The role of DDX41 mutations in the development of myeloid neoplasms (MN) is becoming well established.^1-3 Germline mutations in DDX41 have been shown to predispose to additional somatic DDX41 mutations and confer an increased risk of developing MN, including acute myeloid leukemia and myelodysplastic syndromes. MN with germline DDX41 mutations have many unique clinical and pathologic features and have thus been recognized as a distinct entity in the current World Health Organization classification. Earlier studies, including one from our department, have reported the clinical, pathologic, and molecular characteristics of MN associated with DDX41 mutation.⁴ DDX41 mutations were also identified rarely in non-myeloid hematologic neoplasms in previous studies, although very little information is available in this regard and the extent to which these DDX41 mutations are contributing to non-myeloid malignancies, particularly non-myeloid hematologic malignancies, has yet to be fully determined. The purpose of this study was to further address this question as well as the prevalence and characteristics of DDX41 mutations in non-myeloid hematologic malignancies.

We reviewed our database for all malignancies with a DDX41 mutation detected by our Endleukemia assay V2, an 81-gene clinical next-generation sequencing (NGS) assay performed on all new or suspected acute leukemias or MN. After excluding patients with an established history of MN, we identified a cohort of 20 patients. Clinical, cytogenetic and molecular characteristics were collected. Statistical analysis was performed using GraphPad Prism (GraphPad software, San Diego, CA, USA) with significance set at a p-value <0.05 (two-sided). Of note, out of 164 unique patients with MN, 12 (7.3%) patients had concurrent/history of lymphomas/plasma cell neoplasms; however, none had NGS performed prior to the diagnosis of a MN and were thus excluded.

All 20 patients had a non-myeloid hematologic disorder including 6 (30%) plasma cell neoplasms (PCN), 6 (30%) B-lymphoblastic leukemia/lymphoma (B-ALL), 4 (20%) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 2 (10%) aplastic anemia, 1 (5%) mixed phenotype acute leukemia (B/myeloid), and 1 (5%) EBV-positive cytotoxic T-cell lymphoproliferative disorder.

Eleven (55%) patients had a documented history of cytopenias, including 6 (54.5%) with anemia and thrombocytopenia, 3 (27%) with isolated anemia, 1 (9%) with anemia and leukopenia/neutropenia and 1 (9%) with mild pancytopenia. Of note, 10/11 (91%) had an underlying hematologic malignancy or aplastic anemia that may have contributed to the presence of cytopenias. No patients had a documented family history of MN. Table 1 provides a summary of the clinicopathologic features of the cohort.

All 20 patients harbored only a single DDX41 mutation with 19 unique mutations identified. There was only 1 recurring mutation (DDX41 p.M155I) (Table 1). No p.R525H mutation was identified. Only 5 (25%) mutations had a VAF <20%, which were likely somatic mutations. One (5%) DDX41 p.K187R, had a VAF of 38%, and the origin (germline vs. somatic) could not be definitively determined based on the VAF. The remaining 14 (70%) DDX41 mutations had VAFs 45.6%–54.6% and were presumably germline mutations. Although paired normal control samples are not routinely performed for our hematologic samples, three patients in the total cohort were referred for germline testing using cultured skin fibroblasts which confirmed a germline DDX41 mutation in all three (Supplementary Table 1). Of the canonical DDX41 mutations, a p.M1I mutation occurred in a patient with CLL/SLL, and a p.D140fs occurred in a patient with PCN. There was no significant difference in the types of DDX41 mutation by diagnostic subgroup.

Of the 14 presumably germline DDX41 mutations detected in this study, only 2 (14%) were deleterious (i.e., frameshift or splicing mutations) and the remainder were SNV missense mutations. This is substantially different from the results of our previous study examining DDX41 mutations in MN in which we found that 19 of 30 (63%) presumably germline mutations were deleterious (p = .0025).² Although p.M1I and p.D140fs were more common in our previously reported cohort (13/30, 43% presumably germline DDX41 mutations), this difference was not significant as compared to the current non-myeloid cohort (2/14, 14%; p = .09). We also conducted in silico prediction for pathogenicity of the DDX41 mutations using two online tools and the results can be found in Supplementary Table 1.

