Myelodysplastic syndromes with no somatic mutations detected by next‐generation sequencing display similar features to myelodysplastic syndromes with detectable mutations

To the Editor: Somatic mutations have been reported in 80%-90% of myelodysplastic syndrome (MDS) using next-generation sequencing (NGS) gene panels interrogating at least 30 MDS-associated genes.1, 2 Next-generation sequencing studies are increasingly being used in the diagnosis of MDS, to confirm or exclude a clonal process. However, about 10%-20% MDS cases are reported to have no detectable somatic mutations on typical NGS sequencing panels.1, 2 Given the subjectivity in assessing dysplasia and myriad non-MDS causes of cytopenia and dysplasia, there is skepticism if cases that lack evidence of clonality by both cytogenetics and NGS testing represent true MDS. Indeed, NGS study on peripheral blood may be used to screen cytopenic patients, with the assumption that a negative result makes MDS highly unlikely.3 Most large MDS sequencing studies involve multi-institutional databases in which clinical details to assess the veracity of the diagnosis are limited and thus the clinicopathologic and genetic features and natural history of these mutation-negative MDS cases are largely unknown. We reviewed the pathologic and clinical features of MDS cases (excluding therapy-related disease) from the pathology archives at three large academic medical centers between 2014 and 2020, during which targeted NGS panels (including 48 genes tested in all patients) were performed as a part of the clinical work-up (Table S1). The limit of detection was generally 1% for point mutations and 2%-5% for indels. Only pathogenic or likely pathogenic mutations were included in the analysis. For mutation-negative patients, a diagnosis of MDS required the presence of persistent or progressive cytopenia(s) as well as significant bone marrow (BM) dysplasia. Cases initially called MDS but later proven to be secondary cytopenias due to other processes, or in which cytopenias spontaneously resolved, were excluded. This study was approved by the Institutional Review Boards of all participating institutions. Statistical analysis was performed using GraphPad Prism (GraphPad software, San Diego, CA) and Xlstat, with significance set at a p value < 0.05 (two-sided). In patients with NGS performed after the time of initial diagnosis, overall survival (OS) was measured from the date of NGS; five patients had previously received either hypomethylating agents or lenalidomide, but all showed persistent MDS of the same disease subtype. The cohort consisted of 317 MDS patients, including 61 with no detectable mutations (MDS-NDM). The incidence of mutation-negative MDS was 10.3% based on the consecutively retrieved MDS cases (2016–2018). The mutation heat map (Figure S1) illustrates the mutation distributions and their associations with karyotype and IPSS-R group for the 256 cases with mutations. Details of the 61 MDS-NDM cases are provided in Table S2. Twenty-six (43%) had an abnormal karyotype, including six cases with isolated del(5q) and six with complex karyotype (≥ 4 independent abnormalities). Twenty-three (38%) had increased BM blasts. Of the 38 patients with no increased BM blasts, 18 had a normal karyotype or -Y. In the latter cases, the diagnosis of MDS was based solely on the presence of significant dysplasia in the context of persistent or progressive unexplained cytopenia. Immunophenotypic aberrancies were detected by flow cytometry in 9/10 MDS-NDM cases lacking cytogenetic abnormalities or increased blasts. Follow up sequencing (prior to any SCT) was performed on 26 MDS-NDM patients, of which 18 continued to show no mutations on one or more subsequent NGS tests. The newly detected mutations found in eight patients were mostly present at a low VAF (< 5%), often at or near the limit of detection of the assay. The 61 MDS-NDM patients are compared to the 256 MDS patients with one or more mutation (MDS-mut) in Table 1. The MDS-NDM patients were younger (p < 0.001) and showed different distribution of WHO subtypes, with more frequent MDS-SLD (p = 0.04) and MDS-del5q (p = 0.02) and less frequent MDS-RS (p < 0.001). The incidence of abnormal karyotypes was similar to MDS-mut. When limiting the analysis to the 176 cases with normal/-Y karyotypes, similar differences in patient age (p = 0.009) and WHO subtypes (p = 0.004) were observed between MDS-NDM and MDS-Mut (Table 1). A higher proportion of MDS-NDM patients received SCT (41% vs. 22%, p = 0.005), likely reflecting their younger age compared to MDS-mut patients. During the follow up period (median 26 months), 11% of MDS-NDM (including 5/35, 14% of the patients with normal/-Y karyotypes) and 16% of MDS-Mut patients progressed to AML (p = 0.43). The MDS-NDM patients had significantly longer OS (p = 0.008) and AML-free survival (p = 0.015) compared to MDS-Mut patients (Figure S2A,B), however there was no difference in OS (p = 0.18) or AML-free survival (p = 0.38) between MDS-NDM and MDS-Mut when limiting the analysis to patients with normal/-Y karyotypes (Figure S2C,D). On multivariable analysis, presence of an SF3B1 mutation in the absence of adverse mutations (HR 0.424, p = 0.008) and SCT (HR 0.294, p < 0.001) were favorable factors, whereas, IPSS-R, high/very high (HR 3.585, p < 0.001) or intermediate (HR 1.853, p = 0.004)(compared to IPSS-R low/very low) were unfavorable factors for OS. MDS-NDM (HR 0.592, p = 0.059) showed a trend towards favorable OS, while the presence of adverse mutations (TP53, EZH2, NPM1, or RUNX1)4 showed a trend towards unfavorable OS (HR 1.318, p = 0.131). However, when limiting the analysis to patients with normal/-Y karyotypes, MDS-NDM did not