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Letters to the Editor
No association between ECSIT germline mutations and hemophagocytic lymphohistiocytosis in natural killer/T-cell lymphoma
Hemophagocytic lymphohistiocytosis (HLH) is a clini- cal syndrome of excessive immune activation with fever, cytopenia, and organ infiltration by activated macrophages. Secondary HLH associated with natural killer (NK)/T-cell lymphoma (NKTCL) has extremely poor prognosis,1 and biomarkers that may predict patients who are more likely to develop HLH are lacking. Wen et al.2 recently showed an association between a somatic gene mutation in the evolutionarily conserved signaling intermediate in Toll pathway (ECSIT) gene (c.T419C; p.V140A) and HLH in NKTCL. The variant ECSIT protein triggered NF-κB signaling pathway more potently, leading to aberrant secretion of proinflammato- ry cytokines by ECSIT-T419C-transfected NKTCL cell lines. They found that the ECSIT-T419 mutation was sig- nificantly enriched in individuals with NKTCL-associated HLH, which developed in nine of 17 patients with and five of 36 patients without the mutation, respectively. Patients with ECSIT-T419 had elevated expression of proinflammatory cytokines and poorer prognosis. While intriguing, the prevalence of ECSIT-T419 and relation with HLH has not been assessed in independent cohorts. We therefore sought to examine whether the ECSIT-T419 mutation predisposes to HLH in multiple cohorts of patients with NKTCL and correlates its pres- ence with clinical outcomes.
First, we studied the mutational profile of ECSIT in 25 subjects with sporadic NKTCL from China with available whole exome sequencing of paired tumour-blood sam- ples.3 Samples were sequenced with Illumina HiSeq X and NextSeq 6000, and variant-calling was performed by Strelka2 using default single-sample settings.4 We found the ECSIT-T419 mutation in five of 25 subjects, but they were all germline mutations; heterozygously mutated in both matching tumour (variant allele frequency [VAF], mean, 43.8%, 95% Confidence Interval [CI]: 38.8-48.9) and blood (VAF mean, 53.8%, 95%CI: 51.5-56.2) sam- ples from these five subjects (Figure 1A). The reported prevalence of somatic ECSIT-T419 mutation in Wen et al.’s study was 19.3% (17 of 88), similar to the mutation frequency of Jiang et al.’s cohort, but were all germline mutations.
In order to further verify whether ECSIT-T419 is a germline or somatic mutation, we studied 67 patients with NKTCL who provided written informed consent under respective institutions’ Institutional Review Boards (IRB) from Singapore local hospitals and Sun Yat-Sen University Cancer Center in Guangzhou, China. We Sanger sequenced matched tumor- buccal swab (repre- sentative Sanger sequence in Figure 1B) or peripheral blood (representative Sanger sequence in Figure 1C) sam- ples from NKTCL patients and ECSIT-T419 was validat- ed in 7.5% (five of 67) of both the tumor and matching non-tumor samples. Targeted resequencing using next- generation sequencing method revealed the mean VAF of ECSIT-T419 to be 52.2%, 95%CI: 42.8-61.6 in the five tumors, 52.2%, 95%CI: 48.5-56.0 in four matched blood samples, and 53% in a matched buccal swab sample (P=0.90, 2-tailed Wilcoxon Rank-sum test, VAF of ECSIT- T419 between tumors and non-tumoral samples). The near 50% VAF, and the presence of ECSIT-T419 mutation observed in all matching tumor, blood and buccal swab DNA indicate that this is a germline heterozygous single-nucleotide polymorphism
(SNP), with a report SNP ID of rs145036301. Among the five patients with ECSIT-T419 mutation, HLH informa- tion was available for three patients and none developed HLH, as defined by the HLH 2004 criteria.5
Given the discrepant findings, we re-analyzed the ini- tial discovery cohort of paired tumor-normal exome data (n=5) from Wen et al.2 In the sample where ECSIT-T419 mutation was reported as a somatic mutation, VAF was 52% in the tumor (150 of 288; alternate allele depth/ref- erence allele depth) and 10% (5 of 51) in the matched normal sample (Figure 1D). Furthermore, the VAF exceeded the thresholds of 30% in tumor and 5% in matched normal sample as specified by Wen et al.2 Thus, this variant should not be considered as a somatic muta- tion as based on the authors’ analysis criteria.
Notwithstanding the false somatic call, we wanted to examine whether the germline ECSIT-T419 mutation is associated with HLH in two independent cohorts of patients with NKTCL in Singapore and Taiwan. In Singapore, the cases were identified using local databases from two teaching hospitals and all samples and clinical information were collected after IRB approval. Cases were reviewed by a central pathologist and HLH was defined according to the HLH 2004 criteria. Sixty-four cases of NKTCL were identified between 2007-2017, and ECSIT-T419 mutations were found in 15.4% (two of 13) and 5.9% (two of 51) patients with and without HLH respectively. Out of the 13 patients with HLH, four were women. Median age was 43 (range, 18-60 years). At time of the last follow-up in December 2018, all patients had died. Seven of 12 patients received polychemotherapy, while one was treated with the HLH-2004 protocol (with dexamethasone, etoposide, cyclosporin), and two received steroids. Median survival was only 33 days (range, 1-389 days). Causes of death were lymphoma (n=6), HLH (n=6), and infection (n=1). The two individu- als with ECSIT mutation succumbed at day 1 and day 89. Within these NKTCL patients with HLH in our Singapore cohort, there was no significant association of the ECSIT mutation with them (P=0.18, Fisher’s exact test, Online Supplementary Table S1).
In the Taiwanese cohort of 85 NKTCL cases with clin- ical and sequencing data from the Chang Gung Memorial Hospital, ECSIT-T419 mutation frequency was observed at 11.8% (ten of 85). Nine cases developed HLH, and none of these samples harboured the ECSIT-T419C mutation. When both Singapore and Taiwan cohorts were combined for analysis, we did not find any statisti- cal association between ECSIT-T419C mutation and HLH (OR=1.48, 95%CI: 0.38-5.76, P≈1.0, Fisher’s exact test, Online Supplementary Table S2). There were also no significant associations between the ECSIT-T419C muta- tion with clinical characteristics such as sex, stage, Eastern Cooperative Oncology Group (ECOG) perform- ance status and international prognostic index (Figure 1E; Online Supplementary Table S3). Overall survival (Online Supplementary Figure S1A) and progression-free survival (Online Supplementary Figure S1B) were also not signifi- cantly associated with ECSIT-T419 mutation.
Given the rarity and fulminant nature of malignancy- associated HLH hindering the collection of biopsy speci- mens, we combined data from multiple cohorts to exam- ine associations between ECSIT-T419 and HLH in NKTCL in the largest study to date. Strict diagnostic inclusion criteria were used for both HLH and NKTCL. Some possible explanations for the discordant results between Wen et al. and our study need consideration. Patients in Singapore and Taiwan developed HLH at around the time of diagnosis or relapse, as opposed to
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