CML Patients with Philadelphia-Negative Clones Occurring During Treatment with Tyrosine Kinase Inhibitors Harbor Molecular Mutations Typical for MDS.

In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKIs), so-called Philadelphia-negative (Ph-) clones. The most frequent abnormalities in these Ph- clones reported are monosomy 7 (−7), trisomy 8 (+8) or loss of the Y-chromosome (-Y). This pattern of cytogenetic aberrations is comparable to alterations found in MDS or AML. Previous studies based on clinical parameters and cytomorphology revealed no evidence of a secondary hematologic malignancy in the majority of patients. However, few cases were reported to evolve to MDS or even AML. It has not yet been addressed whether molecular mutations are present in patients with Ph- clones.

Analyse whether CML patients with Ph- clones during TKI treatment harbor molecular mutations pointing to a malignant disease.

We selected 44 patients treated with TKIs (Imatinib: n=29, Dasatinib: n=7, Nilotinib: n=8) who developed Ph- clones. Median time from start of therapy to analysis was 26 months (range 5–78 months). 22 cases were male and 22 female, median age was 59.4 years (range: 34.1–83.9 years). Ph- clones were characterized as follows: +8 (n=19), -Y (n=7), −7 (n=4), +9 (n=2), one case with +8 and del(20q), one case each with: +6; +11; +14; +19; -X; del(4)(p14p15); del(13)(q14q31); t(11;14)(q22;q31),del(20)(q11q13); t(7;18)(q11;q23); ider(13) (q10)del(13)(q14q21); der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11). BCR-ABL1 levels at the time point of analysis were between 0 and 3.1 (median: 0.183) according to international scale. Mutation analyses were performed for ASXL1, RUNX1, MLL- PTD, NRAS, and JAK2. Mutations were analyzed by a combination of amplicon deep-sequencing (RUNX1), direct Sanger sequencing (ASXL1), real time PCR (MLL- PTD, BCR-ABL1) and melting curve analyses (NRAS, JAK2).

In total, in 10 of 44 cases (22.7%) one (n=8) or two (n=2) of the analysed genes were mutated. In five cases we detected an ASXL1 mutation. These were p.Glu635ArgfsX15 (two cases with mutation loads of 50% and 10%, respectively), p.Gly646TrpfsX12 (mutation load 10%), p.Gln592ProfsX27 (10%), and p.Gln748HisfsX24 (10%). RUNX1 was mutated in 4 cases: p.Thr77Ala (50%), p.Asn109X (44%), p.Asn119Asp (3.6%), p.Leu261TrpfsX23 (2.3). In addition, one case harbored a p.Gly12Asp in NRAS (50%) and one an MLL-PTD (10%). The double mutated cases had ASXL1 Glu635ArgfsX15 and MLL-PTD (both 10%) and ASXL1 Gln592ProfsX27 (10%) and RUNX1 Leu261TrpfsX23 (2.3%), respectively. In 9/10 cases a sample from diagnosis of CML was analyzed retrospectively and 10/11 mutations were not detected showing that the mutations, like the Ph- cytogenetic aberrations, evolved during treatment. Only the p.Thr77Ala in RUNX1 was also seen in the diagnostic sample with a mutation load of 50% indicating that this was also present in a Ph+ clone (as a mutation or rare inborn variant). Correlation to the respective cytogenetic aberrations showed +8 in 5 cases (2 with RUNX1 mut and 3 with ASXL1 mut, including the RUNX1/ASXL1 double mutated), +11 in the ASXL1/MLL-PTD double mutated case, +9 (RUNX1 mut), der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11) with RUNX1 mut, t (7;18)(q11;q23) with NRAS mut, del(13)(q14q31) with ASXL1 mut. In 9 cases the load of the respective mutations correlated to the percentage of metaphases positive for the respective chromosome aberration (R=0.82; p=0.004) and to interphase FISH positivity (available in 8 cases) (R=0.86; p=0.007). In one case with a deep-sequencing read count of 50% RUNX1 mut load we detected only 6/20 metaphases with +8 and only 9% +8 as assessed by interphase FISH, so in this case the molecular mutation possibly occurred prior to the gain of +8.

This data shows that a significant number of CML patients treated with TKI not only develop Ph- clones with cytogenetic abnormalities resembling a MDS typical pattern but also harbor molecular mutations in genes that are typically mutated in MDS and AML (ASXL1, RUNX1, NRAS, MLL-PTD). This is a further proof of the malignant character of these Ph- clones. Whether molecular genetic mutations may also occur in Ph- follow up samples of patients without cytogenetic aberration has to be addressed in future studies. These findings have implication for the molecular monitoring in CML patients treated with TKI.

Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.