NG2 Molecule Expression in Acute Lymphoblastic Leukemia B Cells: A Flow-Cytometric Marker for the Rapid Identification of KMT2A Gene Rearrangements.

Background: B-lineage acute lymphoblastic leukemias (B-ALL) harboring rearrangements of the histone lysine [K]-Methyltransferase 2A (KMT2A) gene on chromosome 11q23 (KMT2A-r) represent a category with dismal prognosis. The prompt identification of these cases represents an urgent clinical need. Considering the correlation between rat neuron glial-antigen 2 (NG2) chondroitin-sulfate-proteoglycan molecule expression and KMT2A-r, we aimed to identify an optimized cytofluorimetric diagnostic panel to predict the presence of KMT2A-r. Materials and Methods: We evaluated 88 NG2+ B-ALL cases identified with an NG2 positivity threshold >10% from a cohort of 1382 newly diagnosed B-ALLs referred to the Division of Hematology of 'Sapienza' University of Rome. Results: Eighty-five of 88 (96.6%) NG2+ B-ALLs harbored KMT2A-r and were mainly pro-B ALL (77/85; 91%). Only 2 B-ALLs with KMT2A-r showed NG2 expression below 10%, probably due to the steroid therapy administered prior to cytofluorimetric analysis.Compared to KMT2A-r-cases, KMT2A r+ B-ALLs showed a higher blast percentage, significantly higher mean fluorescence intensity (MFI) of CD45, CD38, and CD58, and significantly lower MFI of CD34, CD22, TdT, and CD123.The study confirmed differences in CD45, CD34, CD22, and TdT MFI within the same immunologic EGIL group (European Group for the immunological classification of leukemias), indicating no influence of the B-ALLs EGIL subtype on the KMT2A-r+ B-ALLs immunophenotype. Conclusions: Our data demonstrate the association between NG2 and KMT2A-r in B-ALLs identify a distinctive immunophenotypic pattern, useful for rapid identification in diagnostic routines of these subtypes of B-ALLs with a poor prognosis that benefits from a specific therapeutic approach.

Multiparametric Flow Cytometry in Newly Diagnosed Multiple Myeloma Patients: An Italian Monocentric Experience.

Multiple myeloma (MM) is a heterogeneous malignancy characterized by the proliferation of abnormal plasma cells in the bone marrow. Multiparametric flow cytometry (MFC) plays a role in the work-up of the disease in view of the aberrant expression of surface antigens. Our study aimed at describing the antigenic profile detected by MFC in a series of newly diagnosed MM patients to correlate the level of expression with other features of the disease. Between April 2018 and June 2022, 84 consecutive MM patients were studied at presentation. CD56 and CD117 were commonly detected, while CD45, CD28, CD20, CD19, CD13 and CD33 were less recurrent. CD20 expression was associated with the type of secretory MM (p=0.041) and with a higher disease burden (p=0.038). CD28 positivity correlated with a lower platelet count at baseline (p=0.005) and with a lower rate of complete response (p=0.038). Furthermore, CD28 positivity and a lower CD138 expression tended to associate with the high-risk chromosomal translocations t(14;16) and t(4;14). The results of this study indicate that in the diagnostic work-up of MM, MFC may help to identify different patient subsets and improve risk stratification. These observations need to be validated in larger series of patients with a longer follow-up.

Sialofucosylation Enables Platelet Binding to Myeloma Cells via P-Selectin and Suppresses NK Cell-Mediated Cytotoxicity.

Multiple myeloma (MM) is a plasma cell disorder that develops in the bone marrow (BM) and is characterized by uncontrolled proliferation and the ability to disseminate to different sites of the skeleton. Sialofucosylated structures, particularly Sialyl Lewis a/x (SLea/x), facilitate the homing of MM cells into the BM, leading to resistance to bortezomib in vivo. Platelets have been shown to play an important role in tumor metastasis. Platelets can bind to the surface of cancer cells, forming a "cloak" that protects them from the shear stress of the bloodstream and natural killer (NK) cell-mediated cytotoxicity. In this study, we showed that the presence of SLea/x induced a strong binding of MM cells to P-selectin, leading to specific and direct interactions with platelets, which could be inhibited by a P-selectin-blocking antibody. Importantly, platelets surrounded SLea/x-enriched MM cells, protecting them from NK cell-mediated cytotoxicity. The interactions between the platelets and MM cells were also detected in BM samples obtained from MM patients. Platelet binding to SLea/x-enriched MM cells was increased in patients with symptomatic disease and at relapse. These data suggest an important role of SLea/x and platelets in MM disease progression and resistance to therapy.

Sialylation regulates migration in chronic lymphocytic leukemia.