show a significant effect on OS independent from IPSS-R, SCT, and SF3B1 or adverse mutations (data not shown). In this study, we examined a cohort of 61 patients diagnosed with de novo MDS based on clinicopathologic data but lacking any pathogenic mutations on standard clinical NGS panels, including 35 with normal/-Y karyotype, of which 18 lacked increased blasts. Putative MDS cases that lack MDS-defining cytogenetics, somatic mutations, or increased blasts are the most challenging to diagnose, since neither cytopenias nor morphologic dysplasia is specific for MDS. We excluded some patients who were initially diagnosed with MDS, but in whom cytopenia(s) and dysplasia resolved at follow-up, underscoring the challenges in initial diagnosis of MDS without genetic evidence of clonality or increased blasts. Although the NGS panels used were large and comprehensive, we acknowledge that the absence of mutations in this study was based on clinical NGS platforms, not whole genome sequencing. Furthermore, mutation detection in clinical NGS platforms might be limited by the regions covered and variable depth of sequencing, the sensitivity of the assays, as well as the individual genes tested. We did identify four cases in which low-level mutated sequences were detected in the initial NGS, but were not callable, reflecting either technical limitations or a very small clone below the limit of reliable detection on clinical NGS panels. Of note, most of the MDS-NDM patients with followup sequencing data continued to show no detectable mutations, in line with the initial negative NGS results. Compared to MDS with at least one mutation (MDS-mut), MDS-NDM patients were younger, less anemic, had lower BM cellularity and included fewer cases with RS. Some of these differences were likely attributed to certain specific mutations, such as SF3B1 mutation associated with RS and anemia. Although the proportion of cases with an abnormal karyotype was not different from MDS-mut, MDS with isolated del(5q) was more common in MDS-NDM than in MDS-mut (8% vs. 2%). Del(5q), either isolated or in a non-complex karyotype, is known to harbor no or fewer mutations than other subtypes of MDS.5 It is possible that leukemogenesis in some MDS-NDM cases might differ from MDS-mut, especially from those carrying well-defined functional driver mutations. In a previous study of MDS cases in which BM CD34+ cells showed preserved B-cell differentiation,6 there was a relatively high proportion of unmutated cases, suggesting that the MDS clone might start in later myeloid-committed cells in MDS-NDM. We found a similar IPSS-R distribution between MDS-NDM and MDS-mut groups and on multivariable analysis, lack of detectable mutations showed a trend towards favorable OS, although this was not seen when limiting the analysis to the subset of patients with normal/-Y karyotype. Of note, an increasing mutation burden has been shown to associate with worsening prognosis in MDS.1, 4 In summary, we show that using clinical NGS platforms covering a large panel of genes commonly mutated in hematopoietic neoplasms, approximately 10% of bona fide de novo MDS cases lack detectable pathogenic mutations. About one-third of these cases lacked cytogenetic abnormalities or increased blasts, and in these cases the diagnoses relied solely on persistent cytopenia(s) and dysplasia. This subset of cases may contribute to the approximately 5% false-negative incidence in peripheral blood NGS screening of cytopenic patients for MDS.3 Flow cytometry can provide additional immunophenotypical support to confirm an MDS diagnosis when genetic proof of clonality is lacking. MDS-NDM has more favorable OS and AML-free survival than MDS-Mut, however, a significant subset of patients do progress to AML or die of BM failure. This similarity to MDS-Mut in clinical presentation and natural history raises the possibility that MDS-NDM cases may harbor mutations in genes not covered in standard sequencing panels, or not detected in clinical NGS panels due to technical limitations. Alternatively, other mechanisms driving altered DNA methylation and/or transcriptional reprogramming warrant further investigation. Our data indicate that a lack of pathogenic mutations detected by standard clinical NGS panels in a patient with persistent cytopenia should not exclude a diagnosis of MDS if there is sufficient morphologic dysplasia and the absence of competing etiologies for persistent cytopenia. A.K. reports consulting income from LabCorp. Some data that supports the findings of this study are available in the supplementary material of this article (Table S2), and the remaining data are available from the corresponding author upon reasonable request. Figure S1 Mutation heatmap of MDS-Mut cases. Figure S2 Outcome of myelodysplastic syndrome with no detectable mutations (MDS-NDM) patients compared to MDS with mutation (MDS-Mut) patients. A. Median overall survival (OS) of MDS-NDM patients is longer than MDS-Mut patients (52.9 versus 32.9 months, p = 0.008). B. Median AML-free survival of MDS-NDM patients is longer than MDS-Mut patients (47.3 versus 30.1 months, p = 0.015). C. Among patients with normal/-Y karyotypes, median OS of MDS-NDM patients is not significantly different from MDS-Mut patients (52.9 versus 44.8 months, p = 0.178). D. Among patients with normal/-Y karyotypes, median AML-free survival of MDS-NDM patients is not significantly different from MDS-Mut patients (47.3 versus 40.2 months, p = 0.38). Table S1 Gene coverage of 4 next-generation sequencing panels used Genes on all 4 panels. Table S2 Clinical, pathologic, and genetic details of 61 MDS cases with no mutations detected on NGS. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.