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Clinical significance of chromatin-spliceosome AML
Introduction
According to the current World Health Organization (WHO) classification,1 secondary acute myeloid leukemia (sAML) is defined either by a previous clinical history of hematological disease, the morphological detection of multilineage dysplasia or specific cytogenetic characteris- tics; the two latter criteria are additive to the clinical his- tory or may be themselves sufficient to diagnose sAML, even in the absence of a known antecedent myelodys- plastic syndrome (MDS) or myeloproliferative neoplasm (MPN).1,2 However, in clinical practice some uncertainty remains regarding the correct classification of potentially high-risk sAML cases, especially when an antecedent his- tory is not thoroughly documented or does not fully sat- isfy the diagnostic criteria of MDS. Moreover, morpho- logical examination to assess blast counts and multilin- eage dysplasia shows inter-observer variability that may impair diagnostic reproducibility.3 Finally, cytogenetic analysis, which usually takes a long turnaround time of 5- 10 days, may not be always informative because of tech- nical failure or a normal result. These features eventually translate into under-recognition of sAML patients and lead to inadequate clinical management, since this high- risk population deems intensive treatment strategies comprising the administration of innovative agents or allocation to clinical trials, which are potentially able to improve the rates of complete remission (CR),4-7 followed by a rapid consolidation with allogeneic hematopoietic stem cell transplant (alloHSCT).7,8 Therefore, more accu- rate diagnostic approaches are warranted. In this regard, studies focusing on the molecular landscape of sAML and preceding conditions have suggested the possibility of defining distinct subtypes of AML based on their muta- tional profiles. Mutations in genes involved in chromatin regulation (ASXL1, EZH2, BCOR, STAG2) and RNA splic- ing (SRSF2, SF3B1, U2AF1, ZRSR2) have shown high specificity for sAML after MDS;9 these mutations, called secondary-type mutations, occur early in leukemogenesis (likely representing the expansion of clones acquired dur- ing previous MDS)10,11 and often persist in clinical remis- sion, presenting as constitutively chemoresistant. Although less characterized, sAML cases progressing after MPN display similar features.12,13 However, a closely related mutational signature can be identified also in some de novo AML cases.14,15 In the seminal study conduct- ed by Papaemmanuil et al.14 on a large cohort of AML patients, overlapping mutations in genes regulating RNA splicing (SRSF2, SF3B1, U2AF1, and ZRSR2), chromatin (ASXL1, STAG2, BCOR, KMT2A-PTD, EZH2, and PHF6), or transcription (RUNX1) constituted an independent genomic class, called the chromatin–spliceosome group. Mutations in RUNX1 and KMT2A-PTD frequently co- occurred with other mutations of the signature and, albeit not universally observed in sAML, have been reported as more significantly associated with high-risk than low-risk MDS,11 and consistently occurred at intermediate time points and not as founding mutations.11, 14-16 Strikingly, 91% of patients in this group were clinically defined as de novo AML; although heterogeneous, mutations in this molecular signature were consistently associated with older age, lower white blood cell and blast counts, lower rates of response to induction chemotherapy and higher relapse rates. While these results warrant prospective val- idation, it can be hyphothesized that the presence of
these mutations represent the trace of a previous, unrec- ognized MDS or MPN phase. However, a formal compar- ison between de novo AML patients carrying chromatin- spliceosome (CS) mutations and sAML patients defined by standard criteria, along with associated outcomes, is currently lacking.
In order to investigate the clinical significance of the CS mutational signature, we reassessed patients’ diagnosis according to the presence of CS mutations in a large cohort of newly diagnosed AML patients enrolled into a prospective trial (NILG AML 02/06) (clinicaltrials gov. Identifier: NCT00495287).17 In this study, we report the characteristics and outcomes of initially defined de novo AML patients carrying CS mutations as compared with other clinically defined de novo AML patients without CS mutations and patients with sAML defined by standard WHO criteria.
Methods
Patients, treatment, cytogenetic and molecular analyses
The NILG-AML 02/06 multi-center Italian trial17 enrolled 574 patients with newly diagnosed AML (≥20% bone marrow [BM] blasts) or high-risk MDS (10-19% BM blasts) between 2007 and 2012. All participants were randomized to receive induction with standard-dose idarubicin, cytarabine and etoposide (ICE) or high-dose cytarabine and idarubicin (sHD). Patients not responding to first induction underwent an intensified re-induc- tion with sHD. Consolidative alloHSCT was performed in high- risk patients based on study-specific risk stratification, as previ- ously reported.17 Written informed consent for inclusion in the clinical trial and genetic analyses was provided by all patients. Study protocols were in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards of each participating center.
Informative karyotype was locally obtained at diagnosis for 413 patients. Molecular analyses were centrally performed on samples collected at diagnosis (see the Online Supplementary Methods). NPM1, FLT3-ITD and point mutations, RUNX1- RUNX1T1, CBFb-MYH11, biallelic CEBPa and KMT2A-PTD mutations were tested on all patients using polymerase chain reaction (PCR), Sanger sequencing and/or fragment analysis. Targeted next-generation sequencing (NGS) was performed on 196 normal karyotype patients using an amplicon-based method (Trusight Myeloid, Illumina, San Diego, California, USA) (n=161) amplifying 54 gene regions and a capture-based method (Sophia Myeloid Solution, Sophia Genetics SA, Saint Sulpice, Switzerland) (n=35) selecting 30 gene regions (Online Supplementary Tables S1 and S2).18
In order to confirm our results in an independent cohort, we also evaluated a single-center series of AML patients (n=50) treated at ASST Ospedale Papa Giovanni XXIII between 2012 and 2020.
Definition of acute myeloid leukemia categories
The following AML categories were defined among patients enrolled into the trial: i) CS-AML: with the specific goal of vali- dating the CS mutational signature, clinically defined de novo AML patients were included in this category based on the pres- ence of at least one variant described according to Papaemmanuil et al.14 including ASXL1, STAG2, BCOR, EZH2, PHF6, SRSF2, SF3B1, U2AF1, ZRSR2, RUNX1 and KMT2A-PTD (or cytogenetic alterations in KMT2A gene in the 11q23 cytoge- netic region), excluding patients with concurrent WHO-recur-
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