Molecular Characterization of Philadelphia Chromosome Positive Acute Myeloid Leukemia - New Provisional Entity?

Abstract

IntroductionPhiladelphia 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.AimPh+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 MethodsWe 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).ResultsWith 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).ConclusionComparing 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. DisclosuresWeber: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.