https://orcid.org/0000-0002-5242-4660
To the Editor:
A 41-year-old male was referred to our hospital with a leukocytosis of 59 000 granulocytes/µL (3900–10 200) in combination with a maculopapular exanthema and mild pruritus. The differential blood count showed a left-shift granulopoeisis and a leukoerythroblastic picture (1 myeloblast, 1 erythroblast), platelet anisocytosis, and polychromasia of red blood cells. A bone marrow biopsy indicated the diagnosis of a systemic mastocytosis with associated hematological neoplasm. The granulopoeisis appeared proliferative and dysplastic, and basophils were almost absent, indicating a myelodysplastic/myeloproliferative neoplasm with neutrophilia. Mast cells were conspicuously increased in number, although spindle-shaped mast cells were rarely detectable. Immunophenotyping confirmed the neoplastic nature of CD117 positive mast cells by CD25 and CD2 positivity. Chronic myeloid leukemia was excluded by negative results for BCR::ABL1. The Department of Laboratory Medicine at Hospital Wels-Grieskirchen performed massive parallel sequencing using the MiSeq System (Ilumina) with a customized myeloid panel (Sophia Genetics) including 30 genes; this detected a KIT p.(Asp816Val) hotspot mutation of systemic mastocytosis with a variant allele frequency of 46.2%. However, mutations associated with myelodysplastic/myeloproliferative neoplasm with neutrophilia could not be determined by this molecular genetic analysis.
The Munich Leukemia Laboratory performed further analysis by whole genome sequencing (WGS) and whole transcriptome sequencing (WTS). WGS was done to identify small nucleotide variants, copy number variants, and structural variants. The library preparation was performed using TruSeq DNA PCR-Free HT sample preparation kit (Illumina) according to the manufacturer’s protocol; 151-bp paired-end sequences were generated on the NovaSeq 6000 Sequencing System with a mean coverage of 101x. In addition to the KIT mutation, WGS identified a structural variant involving chromosomes 1 and 17 (chr1_p36.32_2890797::55500506_q22_chr17), presented in Fig. 1. The translocation t(1; 17)(p36; q22) was also detected by chromosomal banding analysis. This structural variant was not detected in CD3+ T cells purified by magnetic activated cell sorting, indicating that it had been acquired in the hematopoietic clone.
Circos plot of WGS results. Red lines indicate structural variants within chromsomal regions. Pathogenic single nucleotide variants are shown in the outer circle based on their chromosomal localization. Color figure available online at clinchem.org.
In addition, WTS was carried out to investigate gene expression profiles and potential fusion transcripts. RNA sequencing libraries were constructed from ribosomal RNA-depleted RNA using TruSeq Stranded Total RNA Library Prep (Illumina). A total of 62 million 101-bp paired-end reads were produced on a NovaSeq 6000 System. The breakpoint region on chromosome 17 was found to be on RNA binding protein Musashi-2 (MSI2); no additional involved gene locus was detected on chromosome 1. No gene fusion was identified by WTS or WGS. Furthermore, gene expression analysis indicated no overexpression of MSI2.
MSI2 is an RNA-binding protein in the class of heterogeneous nuclear ribonucleoproteins A/B that regulate the post-transcriptional processing of messenger RNA and protein expression. RNA-binding proteins and noncoding RNA are capable of binding double- or single-stranded RNA and forming a ribonucleoprotein complex that influences RNA fate via splicing, alternative splicing, RNA modification, nuclear export, and translation rates (1). In a hemato-oncological context, the MSI2 gene was reported for the first time in 2 patients in which chronic myeloid leukemia in a chronic phase progressed into an accelerated phase to the extent of blast crisis. Initially, HOXA9 was reported as a translocation partner (2).
Further research focused predominantly on the interaction of MSI2 with various binding partners. Transcriptome profiling demonstrated that MSI2 induced increased cyclin D1 and MYC expression. In addition, MSI2 induction resulted in an increased activity of growth factors that regulate crucial signaling cascades involving PI3K-Akt, JAK/STAT, and MAPK/ERK pathways (1, 3, 4).
Beyond the transcriptional regulation mechanisms, Nguyen et al. assessed the cell-type specific binding activity of MSI2 (5). They demonstrated that, while MSI2 expression profiles are similar in normal and leukemia cells, the MSI2-RNA binding activity is increased in leukemic cells. These findings show that MSI2 can contribute to leukemogenesis through different mechanisms without overexpression, providing a pivotal explanation for our case. They also showed that MSI2 strongly interacts with MYB, which is an upstream regulator of the transcription factor MYC. Depletion of MSI2 in leukemic stem cells resulted in a reduction of proteins of HOXA9, IKZF2, and MYB without changes in mRNA. Therefore, the increased RNA binding activity of MSI2 might correlate with the increased requirement of MSI2 in leukemic stem cells. In conclusion, these data suggest that MSI2 differs in binding activity at different cell states of progenitor and leukemic cells, enhanced MSI2 binding activity influences various transcription factors, and, decisively, MSI2 is capable of binding to mRNA targets without overexpression.
The WGS and WTS established a final diagnosis of systemic mastocytosis with associated myelodysplastic/myeloproliferative neoplasm with neutrophilia. Therapy with midostaurin and azacitidine was considered. However, because of the patient's young age, a cytoreductive therapy with hydroxyurea was established, and the patient was prepared for allogenic stem cell transplantation.
Nonstandard Abbreviations
WGS, whole genome sequencing; WTS, whole transcriptome sequencing; MSI2, RNA binding protein Musashi 2.
Author Contributions
The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.
Bernhard Strasser (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Software-Equal, Validation-Equal, Writing—original draft-Equal), Gregor Höermann (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Resources-Equal, Software-Equal, Supervision-Equal, Writing—review & editing-Equal), and Alexander Haushofer (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Resources-Equal, Software-Equal, Supervision-Equal, Writing—review & editing-Equal)
Authors’ Disclosures or Potential Conflicts of Interest
Upon manuscript submission, all authors completed the author disclosure form.
Research Funding
None declared.
Disclosures
The authors state no conflicts of interest.
