For the inconsistent samples, all NGS results were Homdel, and all FISH were deletion except one. Compared the results with FISH, we found that the results of NGS and IHC-P16 were more consistent. The ROC curve determines that the cutoff of deletion and non- deletion by FISH is 39%, and greater than 39% is deletion. The ROC curve showed that the AUC of FISH method with 39% cutoff is 0.962 and the sensitivity and specificity were 95.31% and 90.62%, respectively. Consistency comparison of the four methods showed that the consistency between FISH and NGS in determining Homdel and Non-Homdel, and deletion and non-deletion were the highest, 0.841 and 0.86, respectively. Conclusions: In our study, four methods were used to determine the status of CDKN2A, and results showed that the consistency between FISH and NGS was the highest.