Lineage-Specific Distribution of CALR EXON 9, JAK2V617F, MPLW515L/K, NPM1 and FLT3 Mutations in Myeloid Disorders

• Published in: Blood 2014-12, Vol.124 (21), p.5573-5573
• Main 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
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.5573.5573

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 mu