Pathogenesis and biomarkers of natural killer T cell lymphoma (NKTL)

Epstein-Barr virus (EBV) infection plays a crucial role in the pathogenesis of NKTL, though the actual mechanism remains to be understood. The identification of EBV genomes in sequencing data or the immunohistochemical stains of EBV non-coding RNA (EBER) in NKTL samples provided initial hints of the oncogenic role of EBV in this disease. A causative relationship between EBV and NKTL was established based on the presence of clonal and episomal forms of EBV in tumour cells, in addition to EBV-encoded proteins [8, 9]. In NKTL, EBV-host integration sites were found in various repeat families of the human genome, such as SINE, LINE and satellite [10]. Notably, a 109 bp long EBV genomic fragment was found to integrate into the intron of the human non-homologous end-joining factor 1 (NHEJ1) gene, which led to the downregulation of the gene transcripts [10]. NHEJ1 is a DNA repair factor essential for the NHEJ pathway, which preferentially mediates repair of double stranded breaks (DSBs). Failure to repair DSBs would result in genome-wide instability that could lead to the onset of NKTL.

Infection by EBV induces expression of genes encoding nuclear antigens, membrane proteins and non-coding RNAs in B cells, all of which induce cell proliferation and transformation into lymphoblastoid cell lines. However, in order to escape from T cell mediated immune responses, the infected B cells enter into a latent state. The pattern of gene expression in the various latent phases has been associated with the different EBV-related malignancies. Latency phase II, with expression of EBNA1, LMP1, LMP2A and LMP2B, has been the most implicated in NKTL, in addition to nasopharyngeal carcinoma and Hodgkin lymphoma. The expression of these genes plays an important role in these tumours by modulating cell signalling and forming barriers to apoptotic signals. However, among the Latency II tumours, different mechanisms drive the various gene expressions, contributing to the unique characteristics of each of these malignancies [11]. A novel transcript LMP2 TR was identified at high levels in NKTL, resulting in a high level of expression of LMP2B in the absence of LMP2A. With this, LMP2B has been postulated to play an important role in the pathogenesis of NKTL and may serve as a potential therapeutic target [12]. Consistently, we observed that most of the latent genes were expressed in NKTL, including those of EBNA family, LMP family and BARTs. The BART RNAs are a heterogeneously spliced group of EBV RNAs transcribed rightward from position 138352 to 160531 on the EBV wild-type genetic map [13‐15]. Comparatively, this region is highly transcribed and it consists of about 20 microRNA and the putative proteins RPMS1 and A73 [10]. This highly transcribed region of the EBV genome most probably has an important regulatory role on itself and the host genome, in driving the disease and immune evasion, which warrant further investigation.

The genetic factors that predispose individuals to NKTL were not known until recently. Our genome-wide association study (GWAS) in NKTL identified a common SNP (rs9277378) in the HLA-DPB1 allele which conferred a 2.3 times higher risk of NKTL compared to baseline [16]. The four amino acids within HLA-DPB1, namely Gly84-Gly 85-Pro86-Met87, play a crucial role in CD4 T lymphocytes for antigen presentation, hence contributing to its hereditary susceptibility to NKTL. This study also demonstrated that the HLA locus associated with NKTL is distinct from other EBV-driven malignancies such as nasopharyngeal carcinoma and Hodgkin lymphoma. Besides distinct differences in host genetic susceptibility, we have recently demonstrated that different EBV strains predominate among these diseases [10]. These findings imply that these diseases are driven by distinct biological mechanisms. However, the potential implications of different EBV strains with the host genome for these diseases are still unclear and require further investigation.

Familial NKTL is a rare phenomenon. At the time of this writing, only two sets of familial NKTL cases have been reported. The earlier report was on a father-son pair with known heavy exposure to pesticides, and no specific genetic element was evident in their tumours [17]. In the later report, a novel recessive and homozygous germline mutation in FAM160A1 was found in the tumours of two male siblings from a non-consanguineous Chinese family [18]. In this recent case report, FAM160A1 was also found to be overexpressed in these patients’ tumours harbouring the mutant FAM160A1 when compared to sporadic NKTL tumours. Interestingly, FAM160A1 was found to be expressed predominantly in the CD68-positive histiocytes rather than the lymphomatous cells, which suggests that the mutant FAM160A1 might play a role in the pathogenesis of this disease by altering the microenviromnent of the tumour.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is crucial to haematopoiesis and immune development, in addition to other essential functions. Using targeted sequencing of 188 genes associated with the JAK/STAT pathway approach, we have recently demonstrated that alteration of this pathway is highly prevalent (73%) in peripheral T-cell lymphoma (PTCL) and NKTL [19]. In this study series, STAT3 was identified to be the most frequently mutated gene followed by TP53, JAK3, JAK1 and SOCS1 of the JAK/STAT cascade, suggesting that targeting this pathway might benefit a large portion of NKTL patients. The prevalence of STAT3 mutations in NKTL was reported to range from 8 to 27% in various studies [19‐23].

