Genetic advances in Acute Lymphoblastic Leukemia diagnostics

Acute Lymphoblastic Leukemia (ALL) is the most frequent cancer in childhood. The advancement in ALL characterization by Next-Generation Sequencing (NGS) techniques in molecular genetics, together with the improvements of immunophenotyping and response-to-therapy assessment by measurable residual disease (MRD) monitoring, have determined a finest stratification of patients in risk-based classes. All these factors led to a significant amelioration of patients’ outcomes in the last decades. In this context we aimed to contribute to the genetic characterization of ALL and for this purpose we investigated three main topics. 1. The Ig/TR rearrangements monitoring (PCR-MRD) is the gold standard to evaluate the response to treatment in ALL. We aimed to apply the amplicon-based NGS method for the identification of Ig/TR markers developed by the EuroClonality-NGS study group with the intent to use it also for MRD detection. We screened a cohort of 58 ALL patients (52 at diagnosis and 6 at relapse) and we compared NGS technique with PCR-Sanger method (historically used for this purpose). The NGS approach resulted to be more sensitive, specific, and efficient in identifying clonal Ig/TR rearrangements to be used in MRD monitoring. 2. B-Cell Precursor (BCP) ALL is the most common cancer in childhood. The advancement in omics techniques allows distinguishing different subtypes characterized by specific genetic, transcriptomic and immunophenotypic features. Multiparametric flow cytometry (MFC) plays an important role in blast cell definition for lineage assessment and identification of new BCP-ALL subgroups. Immunophenotype may change during treatment, causing a myelomonocytic switch (mm-SW) that consists of the appearance of a population displaying myelomonocytic antigens together with down-regulation of lymphoid ones. Recently different BCP-ALL subtypes have been demonstrated to undergo this phenomenon during Induction phase, hampering MFC-MRD assessment and risk-based stratification of patients. We analysed the transcriptomic profiles of 77 patients at diagnosis and they were subdivided in 19 mmSW+ 58 mmSW- after the assessment of the presence of monocytosis at Day+15 by MFC. We compared mmSW+ and mmSW- patients to unravel the molecular pathways specific of mmSW+ group. In general, we observed that the majority of mmSW+ patients belong to the ERG-related subtype and they have a more immature transcriptome, committed to myeloid lineage. 3. After the introduction of the myeloid marker CD371 in BCP-ALL diagnostics by MFC, we identified a new BCP-ALL subgroup characterized by the aberrant expression of CD371 marker at diagnosis and a transient mm-SW in up to 60% on Day+15 of Induction therapy. We investigated the molecular basis of the immunoplasticity of mmSW+ patients by a multiomic approach. We performed single cell analysis of 5 BM samples (2 CD371+/mmSW+, 2 CD371+/mmSW- and 1 CD371-/mmSW+) at diagnosis by simultaneously analysing transcriptome and surface antigens. We used the same approach to compare the BM samples at diagnosis and at Day+15 from 3 patients (2 CD371+/mmSW+ and 1 CD371+/mmSW-) to study the clonal evolution of leukemic cells during therapy. Additionally, we analysed the methylation profile of mmSW+ and mmSW- patients to investigate the implication of methylation in the arising of mm-SW. We identified at diagnosis a more immature group of cells among the entire blast population of mmSW+ patients, that is not detected by MFC. The comparison between diagnosis and Day+15 suggested that the more immature population of diagnosis may be responsible for the mm-SW, because of its propensity to myeloid lineage development. Furthermore, preliminary data of methylation analysis of mmSW+ group showed that it has a distinct methylation profile, revealing that epigenetic regulation has a role in the determination of myelomonocytic phenomenon.