DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch.

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Common Variable Immunodeficiency patients with a phenotypic profile of immunosenescence present with thrombocytopenia.

Common variable immunodeficiency (CVID) is a heterogeneous group of diseases. Our aim was to define sub-groups of CVID patients with similar phenotypes and clinical characteristics. Using eight-color flow cytometry, we analyzed both B- and T-cell phenotypes in a cohort of 88 CVID patients and 48 healthy donors. A hierarchical clustering of probability binning "bins" yielded a separate cluster of 22 CVID patients with an abnormal phenotype. We showed coordinated proportional changes in naïve CD4+ T-cells (decreased), intermediate CD27- CD28+ CD4+ T-cells (increased) and CD21low B-cells (increased) that were stable for over three years. Moreover, the lymphocytes' immunophenotype in this patient cluster exhibited features of profound immunosenescence and chronic activation. Thrombocytopenia was only found in this cluster (36% of cases, manifested as Immune Thrombocytopenia (ITP) or Evans syndrome). Clinical complications more frequently found in these patients include lung fibrosis (in 59% of cases) and bronchiectasis (55%). The degree of severity of these symptoms corresponded to more deviation from normal levels with respect to CD21low B-cells, naïve CD4+ and CD27− CD28+ CD4+ T-cells. Next-generation sequencing did not reveal any common genetic background. We delineate a subgroup of CVID patients with activated and immunosenescent immunophenotype of lymphocytes and distinct set of clinical complications without common genetic background.

Leukemia surfaceome analysis reveals new disease-associated features.

A better description of the leukemia cell surface proteome (surfaceome) is a prerequisite for the development of diagnostic and therapeutic tools. Insights into the complexity of the surfaceome have been limited by the lack of suitable methodologies. We combined a leukemia xenograft model with the discovery-driven chemoproteomic Cell Surface Capture technology to explore the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) surfaceome; 713 cell surface proteins, including 181 CD proteins, were detected through combined analysis of 19 BCP-ALL cases. Diagnostic immunophenotypes were recapitulated in each case, and subtype specific markers were detected. To identify new leukemia-associated markers, we filtered the surfaceome data set against gene expression information from sorted, normal hematopoietic cells. Nine candidate markers (CD18, CD63, CD31, CD97, CD102, CD157, CD217, CD305, and CD317) were validated by flow cytometry in patient samples at diagnosis and during chemotherapy. CD97, CD157, CD63, and CD305 accounted for the most informative differences between normal and malignant cells. The ALL surfaceome constitutes a valuable resource to assist the functional exploration of surface markers in normal and malignant lymphopoiesis. This unbiased approach will also contribute to the development of strategies that rely on complex information for multidimensional flow cytometry data analysis to improve its diagnostic applications.