In the present study, we characterized the molecular signature of NKTCL in comparison to normal NK cells and to PTCL, NOS. This led to the identification of deregulated genes and signaling pathways which might be relevant to the pathophysiology and clinicopathologic features of the disease and bring rationale for the development of new therapies.
Unsupervised clustering remarkably segregated NKTCL and PTCL, NOS samples. Notably, the NKTCL case with a T-cell cytotoxic phenotype (sample T5) clustered with those of NK cell origin, providing another molecular argument for grouping nasal “true NK-” and cytotoxic T-cell lymphomas as a single entity as proposed in the current WHO classification.1
Interestingly, the NKTCL case of T-cell derivation also showed the 6q16-q25 deletion. The only mismatch was represented by one PTCL, NOS in the NKTCL cluster. That particular case had a γδ activated cytotoxic phenotype suggesting that derivation from the innate immune system might imprint a peculiar signature.37
By comparison to PTCL, NOS, the molecular signature of NKTCL was significantly contributed by an overexpression of genes associated with cytotoxic functions and NK-cell-associated molecules. Interestingly, the highest fold change of expression was observed for gzm H transcripts. Granzyme H, a gzm family member sharing a 90% amino acid sequence identity with gzm B, is constitutively expressed in NK cells irrespective of their activation status25
but acts differently from gzm B by inducing a caspase-independent cell death program.38
We confirmed a strong gzm H protein expression in all NKTCLs contrasting with its negativity in most PTCLs, NOS, with the notable exception of the γδ T-cell lymphoma. Therefore, gzm H appears to be a novel sensitive marker for NKTCL, although its specificity needs to be delineated with respect to its possible expression in other lymphomas derived from the innate immune system.
Other aspects of the NKTCL signature could be related to some peculiar clinicopathologic features of the disease. Angioinvasion and angiocentricity typical of NKTCL, might be accounted by the high expression of genes such as VCAM1, CXCL9,
encoding proteins involved in the interaction with endothelium or in the pathogenesis of tissue necrosis and vascular damage associated with EBV-positive lymphoproliferations.39
NKTCL, which in most instances arises in the nasal area, is also characterized by a strong tendency to disseminate to other extranodal distant sites. In view of the known roles of CCR7 and SELL/CD62L in peripheral lymph node homing and that of CCL27, CXCL12, in homing to the skin, intestine and bone marrow, it is likely that the lower levels of CCR7 and SELL/CD62L and the overexpression of CXCL12 might explain the pattern of distribution of the disease.
We confirmed here recurrent genomic gains and losses previously reported in NKTCL. Deletion of chromosome 6q reported as the most characteristic but not specific genetic alteration in NKTCL,3–5,40–42
was present in 40% of our cases, including SNK6 cell line. In line with the recent report by Iqbal et al., we also found recurrent gain of 1q21-q44 and loss of 17p13.3 in primary tumor samples.5
These authors found PRDM1, ATG5
, and AIM1
as target genes in the region of del6q21 and reported both mutation and methylation in PRDM1
. Here, we further extended these previous findings by showing low levels of ATG5
transcripts in primary tumors. Conversely, PRDM1
showed a wide range of expression from case to case. In addition, we also found marked reduction in transcripts of HACE1
, a gene encoding a novel E3 ubiquitin ligase, which is the target of epigenetic inactivation in Wilms’ tumor and has been proposed as a tumor suppressor gene in multiple human cancers. Hace1−/−
mice are spontaneously prone to developing multiple malignant tumors in various organs.43
Taken together, it is therefore tempting to speculate that HACE1
might be also involved in the pathogenesis of NKTCL.
The proto-oncogene MET
mapping in 7q31, a region of recurrent gain in our series, was overexpressed in our samples. This receptor with tyrosine-kinase activity is a receptor to HGF, also overexpressed at the mRNA level in our NKTCL series. Interestingly, this pair of ligand-receptor appears to be linked to angiogenesis, tumor formation, invasion, and metastasis.44
These findings, together with the expression of VEGFA and its receptor VEGFR2 might reflect the implication of angiogenesis and/or VEGF signaling pathway in the pathophysiology of NKTCL.
