ASXL1 Mutations at Diagnosis Did Not Impact on the Depth of Molecular Response (MR) and on Treatment-Free Remission (TFR) Eligibility in Chronic Phase (CP) Chronic Myeloid Leukemia (CML) Patients (pts) Receiving either Nilotinib (NIL) First-Line or Imatinib (IM) with Early Switch to NIL in Case of No Optimal Response in the SUSTRENIM Clinical Trial

Whether additional mutations in genes other than BCR::ABL1 harbor predictive or prognostic value and whether they should be incorporated in routine diagnostic workups for newly diagnosed CP CML pts is a controversial issue. Most of the currently available data pointing to a potential negative impact of ASXL1 and/or epigenetic genes on response to therapy come from NGS analyses of selected and/or non-homogeneously treated cohorts of pts, with only 2 studies having so far focused on pts enrolled in prospective clinical trials (TIDEL II and TIGER, respectively).

We undertook a translational study aimed to investigate the impact of molecular profiling at diagnosis on the likelihood to achieve deep MR and stable TFR in CP CML pts enrolled in the GIMEMA/HOVON CML1415 (‘SUSTRENIM’), an international prospective clinical trial that randomized pts to receive either NIL or IM with switch to NIL in case of no optimal response (NCT02602314). All the pts achieving ≥MR4 by 36 months (mo) and maintaining it up to 48 mo of therapy qualified for the TFR phase. SUSTRENIM enrolled a total of 448 pts (IM, n=220; NIL, n=228); median follow-up is 56 mo (range, 42-60). One hundred and twenty-four pts who signed the informed consent for participation in this translational study had peripheral blood samples collected at diagnosis and subject to whole exome sequencing (WES). This subset of pts did not differ from the whole population in terms of age, gender, Sokal and ELTS scores, TFR eligibility; 73 of them were randomized to NIL and 67 to IM. Exome capture was performed with the egSEQ Exome Panel (Edinburgh Genetics), followed by 2×150bp paired-end sequencing on a NovaSeq X Plus (Illumina) (48Gb/sample; average sequencing depth, 300X). Raw sequence reads were aligned on the GRCh38 human reference genome and variants called using Dragen v3.9.5 pipeline. Variants labeled as somatic were retained for downstream annotation using VarSeq v2.5.0. For the purpose of the present report, we focused on somatic single nucleotide variants and frameshift indels in ASXL1 and in a comprehensive set of 123 genes compiled using publicly available databases of epigenetic factors and leukemia-associated genes. Strict filtering criteria were adopted, for uniformity with previous NGS studies in CML, to select for variants predicted to be nonsynonymous or to disrupt essential splice donor or acceptor sites and to be either loss of function or damaging according to ≥4 functional prediction tools. Pts with and without mutations were compared using Wilcoxon rank sum test, Fisher's exact test or Pearson's Chi-squared test; cumulative incidences of progression-free survival (PFS) were compared with the Fine and Gray test.

ASXL1 mutations were detected in 8/124 (7%) pts. These pts were slightly younger (median, 44 vs 58 years, p=0.059); no correlation was observed with gender, Sokal or ELTS, transcript. Depth of MR at 3, 12, 24, 36 and 48 mo was not significantly different between pts with and without mutations, either stratifying into MR levels at each timepoint or categorizing responses as < or ≥MR3, MR4, M4.5 at a given timepoint (of note: ≥MR3 at 3 mo, 0% vs 13%, p=0.6; ≥MR3 at 12 mo, 80% vs 74%, p>0.9; ≥MR4 at 24 mo, 60% vs 60%, p>0.9; ≥MR4 at 36 mo, 100% vs 75%, p=0.6; ≥MR4 at 48 mo, 100% vs 83%, p>0.9 for pts with and without ASXL1 mutations, respectively). A similar percentage of ASXL1-mutated and -unmutated pts became eligible for TFR as per protocol (43% vs 42%, p>0.9). PFS did not differ between pts with or without ASXL1 mutations (p=0.95). Mutations in epigenetic and leukemia-associated genes were detected in 17/124 (14%) pts. Mutated genes were IKZF1, IDH1, IDH2, DNMT3A, BCOR, BCORL1, NSD2, PHF2, KDM1B. As above, no significant differences were observed between pts with and without mutations in terms of MR depth at different timepoints, TFR eligibility, PFS.

In conclusion, presence of an ASXL1 mutation (or of other epigenetic or leukemia-associated gene mutations) did not impact on the depth of MR or on TFR eligibility either in pts treated with NIL or in pts treated with IM with proactive switch to NIL in case of no optimal response. Although algorithms for risk stratification and tailored treatment based on molecular profiling at diagnosis are eagerly awaited, our data warn against the premature incorporation of ASXL1 mutations among high risk features in treatment recommendations, and highlight the need for further studies in prospective series of uniformly treated pts.