Molecular Characterization of Philadelphia Chromosome Positive Acute Myeloid Leukemia - New Provisional Entity?
Authors: Weber Simone , Meggendorfer Manja , Nadarajah Niroshan , Perglerová Karolína , Schnittger Susanne , Haferlach Claudia , Kern Wolfgang , Haferlach Torsten 
Published: October 2019

Abstract
Introduction
Philadelphia chromosome positive (Ph+) acute myeloid leukemia (Ph+AML) is discussed to be a new provisional entity for the upcoming WHO classification. Whether Ph+AML represents a distinct entity or rather embodies chronic myeloid leukemia in myeloid blast crisis (CML-BC) without preceding clinical manifestation is under debate mostly due to lack of robust criteria to reliably differentiate these two diseases. Further, while Ph+AML is clearly distinguishable from Ph+ acute lymphoblastic leukemia (Ph+ALL) based on immunophenotyping, recent studies demonstrated that Ph+AML retain typical characteristics of lymphoid disease.
Aim
Ph+AML, CML-BC and Ph+ALL were analyzed by a panel of 24 genes and array CGH to get more insights into these Ph+ leukemias and to potentially define delimiting genetic features.
Patients and Methods
We examined 24 pts with Ph+AML (11 females/13 males, median age: 58 (24-83)), 11 CML-BC (7 females/4 males, median age: 60 (32-79)) and 11 Ph+ALL (7 females/4 males, median age: 67 (44-77)). AML and ALL were diagnosed according to WHO classification by morphology, MPO and flow cytometry. CML-BC all were diagnosed as CML before and treated accordingly. All cases revealed the BCR-ABL1 fusion gene. Next generation sequencing was performed for ASXL1, BCOR, CBL, CSF3R, DNMT3A, ETV6, FLT3 tyrosine kinase domain(FLT3 -TKD), IDH1/2, JAK1/2/3, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1 and ZRSR2 using the MiSeq Instrument (Illumina, San Diego, CA). Partial tandem duplications in MLL (MLL- PTD), internal tandem duplications in FLT3 (FLT3- ITD)and deletions in IKZF1 were analyzed by quantitative real-time PCR or genescan analysis. 45 cases were investigated by array CGH (Agilent, Waldbronn, Germany).
Results
With respect to cytogenetic abnormalities besides Philadelphia chromosome, several unbalanced abnormalities were found in Ph+ALL (mean: 9, range: 2-31), while less aberrations were found in Ph+AML and CML-BC (mean: 4, range: 0-28 and mean: 4, range: 0-16, respectively; p=0.03). Of these the most prominent aberrations which were present in all three groups included loss of 7p encompassing IKZF1 (Ph+AML: 7/23, 30%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%), loss of 9p encoding CDKN2A/B (Ph+AML: 2/23, 9%; Ph+ALL: 6/11, 55%; CML-BC: 2/10, 20%) as well as gain of 8q (Ph+AML: 6/23, 26%; Ph+ALL: 3/11, 27%; CML-BC:4/11, 36%). Loss of 5q (5/23, 22%), gain of 13q (4/23, 17%) and loss of 21q (3/23, 9%) was exclusively present in Ph+AML. While in Ph+ALL loss of 10q (3/11, 27%), 2p (3/11, 18%), 11q (3/11, 18%) and gain of 4q (3/11, 18%) was exclusively found. Regarding recurrent balanced aberrations no rearrangements were found in Ph+AML and Ph+ALL, while 3/11 (27%) CML-BC pts harbored balanced 3q26-rearrangements.
With respect to molecular genetics, alterations were found in 15/24 (63%) Ph+AML, 8/11 (73%) CML-BC and 8/11 (73%) Ph+ALL pts. Commonly shared molecular aberrations were deletions in IKZF1 (Ph+AML: 3/24, 13%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%) as well as mutations (mut) in RUNX1 (Ph+AML: 5/19, 26%; Ph+ALL: 1/7, 14%; CML-BC: 5/10, 50%). Further, mut found in Ph+AML and CML-BC affected ASXL1 (Ph+AML: 2/22, 9%; CML-BC: 2/11, 18%) and IDH1 (Ph+AML: 2/22, 9%; CML-BC: 1/11, 9%). Additionally, Ph+AML harbored alterations in TP53 (3/21, 14%), TET2 (2/21, 10%) and DNMT3A (1/20, 5%). For CML-BC, additional mut were found in WT1 (2/9, 22%), ETV6 (1/9, 11%) and KRAS (1/9, 11%). Regarding FLT3 -ITD, NPM1 and the remaining genes no alterations were found. Overall, Ph+ALL differed from the combined cohort of Ph+AML and CML-BC in that mut in ASXL1, DNMT3A, ETV6, IDH1, KRAS, TET2 and WT1 as well as MLL -PTD occurred not in the former but only in the latter (12/31 cases with at least one gene mutated, p=0.07). Intriguingly, the mean±SD number of mut in these genes did not significantly differ between Ph+AML and CML-BC cases (0.36±0.58 vs. 0.67±0.71, p=0.23).
Conclusion
Comparing cytogenetic alterations Ph+AML could be clearly distinguished from CML-BC or Ph+ALL by harboring loss of 5q and gain of 13q, which are typically found in myeloid diseases. Beside, Ph+AML and CML-BC showed a high frequency of molecular mutations which were hardly found in Ph+ALL. This data supports the concept discussed by the WHO that Ph+AML is a specific entity and can be distinguished from CML-BC and Ph+ALL. However, further studies are warranted to define the most appropriate parameters to distinguish Ph+AML from CML-BC.

Disclosures
Weber: MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.