JAK2 Mutation Screening and Chromosome Analysis Are Necessary for a Comprehensive Diagnostic Work up in CMPD: A Study on 469 Cases.
Authors: Schnittger Susanne , Haferlach Torsten , Petrides Petro E. , Kern Wolfgang , Schoch Claudia 
Published: April 2020

Abstract
The diagnosis of BCR-ABL negative chronic myeloproliferative disorders (CMPD) is still a challenge for the morphologist and clinician, mainly because of overlapping phenotypes of essential thrombocythemia (ET), polycythemia vera (PV), idiopathic myelofibrosis (IMF) and non-malignant reactive phenotypes. Recently, a new mutation in JAK2 leading to V617F exchange in exon 12 was described in these entities. Therefore, we developed a rapid and easy LightCycler based melting curve assay and screened 469 patients with various malignancies for the respective JAK2 mutation using cDNA prepared from mononucleated cells. All cases tested positive were confirmed by sequencing and without any exception all mutations were G to T exchanges at nucleotide 1849. In total, 61 cases with PV were analysed. 56 (91.8%) were mutated (see table). A karyotype was available in 36 cases. Of the JAK2- cases 4 had normal karyotypes and one 20q-. Of the JAK2+ cases 21 had normal karyotype; four (7.1%) had +9, one +8, one +22, one 20q-, and three showed a complex aberrant karyotype. Of the 47 analyzed ET 26 (55%) were mutated. Cytogenetics was available in 20 cases (9 JAK2-, 11 JAK2+). All JAK2- cases all had normal karyotypes. Of the JAK2+ 10 had a normal karyotype and one a t(9;13)(p24;q22) involving the JAK2 locus. Of the 25 IMF cases 14 (56%) were mutated. Karyotype was available in 17 cases (7 JAK2-, 10 JAK2+). All 7 JAK2- had normal karyotype. Of the 10 JAK2+ 6 had normal karyotype and four were aberrant (+9:n=2; 20q-: n=2). In addition, 2/7 cases (28.6%) with CMML were JAK2+. Furthermore, we analysed 89 BCR-ABL negative MPS that were not further specified. V617F was detectable in 43 patients (48.3%). At next 128 AML were analyzed (84 novo AML, 11 t-AML after a previous malignancy, 15 secondary to MDS, and 16 secondary to MPS (PV:n=7, ET:n=5, OMF:n=2). No V617F was detected in all cases with t-AML and s-AML after MDS. In contrast, in de novo AML 6/84 (7.1%) were JAK2+, of which 4 were proven homozygous. Surprisingly, 4 of these six cases had a +9. In AML secondary to a previous CMPD 11/16 (68.8 %) were JAK2+. Of these 2 had +9, one a normal karyotype and 12 a complex aberrant karyotype. These data suggest that acquisition of +9 may lead to progress or blast crisis in JAK2 mutated MPS and supports the gain of function mechanism of the mutation.
In conclusion, more than half of all BCR-ABL-negative MPS harbour a V617F mutation. This helps in the differential diagnosis of MPS versus reactive disorders. V617F is more frequently associated with aberrant karyotype than wildtype JAK2. In addition, there is an increasing level of aberrant cytogenetics and mutation rate with respect to incidence and status from ET&lt;IMF&lt;PV indicating that these diseases might be overlapping or even a continuum. It demonstrates that the present classification is artificial and a new classification of “JAK2” positive diseases may be more adequate. In addition, the same mutation was observed in most cases of AML secondary to CMPD. It was also found in few cases with de novo AML and correlated with +9. JAK2V617F is a new and promising target for therapy as well as for molecular monitoring of therapy response in CMPD.
Distribution of JAK2 mutation in various malignancies ET IMF PV MPS* CMML de novo AML AML after MPS *not further specified total 47 25 61 89 7 84 16 mutated (n=) 26 14 56 43 2 6 11 mutated (% of all) 55.0 % 56.0 % 91.8 % 48.3 % 28.6 % 7.1 % 68.8 % homozygous (n=) 3 9 33 19 1 4 7 homozygous (% of all) 6.4 % 36.0 % 54.1 % 21.3 % 14.3 % 4.8 % 43.8 %