Tyrosine Kinase Inhibitor Treated CML Patients Harboring Philadelphia-Negative Cytogenetically Aberrant Clones Show Molecular Mutations In 31% Of Cases Not Present At Diagnosis: A High-Throughput Amplicon Sequencing Study Of 29 Genes
Authors: Schnittger Susanne , Kuznia Sabrina , Meggendorfer Manja , Nadarajah Niroshan , Jeromin Sabine , Alpermann Tamara , Roller Andreas , Albuquerque Andreia , Weissmann Sandra , Ernst Thomas , Eder Christiane , Dicker Frank , Kern Wolfgang , Kohlmann Alexander , Haferlach Torsten , Hochhaus Andreas , Haferlach Claudia 
Published: October 2019

Abstract

Background
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 -7, +8 and -Y. This pattern of cytogenetic aberrations is comparable to alterations found in MDS. In the majority of patients no clear evidence of a secondary hematologic malignancy is observed. However, few cases especially with -7 were reported to evolve to MDS or even AML.

Aim
1) Analyze whether TKI-treated CML patients who developed Ph- clones also harbour molecular mutations. 2) Evaluate whether these mutations were already present at diagnosis of CML.

Patients and Methods
We selected 42 patients (pts) treated with TKIs (Imatinib: n=27, Nilotinib: n=2, and 13 pts who were treated with 2 or 3 different TKIs) who developed Ph- clones. Median time from start of therapy to analysis was 3.2 years (range 8.9 months-13.5 years). 23 cases were male and 19 female; median age was 63.3 years (range: 34.1-83.9 years). Cytogenetics in pts with Ph- clones were as follows: +8 sole (n=18), -Y (n=7), -7 sole (n=2), +9 (n=2), an 10 cases with various abnormalities. Additional three cases had two different clones: 1) -7 and +8; 2) +8 and +Y, 3) -Y and +Y. Median clone size was 37.5% (range 10-100%) of all metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 4.5 (median: 0.019) according to international scale. All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sciences NGS platform and the MLL-PTD was analyzed by quantitative real time PCR. Median coverage per amplicon was 2215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%.

Results
In total, in 13/42 pts (31.0%) one (n=9) or two (n=4) of the analyzed genes were mutated. Median mutation load was 15% (range: 3-50%). In detail, mutations in the following genes were detected: ASXL1 (n=5), DNMT3A (n=4), NRAS (n=2) and each one in BCOR, CBL, MLL-PTD, RUNX1, TET2 and ZRSR2. 4 pts had combinations of: 2 ASXL1mut, 2 NRASmut, BCORmut and RUNX1mut, or ASXL1mut with MLL-PTD, respectively. Subsequently, the mutations were tracked as far as serially collected samples were available (12 pts with a total of 125 samples, range: 3-20 samples/pt) from earlier or later time points. In 6 cases it clearly could be shown that the mutations were not present at diagnosis and arose 5 months, 7 months, 14 months, 15 months, 5 years, and 7 years after start of TKI therapy, respectively. In all these cases the mutation loads increased in parallel to decreasing BCR-ABL1 levels. In one of these cases one mutation was found 3 years and a second 11 years after start of therapy. Importantly, in three of these cases the molecular mutations were detectable before the cytogenetic aberration in the Ph- clone. In six further cases mutations were present with similar levels in several samples but due to lack of samples from initial diagnosis no estimation of the time to their occurrence was possible. In one of these cases the BCR-ABL1 load increased again during therapy and the DNMT3A mutation decreased showing that the BCR-ABL+ clone and the DNMT3Amut clone were competitive clones.

Conclusions
1) For the first time we show that more than 30% of CML patients treated with TKI who develop Ph- clones as defined by cytogenetic abnormalities, also harbour molecular mutations. 2) For most of the cases it was shown that these mutations arose during treatment. 3) In some cases the molecular mutations were detectable before the cytogenetic aberrations. This may be regarded as further proof of the malignant character of these Ph- clones. However, none of the analyzed cases, so far, developed a secondary disease, i.e. MDS or AML, thus the relevance of these findings is still unclear and needs further attention.

Disclosures:
Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kuznia:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Novartis: Research Funding. Hochhaus:Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.