Among the six patients with PCN, five had conventional plasma cell myeloma and one had plasma cell leukemia. The most common co-occurring mutation was TP53, seen in two (33.3%) patients. Among the six patients with B-ALL, four harbored t(9;22)(q34.1;q11.2)/BCR-ABL1 and the most common co-occurring mutations were KRAS seen in two/six (33.3%) patients followed by TP53 in one (16.6%) patient. There were four patients with CLL/SLL. One patient also harbored a NOTCH1 mutation, another also had ASXL1 and DNMT3A mutations. Interestingly, one patient with CLL/SLL and a presumably germline DDX41 pM1I mutation subsequently developed AML, along with a second p.R525H mutation.

The mutational landscape of the DDX41 mutations in patients without a MN appears different than that of patients with MN. The frequencies of p.D140fs and p.M1I were far less than that seen in MN, and most mutations, presumably germline, were missense mutations. With the exception of one patient who subsequently developed AML (see below), no other patients harbored two DDX41 mutations, and no patients harbored p.R525H somatic mutations.

This study included 20 patients each with a single DDX41 mutation but no documented MN. We also identified a patient with CLL, pancytopenia and mild dyserythropoeisis in whom a p.M1I mutation was initially detected and who subsequently developed AML 6 months later along with a second p.R525H mutation. Both of these mutations have been very well documented to occur in tandem in MN with DDX41 mutations.^{2, 4} These findings suggest that two mutations in DDX41, although not necessarily required, appear more likely to promote the development of MN.

The results of this cohort suggest that DDX41 mutations may not have exactly the same etiologic role as they do in MN. Our current understanding of the molecular mechanism is that bi-allelic DDX41 mutations may further predispose to MN. The finding that none of the patients in the current cohort have two DDX41 mutations could be a possible explanation as to why these patients did not develop a MN. However, this observation also argues against DDX41 being a predisposing factor for these non-myeloid malignancies. Only one patient in this cohort subsequently developed a second somatic DDX41 mutation and this same patient also developed AML. Furthermore, although the remainder of the cohort did not develop a MN, 11 patients had cytopenias or morphologic evidence of dyspoeisis which could represent the early stages of a MN or could simply be the result of the patients' underlying conditions. In addition, only two patients in this cohort had canonical DDX41 mutations. These results, together with those reported elsewhere, further support the leukemogenic models for DDX41 mutations in MN, namely that germline DDX41 mutations predispose to MN, and there is likely a strong interplay between the germline mutation and the subsequently acquired somatic mutations for leukemogenesis. However, functional studies would be required to determine the etiologic role, if any, that DDX41 mutations have in non-MN.

A recent study by Zhang et al. identified six patients with DDX41 mutations in B-ALL, but all the mutations were distinct from those identified in the cohort we report.⁵ However, similar to our study, Zhang et al. also did not identify any co-occurring germline and somatic mutations, further supporting the concept that DDX41 mutations may not be playing the same role in B-ALL as they do in MN.

The precise role of DDX41 in non-myeloid malignancies remains to be more fully elucidated. For example, thus far, a single reported case of a B-ALL with t(9;22)(q34.1;q11.2) harboring both germline (p.Thr214Profs*8) and somatic mutations (p.Leu87Phe) has been reported. Shin, et al. suggested that DDX41 could be an underlying event in Philadelphia chromosome (Ph) positive B-ALL.⁶ They identified different pathways enriched in the Ph + B-ALL with DDX41 mutation as compared to Ph + B-ALL without DDX41 mutation. Although neither of the mutations were detected in our cohort, we did see DDX41 mutations present disproportionally in patients with Ph + B-ALL, in about two thirds of cases. Whether DDX41 mutations may be involved in the pathogenesis of Ph + B-ALL still requires further investigation.

Shin et al. also reported a high-level expression of genes involved in the p53 signaling pathway.⁶ In our previously reported study of MN with DDX41 mutation,⁴ we also found a significant association with TP53 mutation. In the present study, TP53 mutation was seen in one third of patients with plasma cell neoplasms and DDX41 mutation. However, the precise interplay between DDX41 and TP53 mutations remains to be further studied.

In conclusion, we identified DDX41 mutations in a variety of non-myeloid hematologic malignancies, including B-ALL, PCN, CLL/SLL, and MPAL as well as aplastic anemia. We found that t(9;22) was common in DDX41 mutated cases of B-ALL. Because none of these cases harbored more than one DDX41 mutation, our data suggest that DDX41 may not be contributing to these conditions in the same manner as in MN. However, further studies are necessary to better understand the role of DDX41 mutations in non-MN, particularly in Ph + BALL.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.