In addition to the frequent STAT3 activating mutations, JAK3 activating mutations were also identified in NKTL, in 34% of cases [24]. An additional study reported novel JAK3 mutations, residing in the pseudokinase domains of JAK3 [25]. The frequency of JAK3 mutations varies across different populations, ranging from 0 to 35% [26, 27]. Functional overexpression of JAK3^A572V resulted in the phosphorylation of downstream effector proteins, STAT3 and STAT5. Importantly, preclinical study with Tofacitinib, a pan-JAK inhibitor, could effectively reduce tumour growth and metastatic spread of NKTL [26] indicating that JAK3 is a promising therapeutic target for NKTL. A novel JAK3-specific inhibitor (PRN371) was further developed, to supersede tofacitinib in terms of specificity and durability in inhibiting JAK3 in NKTL [28]. However, the single-agent regime with PRN371 did not confer complete response in mice. While this observation could partially be attributed to suboptimal dosing or delivery of the drug to the target tumour site, this could also reflect the complexity of the JAK/STAT signalling pathway that has yet to be understood fully and hence may warrant combination therapeutic approaches for improved efficacy.

Activating STAT3 mutations was also demonstrated to confer resistance to PRN371 by rescuing the inhibition STAT3/5 phosphorylation in NKTL [28] suggesting that the activation of STAT3 could be a biomarker of resistance to JAK3-inhibition therapy. In activated B cell subtype of diffuse large B cell lymphomas (DLBCL-ABC), in vivo inhibition of STAT3 was found to be a more effective strategy in suppressing tumour growth than targeting upstream JAK inhibition [29]. Hong et al. demonstrated that AZD9150, an antisense oligonucleotide (ASO) inhibitor of STAT3, achieved an almost complete inhibition of STAT3 in lymphoma and lung patient-derived explant models [30]. This provided the proof-of-concept that STAT3, a notoriously difficult protein to inhibit therapeutically, can now be better positioned for better treatment outcome.

Another interesting role of STAT3 in NKTL is its relationship with immune surveillance and evasion. We demonstrated that activated STAT3 could upregulate PD-L1 by binding directing to the proximal promoter of PD-L1 [19] Inversely, blocking STAT3 with ASO or Statiic downregulated PD-L1 expression effectively. It was further shown that NKTL tumours with high expression of phosphorylated STAT3 correlated significantly with PD-L1 levels highlighting its clinical importance in immune checkpoint inhibition. STAT3 contributes to tumour immune evasion through the accumulation and activation of tolerogenic dendritic and Treg cells, as well as the upregulation of immune checkpoint proteins such as CTLA-4, programmed cell death protein 1 (PD-1), and programmed death ligand 1 (PD-L1) [31]. PD-L1 expression in NK/T cell lymphoma has been reported to be 56–93% in various studies and this has stirred excitement in the use of PD-1/PD-L1 inhibitors in NKTL [19, 32‐34]. These studies suggest that inhibiting STAT3 could effectively challenge the survivability of NKTL by simultaneously disrupting its immune evasion pathway. A combination strategy, using PD1/PDL1 antibodies and STAT3 inhibitors, may be yet another therapeutic strategy that can be explored.

Anti-PD1 therapy has shown promising activity in relapsed/refractory (R/R) NKTL [35]. This was echoed in another study that demonstrated 57% response rates in R/R NKTL with pembrolizumab (PD1 blockade) [36]. In an attempt to understand the mechanism of response or resistance, we performed whole genome sequencing on the tumour of 11 NKTL patients treated with pembrolizumab [37]. Our data revealed that PD-L1 structural variations (SV) in the 3′UTR region of the PD-L1 gene were seen exclusively in 4 out of 7 patients who achieved complete response (CR) to pembrolizumab (PD-1 antibody) and not seen in any of the non-responders. Structural variation in 3′UTR region of the PD-L1 gene was also demonstrated in a patient with chemorefractory ovarian cancer who had CR to pembrolizumab [38]. While these findings need to be validated in a larger dataset, this illustrates a molecular mechanism that would explain the efficacious activity of anti-PD-1 therapy in NKTL patients.

In the same study, we have also identified recurrent JAK3-activating mutations in two of the seven patients who had achieved CR to pembrolizumab [37]. JAK3 mutations were also seen in one patient who had prolonged benefit from anti-PD-L1 therapy in refractory lung adenocarcinoma [39]. JAK3 activation deregulated the cytokine receptor signal transduction in lung cell lines, led to the upregulation of PD-L1, and provided the explanation to the durable response seen in this patient with anti-PD-L1 blockade therapy. Taken together, JAK3-activating mutation and PD-L1 3′UTR SV are potential biomarkers which could better select NKTL patient for immune checkpoint blockade therapy.

The recent data on the various driving mechanisms behind NKTL attempt to unravel the complex pathogenesis driving this disease, which at present has poor treatment outcomes. A summary of current therapeutic targets is presented in Table 1. With a better understanding of the underlying pathogenic mechanisms, biologically sound therapeutic strategies can be employed to potentially overcome this disease.