Advanced Flow Cytometry Application in Childhood Acute Leukemia

Research Activity

Acute Lymphoblastic Leukemia (ALL) is the most frequent cancer in childhood and the second cause of death for tumor in pediatric age, mainly after relapse. ALL is a heterogeneous group of neoplasms with different clinical, morphologic, immunophenotypic, and genetic features and variable response to therapy.
Improvements of immunophenotyping and response-to-therapy assessment by measurable residual disease (MRD) monitoring, together with the advancement in ALL characterization in molecular genetics by Next-Generation Sequencing (NGS) techniques, have determined a finest stratification of patients in risk-based classes.
In the last decades, our Laboratory has been playing a central role in multiparametric flow cytometric application at diagnosis and MRD monitoring in pediatric ALL. One important achievement has been the publication of common guidelines for immunophenotyping at diagnosis published in 2017 after an extensive standardization and validation effort by our Lab together with other 9 referral Laboratories in Europe belonging to the AIEOP-BFM (Associazione Italiana Ematologia Oncologia Pediatrica and Berlin-Frankfurt-Munster) Flow Network.
At diagnosis, multiparametric flow cytometry (MFC) has an important role in blast cell definition for lineage assessment and identification of different subgroups. Moreover, immunophenotype may change during treatment causing a switch (SW) that consists of the appearance of a blast population with different phenotype from that detected at diagnosis.
Our group, recently described a subgroup of pediatric B-cell Precursor ALL (BCP-ALL) characterized by the aberrant expression of the myeloid marker CD371, a transient switch towards the myelomonocytic lineage during the first phase of chemotherapy, and a worse response to Induction therapy. We deeply characterized the immunophenotypic switch in order to provide standardized and reproducible operating procedure for blast identification during therapy, pointing to a proper identification of blast cells for a correct risk-based stratification of patients. We are actually running a project that aims to characterize the biological bases of the immunophenotypic switch through a multiomic single-cell application to bone marrow specimens collected at diagnosis and during therapy. Moreover, by the analysis of the genes and the surface proteins expressed in each single cell isolated from these patients at diagnosis we were able to distinguish a unique immature population never recognized with current standard technology. To better define this subgroup of patients we also adopted a very large study on genomic alteration and epigenetic modification characterizing these cells. The integration of all these omic approaches will give a complete description of this subtype of leukemia to potentially identify new targets for a tailored personalized therapy and improve the response to Induction therapy.
There is a peculiar ALL subtype associated with hypereosinophilia, in which eosinophils may be the prevalent population in the bone marrow with an amount of blast cells much less than the one required by international classifications as the blast cut-off (25%) to begin treatment. Nevertheless, the collateral effects associated with hypereosinophilia may lead patients to death, this requiring a precocious chemotherapy starting. We are actually running and leading an international study of the Ponte di Legno consortium with the aim of refining blast cell cut-off for ALL definition in the presence of hypereosinophilia at diagnosis to initiate treatment.
During treatment, MFC is critical for a fast identification of residual amounts of blast cells with a sensitivity level up to 10-5 nucleated cells for a peculiar immunophenotype. Additionally, MRD by MFC replaces quantitative PCR-MRD of IG/TR rearrangements for therapeutic risk group stratification in absence of appropriate molecular markers to be used. This phenomenon occurs more frequently in T-ALL, in which MFC-MRD by flow is particularly challenging due to the heterogeneity of blast immunophenotype at diagnosis, and possible antigen modulation during treatment. In the effort to improve MFC-MRD sensitivity, we recently described the usefulness of CD48 and CD99 markers in MFC-MRD in T-ALL. We are actually running a project with the purpose of refining the MFC-MRD panels for T-ALL with the introduction of new markers (e.g., CD371) and the adoption of personalized panels built up on the immunophenotypic features identified at diagnosis. Moreover, we are joining an international project to produce a custom combination of antibodies to be applied in the iBFM Consortium for MRD detection in T-ALL.
Antigen downregulation during therapy is a well-known phenomenon and is becoming even more frequent after the introduction of targeted immunotherapy. This phenomenon can have critical consequences on MRD assessment by flow. Indeed, a downregulation of target antigens, that are also the pivotal antigens of MFC-MRD detection (CD19 and CD7 for B- and T-ALL, respectively), requires a substantial modification of the gating strategy for leukemia associated immunophenotype (LAIP) detection. Additionally, CD19 and CD7 expression may also change in the normal regenerating counterpart of bone marrow, impacting on the leukemic cell discrimination through the “different from normal” approach in MRD detection. Therefore, it is mandatory to identify new markers to improve MFC-MRD monitoring in this setting, both for T- and B-lineage ALL. In this context, we recently described CD72 as a potential new marker for MFC-MRD monitoring in B-ALL, being expressed also in CD19 negative B-lineage ALL. We are actually carrying out a study to evaluate the role of CD72 in MFC-MRD monitoring in B-lineage ALL.
Pediatric refractory/relapsed (R/R) acute lymphoblastic leukemia (ALL) is still associated with a dismal prognosis. Traditionally, pediatric R/R ALL treatment relied on conventional chemotherapy and consolidation with hematopoietic stem cell transplant once deep remission was obtained. Regardless, modern oncology aims toward a personalized medicine approach based on disease and patient-specific molecular and cellular features.
In this context, we are working on a project with the aim to develop a real-time drug response profiling (DRP) platform to screen patient-derived bone marrow (BM) and peripheral blood (PB) samples with a multi-drug library, including from 20 to 100 compounds already tested at least in phase 2 clinical trials, to have them available for patient treatment on compassionate use, if yet to be approved for pediatric use.
This study may potentially offer a concrete plan of treatment for those R/R ALL pediatric cases who would not have an option to be cured so far. The real-time DRP platform will contribute to a patient-tailored treatment, limiting the use of ineffective drugs and drug-related toxicity and potentially impacting pediatric patients’ quality of life and outcomes.