Lineage-Specific Distribution of CALR EXON 9, JAK2V617F, MPLW515L/K, NPM1 and FLT3 Mutations in Myeloid Disorders
Authors: Burnworth Bettina , Bennington Richard , Fritschle Wayne , Nguyen Phung , Verkamp Stephanie , Angela Bennington , Wentzel Collette , Broderson Lisa , Loken Michael R. , Wells Denise A. , Zehentner Barbara Katharina 
Published: October 2019

Abstract
Introduction:
Myeloid malignant disorders are clonal diseases arising in hematopoietic stem or progenitor cells. Several somatic mutations involved in these diseases are currently known and routine molecular testing involves screening genes of therapeutic and prognostic significance. Mutational analysis of FLT3 in combination with NPM1 can be used to predict outcome and direct therapy in normal karyotype acute myeloid leukemia (AML). JAK2, MPL and CALR mutation detection complements the molecular diagnostic testing menu for myeloproliferative neoplasm’s (MPN): polycytemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In addition, these molecular markers are utilized for minimal residual disease (MRD) detection, e.g. following stem cell transplants. Little is currently known about the lineage-specific distribution of some of these markers. In this study we aimed to assess the distribution of common genetic mutations in multiple lineages (lymphoid, myeloid, monocyte, multipotent progenitors, myeloblast and erythroid) of MPN and AML utilizing fluorescent activated cell sorting (FACS).
Method:
Different cell lineage fractions (lymphoid (CD3+), mature (CD16+) and immature (CD16dim) granulocytes, monocyte (CD14+), erythroid(CD36+), multipotent progenitors (34+) and/ or myeloblasts (CD117+) of unseparated bone marrow and peripheral blood specimens of myeloid disorders were sorted on a BD Aria 2. The patient specimens selected were positive for either JAK2V617F, MPLW515L or CALR Exon9 insertion/ deletion (MPN’s) or for NPM1 and/or FLT3 mutations (AML; diagnostic, relapse and minimal residual disease (MRD)). Fractions were subsequently analyzed for the presence of the respective mutation by PCR and/ or bi- directional sequencing.
Results:
All FACS purified CD34+ progenitors, myeloid and erythroid cell fractions of MPL W515L (3) or CALR exon9 (12) positive MPN specimens demonstrated the presence of mutations, respectively. Interestingly, JAK2V617F was present in the sorted erythroid cell fraction in 5/6 MPN cases tested. However, the granulocyte cell and blast cell fraction of one polycythemia vera specimen tested negative for the presence of Jak2V617F. All lymphoid CD3+ T-cell fractions were negative.
The NPM1 exon 12 mutation was uniformly detected in progenitors and all myeloid cell fractions of 3/3 diagnostic, 2/2 relapse and 1/8 MRD AML specimens. For 5/8 MRD cases all lineages tested negative. Surprisingly, for two MRD cases the mutation was observed only in the unseparated and myeloid lineages but not in CD34+ blast fraction. Similar to the above findings, FLT3 mutations were detected in multipotent progenitors, and/ or myeloblasts collections of 4/4 diagnostic specimens. However, the mutation was absent in the granulocyte and monocyte fraction of one case. No detectable signals were observed in the cell fractions of 5 MRD specimens and in the CD3+ lymphoid cell fractions of all AML cases.
Conclusion:
We conclude that CALR and MPL mutations are uniformly detectable in the unseparated bone marrow specimens of MPN’s as well as separated progenitor, erythroid, granulocyte and monocyte fractions. Interestingly, JAK2 mutations can be exclusively found in the erythroid lineage in PV, whereas it can be absent in the granulocyte and blast compartment. This finding may have implications on specimen processing to ensure that erythroids are retained for clinical Jak2 testing. In addition, our results support the hypothesis that CALR Exon9 mutations are early event driver mutations in comparison to JAK2V167F.
Both NPM1 and FLT3 mutations, in AML, were detected in unseparated specimens as well as in the multipotent progenitors or myeloblasts at diagnosis and relapse. However, the NPM1 mutation was observed in unseparated specimens and granulocyte cell fractions of 2 residual disease cases, whereas it was surprisingly absent in the CD34+ cell lineage fractions. Conversely, FLT3-ITD was exclusively found in the progenitor cells and absent in the granulocyte lineage of one case at diagnosis.
Our findings reported here may be able to assist assay development efforts for diagnostic and residual disease mutation detection in myeloid disorders. In addition, flow cytometric assessment of monitoring specimens prior to molecular analysis may be beneficial to decide if cell enrichment steps can give additional evidence for the presence of residual disease.

Disclosures
Burnworth: HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Angela:Hematologics: Employment. Wentzel:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.