The molecular pathways involved in the pathogenesis of NKTCL are largely unknown. Our study identified deregulated pathways in NKTCL, in comparison to normal NK cells and PTCL, NOS. Among growth factor receptors, the receptor tyrosine kinase PDGFRα was expressed at a higher level than in normal NK cells, both at the mRNA and protein levels, in its activated phosphorylated form, a feature which appears to be shared with PTCL, NOS.34
PDGF signaling pathway is known to be associated with both JAK-STAT and AKT pathways. The AKT protein kinases play a critical role in cell proliferation, survival and programmed cell death, transcription, and cell migration via phosphorylation of a multitude of substrates. Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. STAT3 in particular plays a crucial role in regulating cell growth and apoptosis.32
Several solid or hematological malignancies including ALK-positive anaplastic large cell lymphomas show constitutive STAT3 activation. Using statistical methods, we showed here that AKT and JAK-STAT pathways were differentially expressed in comparison to normal NK cells and/or PTCL, NOS. In addition, we evidenced the nuclear expression of the phosphorylated forms of STAT3 and AKT in most NKTCLs implying constitutive activation of these pathways in this disease. Our results expand recent findings that AKT was phosphorylated in NK-92 cell line and in a few NKTCL primary tumors, probably through involvement of IL-2 or IL-15.45
Many genes related to proliferation and survival, angiogenesis, and immunosuppression are known to be regulated by STAT3.32
The high transcript levels of several genes regulated by STAT3 in NKTCL compared to normal NK cells such as MYC, VEGFA, BCL2L1
, - and also less significantly BIRC5, HGF, IL6, MMP2, MMP9, IL10
, and CDK5
- suggests its implication in the pathogenesis of NKTCL. Among these, MMP2, MMP9 and IL-10 proteins have already been evidenced in NKTCL tumor cells and we show here immunohistochemical expression of VEGFA.46,47
Recently, we demonstrated that the inhibition of STAT3 activation leads to underexpression of two STAT3 target gene products, BCL-XL
and MYC in MEC04 cells.17
Altogether, these data provide strong arguments supporting the involvement of JAK-STAT and AKT pathways in NKTCL ().
Hypothetical representation of signaling pathways involved in NKTCL
EBV, constantly present in NKTCL, is suspected to play an important role in oncogenesis. Here we showed that, in comparison to EBV-negative normal NK cells, NKTCL overexpressed several EBV-induced genes.15,30
In agreement with a previous report,24
one of these genes, EBI3
, was validated at the protein level. Among the various mechanisms involved in NF-κB pathway, EBV is known to activate NF-κB pathway through LMP-1 and/or TRAF,48
especially in Hodgkin lymphoma and in EBV-positive B-cell lymphoproliferative disorders. Here, we showed differential expression of this pathway in NKTCL and further demonstrate expression of RelA, supporting activation of NF-κB in this entity. Interestingly, TNFAIP3
gene, encoding an inhibitor of the NF-κB pathway, maps to the region of recurrent loss in 6q16-q25 and was underexpressed in our study. Deletions and/or somatic mutations of this gene have been recently reported in classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma as well as in MALT lymphoma49,50
supporting the role of this key regulator of NF-κB activity as a novel tumor suppressor gene in these lymphomas. Altogether, these findings suggest the involvement of NF-κB pathway in the pathogenesis of NKTCL. The respective role of EBV and/or TNFAIP3
inactivation in NF-κB activation in NKTCL needs further investigation.
The demonstration of pPDGFRα by immunohistochemistry prompted us to test the sensitivity of NKTCL cell lines in vitro to imatinib mesylate. The effect of the drug already significant at a 1μM concentration with a 50% growth inhibition on MEC04 cells, was most prominent (90% inhibition of growth) at 6μM. Conversely, there was no substantial cytotoxic effect on the SNK6 cell line, which might be related to a lower expression of PDGFRA, as suggested at the RNA level (). Although our results do not preclude the precise mechanism of action of imatinib and suggest heterogeneity in the sensitivity to the drug, the dramatic effect on MEC04 cells shed light on the possible use of tyrosine kinase inhibitors as a novel therapeutic option in some patients with NKTCL refractory to conventional therapies.
The cause of PDGFRA
deregulation in NKTCL remains to be determined. We did not evidence either genomic imbalances or gene mutations. Furtheremore, mutations in the promoter region were absent in NKTCL primary tumors and cell lines, and we did not find overrepresentation of H2α haplotype, known to result in upregulation of PDGFRA
In conclusion, this integrative genomic and transcriptomic study characterizes the molecular signature of NKTCL, highlights emerging oncogenic pathways in this disease entity and offers rationale for exploring new therapeutic options such as tyrosine kinase inhibitors in patients with this aggressive malignancy.