Comparative Analysis of Clinicopathological Features and Genetic Landscape of Relapsed/ Refractory and Effectively Treated Extranodal Nasal NK/ T Cell Lymphoma
Authors: Gao Limin , Li Huifang , Jian Qi-Jie , Zhang Wenyan , Zhao Sha , Wang Kai , Liu Weiping 
Published: November 2021

Abstract
Background
Extranodal natural killer/T-cell lymphoma (ENKTL) is an Epstein-Bar virus (EBV)-associated aggressive lymphoma that is endemic in East Asia, Central and South America, and sporadic in Western countries. L-asparaginase regimens have significantly improved prognosis and are the current most commonly used initial therapy. Unfortunately, about 20% to 40% patients had inadequate responses to L-asparagine regimens, and diagnosed as relapsed or refractory (R/R). Due to the lack of study investigating on R/R-ENKTL, the differences in clinicopathological features and gene mutations between R/R-ENKTL and effectively treated ENKTL (ET-ENKTL) remain unclear.
Material and method
This study included 21 R/R-ENKTL cases and 10 ET-ENKTL cases from June 2015 to December 2020. The clinical data, pathological features, and the genomic variations detected by whole exome sequence (WES), were compared between the two groups.
Result
The clinical characteristics of R/R and ET ENKTL were summarized in Table 1.
Compared with ET patients, more R/R-ENKTL patients had B symptoms (P = 0.038) and elevated LDH (P = 0.041). Pathological features such as the diffuse distribution of medium-sized cells and necrosis were observed in both groups. It is noteworthy that angiocentric and angiodestructive growth pattern is more frequently present in the R/R group (P = 0.032). Immunohistochemistry showed that R/R-ENKTL patients had higher CD30 positive rate (P = 0.045) and lower PD-L1 expression rate (P = 0.030)（Figure 1. A）.
A significant difference in WES-based tumor mutation burden (TMB) between the two groups was observed, with a median TMB of 3.7 Muts/Mb in the R/R group and 2.3 Muts/Mb in the ET group. Mutated genes were mainly involved in the biological processes (BP) with extracellular structure/ matrix organization in the R/R group, but not the ET group, in the gene ontology (GO) analysis (Figure 1. B). As the change of extracellular matrix will restrict the entry of drugs into target tissues, this may be one of the reasons that led to the resistance of R/R-ENKTL.
Among all the 1738 mutant genes in the R/R-ENKTL groups, none of substitution mutations were found significantly higher frequency than the ET group. However, when all of the epigenetic regulatory genes were gathered and compared, the mutation frequencies of them were significantly higher in the R/R-ENKTL group, especially for the genes encoding histone methylation including KMT2D, KMT2C and KDM4C etc. Mutations of KMT2D were found in 5 patients (23.8%), including three substitutions and two truncations in the R/R group. In contrast, only a truncation of KMT2D was found in a patient of the ET group (10.0%) (Figure 1. C). It suggested that histone methylation abnormality might play a role in ENKTL resistance, which is worthy of further study.
Compared with R/R groups, the mutation frequencies of three mutant genes (RELN, LAMA5 and TPTE, all with P = 0.027 respectively) were more prevalent in the ET-ENKTL (Figure 1. C). Whether the abnormalities of these genes were related to the effectiveness of drugs like L-asparagine and acted as the potential biomarker in ENKTL needs to be further investigated.
The patterns of structural variations (SVs) were also different between the two groups. NRG1 was amplified in 19.0% (4/21) patients of the R/R group, while no abnormality was found in the ET group. Besides, SVs that only appeared in the R/R group included homozygous deletion of CDKN2A (9.5%) and CDKN2B (4.8%), and so on. In addition, the PD-L1 encoding gene CD274 had a higher rate of fusions in the R/R-ENKTL group (n=3) than the ET group (n=1).
Discussion and Conclusion
Our results showed that more R/R-ENKTL patients had B symptoms and elevated LDH. For pathological features, R/R-ENKTL patients was more likely to have angiocentric and angiodestructive growth pattern, higher CD30 positive rate and lower PD-L1 expression rate. Based on tumor genetics, frequently mutated genes encoding histone methylation and enriched in the GO term of extracellular structure/ matrix organization might play a role in the drug resistance of R/R-ENKTL. Furthermore, the high prevalence of NRG1 amplification and CD274 fusions suggested their potential as therapeutic targets in R/R-ENKTL treatment.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.