Contribution of the TIME in BCP-ALL: the basis for novel approaches therapeutics.

The bone marrow (BM) niche is a microenvironment where both immune and non-immune cells functionally interact with hematopoietic stem cells (HSC) and more differentiated progenitors, contributing to the regulation of hematopoiesis. It is regulated by various signaling molecules such as cytokines, chemokines, and adhesion molecules in its microenvironment. However, despite the strict regulation of BM signals to maintain their steady state, accumulating evidence in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) indicates that leukemic cells can disrupt the physiological hematopoietic niche in the BM, creating a new leukemia-supportive microenvironment. This environment favors immunological evasion mechanisms and the interaction of these cells with the development and progression of BCP-ALL. With a growing understanding of the tumor immune microenvironment (TIME) in the development and progression of BCP-ALL, current strategies focused on "re-editing" TIME to promote antitumor immunity have been developed. In this review, we summarize how TIME cells are disrupted by the presence of leukemic cells, evading immunosurveillance mechanisms in the BCP-ALL model. We also explore the crosstalk between TIME and leukemic cells that leads to treatment resistance, along with the most promising immuno-therapy strategies. Understanding and further research into the role of the BM microenvironment in leukemia progression and relapse are crucial for developing more effective treatments and reducing patient mortality.

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients: Association with MRD Status and Patient Outcome.

For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort­hazard ratio (95% confidence interval) of 2.50 (1−9.66); p = 0.05­together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Altered neutrophil immunophenotypes in childhood B­cell precursor acute lymphoblastic leukemia.

An increasing number of evidences suggest a genetic predisposition in acute lymphoblastic leukemia (ALL) that might favor the occurrence of the driver genetic alterations. Such genetic background might also translate into phenotypic alterations of residual hematopoietic cells. Whether such phenotypic alterations are present in bone marrow (BM) cells from childhood B-cell precursor (BCP)-ALL remains to be investigated. Here we analyzed the immunophenotypic profile of BM and peripheral blood (PB) maturing/matured neutrophils from 118 children with BCP-ALL and their relationship with the features of the disease. Our results showed altered neutrophil phenotypes in most (77%) BCP-ALL cases. The most frequently altered marker was CD10 (53%), followed by CD33 (34%), CD13 (15%), CD15/CD65 (10%) and CD123 (7%). Of note, patients with altered neutrophil phenotypes had younger age (p = 0.03) and lower percentages of BM maturing neutrophils (p = 0.004) together with greater BM lymphocyte (p = 0.04), and mature B-cell (p = 0.03) counts. No significant association was found between an altered neutrophil phenotype and other disease features. These findings point out the potential existence of an altered residual hematopoiesis in most childhood BCP-ALL cases.

Evaluation of multiplex ligation dependent probe amplification (MLPA) for identification of acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21 (iAMP21) in a Brazilian population.

BACKGROUND: An intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define an iAMP21. Genomic profiling of chromosome 21 has been suggested for assisting diagnostic case identification. Due to limitations of comparative genomic hybridization, in terms of a routine application as first line-screening tests we evaluated the multiplex ligation-dependent probe amplification (MLPA) SALSA P327_A1 and P327_B1 probe sets for detecting chromosome 21 copy number alterations in Brazilian childhood BCP-ALL. RESULTS: In 74 out of 368 patients gain of genetic material was detected. For data confirmation RUNX1 directed FISH was performed. Cells with ≥5 RUNX1 signals (n = 9) were considered as "true iAMP21" while <5 RUNX1 signals (n = 41) were counted as evidence for additional copies of intact chromosomes 21. All patients with an iAMP21 had high MLPA peak ratios (≥1.8), while the majority of patients with <5 RUNX1 presented low MLPA peak ratios (<1.8). Observed differences gained statistical strength by comparing probes located within the common region of amplification. Next, a principal component analysis was performed in order to illustrate distribution of cases according to their MLPA peak profile in two dimensions. Cases with an iAMP21 mostly clustered together, however additional cases with <5 RUNX1 signals or no available FISH data located in proximity. CONCLUSIONS: MLPA qualified as a high throughput technique that could be employed in future studies for a critical comparison with data obtained by FISH, especially in cases where metaphase nuclei are not available. Taking submicroscopic aberrations into account examined by MLPA, cases exhibiting an "iAMP21 like" peak ratio profile but <5 RUNX1 signals should be considered as candidates for this chromosomal abnormality.