Impact of the Association between Tgl and EZH2 Mutations (Y641N/Y641F/Y641H/Y641S) on Disease Free Survival of Patients with Diffuse Large B-Cell Lymphoma
Authors: Candelaria Myrna , Palacios Campos Adriana , Gutierrez Hernandez Olga , Aviles Alejandro , Cancino-Ramos Uvi , Taja Chayeb Lucia , García-Perez Osvaldo , Cantú de León David 
Published: November 2019

Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma. Although PET/CT has allowed a better diagnostic evaluation in these patients, none metabolic index has been incorporated into prognostic scores, neither molecular markers have been associated with PET/CT findings.
Aim: To define the association between PET/CT findings and EZH2 mutations, and their impact on survival of patients with DLBCL.
Methods: Patients: A cohort study of newly diagnosed DLBCL patients. Cohorts were defined according to the presence of EZH2 (Y641N/Y641F/Y641H/Y641S) mutations. Clinical variables were: age, comorbidities, IPI score, bulky disease, clinical stage, serum albumin, LDH and B2-microglobulin levels, and ECOG. Histopathologic variables were GC (Germinal center) versus no-GC by Hans nomogram, BCL2, BLC6, & MYC expression, as well as double-hit. All patients were treated with six cycles of standard RCHOP. PET/CT assessed parameters were: Total metabolic tumor volume (TMV), Total lesion glycolysis (TLG) and SUV max. Clinical response was evaluated by PET/CT, using Deauville's criteria. All 18F-FDG PET/CT scans were performed using the Biograph 16 PET/CT scanner (Siemens AG, Munich, Germany). Patients fasted for at least 6 h prior to intravenous (IV) administration of 18F-FDG (5.5 MBq/kg body weight) to ensure a serum glucose level of <10 mmol/l.
Methods: DNA was extracted from paraffin-embedded tissue. PCR analysis was carried out in a 2700 Thermalcycler (Applied Biosystems) to detect mutations in Exon 16 of EZH2; the sequencing and electrophoresis of PCR products was performed using ABI3100 genetic analyser. Sequences were compared with the EZH2 reference sequence (GenBank NG_032043.1). The protocol was approved by IRB (Approval number CEI/966/15). Statistics: After descriptive analysis, mean metabolic indexes were compared by ANOVA between patients with wt and any of the EZH2 mutations . Kaplan-Meier method was used to construct Disease-free survival (DFS) curves and the Log-rank test was used for comparisons. COR curves were used to evaluate metabolic indexes as risk factors infuencing on DFS.
Results: Results: 230 patients (120 male), were included. Median age was 58.3 years (SD 14.25; range: 21-91); 169 of them (73.4%) had advanced disease and 224 (97.3%) had ECOG <2. Absence of B symptoms or bulky disease was documented in 154 (67%) and 136 cases (59.1%), respectively. GC-type was found in 65%. Although sixty cases (26%) were considered to be DLBCL double expressor (MYC +BLC2 overexpression), only 15% were double-hit DLBCL. Mutations at codon 641, exon 16 of EZH2 were described in 15% of cases, as follows: Tyr 641 Asn (Y641N) [6 %],Tyr 641 Phe(Y641F) [6%], Tyr 641 His (Y641H) [3%] and Tyr 641Ser (Y641S) [1%]. Any clinical parameter was different between wt and any EZH2 mutations. However, regarding metabolic indexes by PET/CT, TLG was higher in EZH2 mutated patients (mean: 8314.02g [95 %CI: 5623-11004g]), in comparsion with wt patients (mean: 5228.99g, [ 95 % CI: 4114-6316g], p=0.02). Response was classified as complete in 75% of cases, partial in 3.5%, and progressive disease in 11%. The association of these mutations on DFS is shown in table 1.
In addition, mean TLG and TMV indexes were lower in patients without relapse. (Table 2)
Summary/Conclusion: These preliminary results suggest that patients with any of EZH2 (Y641F) mutations have a higher TLG by PET/CT at diagnosis, and both values have a negative impact on DFS of patients with DLBCL. Our results need to be confirmed on a larger sample and a longer follow-up.

Disclosures
No relevant conflicts of interest to declare.