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A. Bidet et al.
Cytogenetic Nomenclature. Molecular monitoring was performed according to the ELN recommendations.1 A morphological central review was used to screen for myelodysplastic features at the time of CCA/Ph- emergence in 48 cases. Morphological dysplasia was considered significant when it was observed for 10% or more cells in any hematopoietic lineage with or without excess of blasts (>5%). Erythroid lineage dysplasia criteria include nuclear and cytoplasmic abnormalities (multinuclearity, laminated cytoplasm, macroerythroblasts). Dysgranulopoiesis also includes hypogranu- lar or hypergranular precursors and/or neutrophils and/or lack of nuclear segmentation. Micromegakaryocytes, multinuclear or hypolobulated megakaryocytes were the main abnormalities observed in the megakaryocyte lineage. Patients were stratified according to the presence or absence of chromosome 7 abnormal- ities, whether isolated or not, leading to -7/del(7q) CCA/Ph- iden- tified by conventional cytogenetics. In the case of CCA/Ph- detec- tion after the diagnosis of CML, time of emergence was retrospec- tively evaluated on prior samples by fluorescence in situ hybridiza- tion when possible. Complex karyotypes (≥3 anomalies) affected only five of the 102 patients; since this precluded statistically meaningful analyses, these abnormalities were not considered as a separate category. Underlying MDS was documented both by cen- tralized morphological analysis of bone marrow smears and by NGS for a targeted panel of 27 genes frequently altered in MDS and AML (ASXL1, CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, PTPN11, RIT1, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, ZRSR2). NGS data obtained for 45 CML patients were compared with a MDS control group of patients with (n=4) or without (n=7) cytogenetic chromosome 7 anomalies. NGS was performed as previously described15 on follow-up bone marrow samples at complete cytogenetic response and preferably at best molecular response, to limit the risk of residual Ph+ clone contam- ination.
The quality of response to treatment was classified by the best cytogenetic or molecular response obtained at any point after one, two, three or more lines of treatments into no cytogenetic response, partial cytogenetic response, complete cytogenetic response, major molecular response and deep molecular response ≤0.0032% (MR4.5). The response criteria were according to the standard ELN definitions.1
Statistical analysis
Bivariate analyses were performed to compare -7/del(7q versus other CCA/Ph-. Quantitative variables were described by their mean and standard deviation in both groups and compared by a Student t test if the distribution was normal or with their median, range and quartiles along with a non-parametric Mann-Whitney test when the distribution was not normal. Cumulative incidences from diagnosis or initiation of first-line TKI treatment until the achievement of the considered response were calculated with death without response as a competing risk. Gray tests were per- formed to compare cumulative incidence curves. Analyses from time of CCA/Ph- emergence were not performed because detec- tion time may be delayed and underestimated for some patients depending on the clinical context and requirement of additional cytogenetic controls despite complete cytogenetic response. However, the effect of type of CCA/Ph- on response, progression, or late events was considered by landmark analyses 3 years after the initiation of TKI therapy. Probabilities of overall survival, pro- gression-free survival and event-free survival since initiation of first-line TKI therapy were calculated and illustrated by the Kaplan-Meier method: until death at any time and for any reason (overall survival); until death or progression to accelerated phase or blastic transformation (progression-free survival); and until intoler-
ance, loss of response, resistance, treatment switch, progression, or death (event-free survival). Survival curve comparisons, including landmark analyses, were performed using log-rank tests. The level of statistical significance was set at 5%. All the statistical analyses were performed with R V3.2.3.
Results
Clinical and cytogenetic presentation of patients with chronic myeloid leukemia
The patients’ baseline characteristics are summarized in Online Supplementary Table S1). Twenty-six out of the 102 (25.5%) patients had CCA/Ph- affecting chromosome 7, which was an isolated abnormality in 13 out of the 26 cases (50%) and associated with +8 in 19.5% cases. In one patient with -7 CCA/Ph-, +8 was detected in a separate clone. Among other CCA/Ph- cases, +8 was the most fre- quent other abnormality, affecting 31 out of the 46 patients (67.4%); 13.7% (n=14) patients had -Y CCA/Ph-. Patients who developed -7/del(7q) CCA/Ph- were significantly younger (mean 48 vs. 55 years old; P=0.035) and mostly benefited from second-generation TKI as first-line treat- ment (mainly dasatinib: 15.4% vs. 2.6% cases in the other CCA/Ph group; P=0.027). Other baseline characteristics were similar between the -7/del(7q) CCA/Ph- cases and the other CCA/Ph- cases. The median follow up from the diag- nosis of CML was 6.47 (range, 1-19) years. The median time of CCA/Ph- detection after starting TKI therapy was 2.08 (range, -0.8 to 12.65) years for -7/del(7q) CCA/Ph- and 1.02 (range, -3.32 to 11.02) years for other CCA/Ph-. The -7 CCA/Ph- was present at diagnosis in only one case, as deter- mined by retrospective fluorescence in situ hybridization analysis. Twelve out of the 26 patients who developed - 7/del(7q) CCA/Ph- were in complete cytogenetic response at the time the abnormality was detected. The median fol- low up since CCA/Ph- detection was 5.35 (range, 1-14) years for -7/del(7q) and 7.41 (range, 0-15) years for other CCA/Ph- (P≥0.05). The trend approached statistical signifi- cance for the number of treatment lines required after the first 3 years of follow up, as patients with -7/del(7q) CCA/Ph- more frequently needed two or more treatment lines (30% vs. 13.10% of patients with other CCA/Ph-; P=0.049, chi-square).
Biological analysis of underlying associated myelodysplastic syndrome
The presence of MDS features at the time of CCA/Ph-, determined by morphological analysis of the bone mar- row, was evaluated in 48 patients and appeared to alter the quality of response to treatment, as only 9.4% of patients with MDS signs (24 patients) reached a major molecular response or better versus 47% of patients with- out MDS signs (P<0.0001, chi-square). Morphological MDS features were more frequent among the -7/del7q CCA/Ph- group [50% of patients with -7/del(7q) CCA/Ph- vs. 20% with other CCA/Ph-], and only 33% (4 out of 12) of the -7/del(7q) patients with MDS signs achieved a major molecular response or better compared to 75% (9 out of 12) of these patients without MDS signs. For patients with other types of CCA/Ph-, only six out of 49 patients (18.8%) with an optimal response had morpho- logical features of MDS (P<0.00001, chi-square). NGS was performed for 45 CML patients with available DNA at the time of detection of CCA/Ph- and 11 MDS patients as a
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haematologica | 2019; 104(6)