This Study Aims to Improve the Stratification of Patients With Acute Myeloid Leukemia to Enable More Personalized and Effective Treatments, and to Identify and Functionally Validate, at the Single-cell Level, Biomarkers That Can Predict Response to Venetoclax.

The investigators propose to enroll approximately 420 patients (of which 120 patients at the Foundation). The samples, consisting of mononuclear cells purified from bone marrow blood (obtained from bone marrow aspirate) through density gradient, will be collected and frozen under vital conditions. For 4 patients selected for in situ spatial transcriptomic analysis, the investigators will use leftover tissue sections obtained from the bone marrow biopsy performed in routine clinical practice for histopathological analysis. All samples will follow routine diagnostics, with standard karyotyping and conventional sequencing of FLT3 and NPM1 genes in line with current clinical practice. Clinical and laboratory parameters at diagnosis, during treatment, and at any relapse will be recorded in dedicated case report forms (CRFs). A second bone marrow sample will be taken in case of recurrence or refractory disease.

1. Bulk genotyping of AML samples at diagnosis and relapse. All patients will be genotyped at diagnosis by NGS of a 69-gene panel involved in myeloid neoplasms and including targets relevant from a diagnostic, prognostic, and therapeutic standpoint. This will allow sub-classification of patients into genomic subgroups with biological and prognostic relevance. Cases will also be sequenced at relapse or in case of refractory disease. In this way, it will be possible to identify genomic lesions associated with chemoresistance and gene mutations that serve as markers for personalized targeted treatments and that could emerge during therapy.
2. Single-cell analysis at diagnosis. Since AML is clonally heterogeneous, bulk sequencing might not be able to identify genetic or transcriptomic features associated with treatment response, nor to determine the role of the microenvironment. The investigators will therefore select 40 patients (of which 10 at the Foundation) undergoing treatment with venetoclax, provided by normal clinical practice, according to the following criteria: 1) cases with the worst prognosis and not recognized by current routine diagnostics (complex karyotype and/or TP53 abnormalities; RUNX1 gene mutations or mutations in genes encoding spliceosome/chromatin components); 2) NPM1-mutated cases, generally with good prognosis. In these cases, the investigators will perform transcriptome analysis by RNAseq in approximately 5000 single cells using the Chromium Controller instrument from 10x Genomics. Single-cell RNAseq (scRNAseq) libraries will be sequenced on Illumina platforms at a coverage depth of 30,000 reads per cell. This will allow a detailed dissection of heterogeneity at the single-cell level, and in particular: i) the identification of blasts and their distinction from stromal cells and immune microenvironment cells; ii) within blasts, the identification of their transcriptomic profiles and their heterogeneity at the single-cell level; iii) through analysis of expressed mutations and chromosomal copy number alterations, the evaluation of transcriptomic consequences of specific genomic lesions at the single-cell level; iv) as a consequence of the above, the creation of a hierarchy of blasts in these heterogeneous cases; v) analysis of the immune microenvironment; vi) through RNA velocity analysis, the prediction of the state of these cells both in the blast population and in the microenvironment.
3. scRNAseq and genotyping analysis at day 28 (d28). All 40 patients will undergo a second bone marrow aspirate, as per normal clinical practice, and it is foreseeable that approximately one-third will show chemoresistance. The investigators will perform scRNAseq and genotyping analysis 28 days from the start of therapy to identify subclones that survive or do not survive therapy and correlate their genomic and transcriptomic characteristics with clinical response. The investigators will also determine the role of the microenvironment, and correlate early modifications found in the bone marrow with longer-term clinical outcome.
4. Spatial transcriptomic analysis at day 0 and day 28. In 4 selected patients, the investigators will perform in situ spatial transcriptomic analysis (Xenium 10X Genomics) on osteo-myeloid biopsy, in order to evaluate the expression of target RNA molecules within the bone marrow architecture at single-cell resolution. The investigators will analyze the leftover tissue sections obtained from the BOM performed according to normal clinical practice at diagnosis and after treatment.