NUP-98 Rearrangements Led to the Identification of Candidate Biomarkers for Primary Induction Failure in Pediatric Acute Myeloid Leukemia.

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for "Differentially Expressed Genes" (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2, TMA7, SPCS2, CDCP1, CAPZA1, FGFR1OP2, MAN1A2, NT5C3A and SRP54. In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

"Feasible and effective administration of Bortezomib with Rituximab in children with relapsed/resistant B-cell precursor acute lymphoblastic leukemia (BCP-ALL): A step toward the first line".

Despite a high cure rate in childhood BCP-ALL, 20% of children still presents with relapse, mostly due to a persistent leukemic clone during the first-line treatment. In this context, obtaining a molecular remission is crucial for reaching a successful allogeneic hematopoietic stem cell transplantation. Bortezomib was effectively administered to children with resistant/relapsed (r/r) BCP-ALL. Moreover, high risk ALL is characterized by the increasing expression of CD20. For the first time we reported two children with r/r BCP-ALL who received a treatment schema including Bortezomib and Rituximab, achieving morphological and molecular remission. Children with high risk features, such as persistent minimal residual disease during induction, will benefit from this combination. Is it time to move toward the first line?

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Temporary serine protease inhibition and the role of SPINK2 in human bone marrow.

Protease temporary inhibitors are true substrates that bind the catalytic site with high affinity but are slowly degraded, thus acting as inhibitor for a defined time window. Serine peptidase inhibitor Kazal type (SPINK) family is endowed with such functional property whose physiological meaning is poorly explored. High expression of SPINK2 in some hematopoietic malignancies prompted us to investigate its role in adult human bone marrow. We report here the physiological expression of SPINK2 in hematopoietic stem and progenitor cells (HSPCs) and mobilized cluster differentiation 34 (CD34)+ cells. We determined the SPINK2 degradation constant and derived a mathematical relationship predicting the zone of inhibited target protease activity surrounding the SPINK2-secreting HSPCs. Analysis of putative target proteases for SPINK2 revealed the expression of PRSS2 and PRSS57 in HSPCs. Our combined results suggest that SPINK2 and its target serine proteases might play a role in the intercellular communication within the hematopoietic stem cell niche.

Single-cell profiling of pediatric T-cell acute lymphoblastic leukemia: Impact of PTEN exon 7 mutation on PI3K/Akt and JAK-STAT signaling pathways.

BACKGROUND: The PI3K/Akt/mTOR (PI3K) signaling pathway has a crucial role in T-cell acute lymphoblastic leukemias (T-ALLs). Although loss-of-function of phosphatase and tensin homolog (PTEN) is a common event in pediatric T-ALLs, the exact role of this tumor suppressor in T-ALL development has yet to be defined. METHODS: Here, we report an optimized cytometric method for accurate proteomic profiling of T-ALL leukemic blasts at single-cell level. We determined the expression of PI3K and JAK-STAT signaling components in both primary and immortalized T-ALL cells as well as in normal T cells. RESULTS: We observed that PTEN exon 7 mutated T-ALL cells retain a distinct PI3K activation; in particular, these cells show higher pAkt levels and a lower pS6 expression. Interestingly, we demonstrated for the first time that PTEN exon 7 mutated T-ALL are nonresponsive to IL7 in vitro as assessed by lack of pSTAT5 activation, although they do express IL7R. CONCLUSIONS: Phosphoflow analysis represents a fast, reliable, and accurate method to study the signaling profile of T-ALL. PTEN exon 7 mutated T-ALL cells are nonresponsive to IL7 in vitro suggesting that they may activate other mechanisms to support their viability and proliferation such as a higher constitutive PI3K/Akt signaling. Further investigations are necessary to elucidate the significance of this peculiar signaling behavior. Our observations should be taken into account in future studies aiming at molecular targeting of PI3K and/or JAK/STAT pathways for pharmacological intervention in T-ALL.