Sialylation is the terminal addition of sialic acid to underlying glycans. It plays a prominent role in cell adhesion and immune regulation. Sialylated structures found on adhesion molecules, such as CD49d, mediate the interactions between cancer cells and the microenvironment, facilitating metastatic seeding in target organs. Chronic lymphocytic leukemia (CLL) is a clonal B-cell malignancy characterized by the accumulation of CD5-positive B cells in the peripheral blood, bone marrow and lymph nodes. CLL cells proliferate mainly in the lymph node "proliferation centers", where the microenvironment provides pro-survival signals. Thus, migration and homing into these protective niches play a crucial role in CLL biology. In recent years, therapeutic strategies aimed at inducing the egress of CLL cells from the lymph nodes and bone marrow into the circulation have been highly successful. In this study, the sialylation status of 79 untreated and 24 ibrutinib-treated CLL patients was characterized by flow cytometry. Moreover, the effect of sialic acid removal on migration was tested by a transwell assay. Finally, we examined the sialylation status of CD49d by Western blot analysis. We found that CLL cells are highly sialylated, particularly those characterized by an "activated" immune phenotype. Notably, sialylation regulates CLL migration through the post-translational modification of CD49d. Finally, we showed that therapeutic agents that induce CLL mobilization from their protective niches, such as ibrutinib, modulate sialic acid levels. We propose that sialylation is an important regulator of CLL trafficking and may represent a novel target to further improve CLL therapy.

Neoplastic bone marrow invasion:rapid exclusion of hematological disease by flow cytometric routine panels.

Multiparametric flow cytometry is an extensively used technique to assess the presence of different cellular populations in immunology and hematology. During routine immunophenotyping analysis, it is not uncommon to face cells of non-hemopoietic origin, negative for CD45 and other myeloid, megakaryocytic, B and T lineage antigens and positive for at least one antibody among CD56, CD117 and CD138. If cytology cannot identify cell origin, especially in cases of unclear interpretation, the contribution of multiparametric flow cytometry analysis can be crucial. We report 6 patients with a clinical suspicion of hematological disease in which multiparametric flow cytometry was extremely useful to quickly exclude blood disorders in order to initiate patients to the most appropriate diagnostic process.

Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases.

Monoclonal antibody (MoAb)-based therapies have opened innovative treatment avenues that have impacted on the management of patients with both neoplastic and non-neoplastic hematological diseases. The aim of our study was to evaluate in a large series of cases of acute lymphoblastic leukemia (ALL) the expression of specific antigens, CD19, CD20, CD22, and CD33, for which MoAbs are available for clinical use. For each antigen, evaluation was based on the percentage of positive leukemic cells and the degree of antigen expression by mean fluorescence intensity (MFI) and antibody binding capacity (ABC) that were correlated with age, immunophenotype, and presence/absence of particular molecular markers. We can document that some of the analyzed antigens showed a degree of expression related to the B-cell maturation profile, and that the antigen expression intensity appeared to vary according to the presence of specific genetic markers. These findings suggest that the possible clinical use of a given MoAb in patients with ALL should take into account both the maturation profile of the leukemic cells and the presence of a given molecular transcript. Only clinical studies will conclusively demonstrate whether the differences in antigenic expression truly correlate with the different therapeutic efficacies of the various clinical grade MoAbs.

Immunocompetent cell functions in Ph+ acute lymphoblastic leukemia patients on prolonged Imatinib maintenance treatment.

Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively used for the treatment of malignancies characterized by the constitutive activation of these tyrosine kinases, such as Philadelphia chromosome-positive (Ph(+)) leukemias and gastrointestinal stromal tumors. Suppressive as well as stimulating effects of this drug on T lymphocytes or dendritic cells (DC), which play a major role in immune tumor surveillance, have been reported. For this reason, we questioned whether Imatinib could also affect the phenotypic and functional properties of these subpopulations in Ph(+) acute lymphoblastic leukemia (ALL) patients on prolonged Imatinib maintenance treatment. Circulating T lymphocytes and NK cells from Imatinib-treated Ph(+) ALL patients showed a subset distribution comparable to that of healthy donors. In addition, T-cell immunomodulant cytokine production (IFN-γ, TNF-α) and proliferative responses were not impaired. A normal monocyte-derived DC differentiation and apoptotic body loading capacity was also observed in the majority of Imatinib-treated patients. In contrast, an impairment in the DC intracellular production of IL-12 was recorded, although this was not observed when normal DC were exposed in vitro to Imatinib. Finally, in vivo Imatinib treatment did not affect the T-lymphocyte proliferation and IFN-γ production induced by leukemic apoptotic body-loaded DC, underling the potential capability of these cells to generate a specific immune response against tumoral antigens. Taken together, these findings provide evidence that immunotherapeutic approaches aimed at controlling residual disease in Ph(+) ALL patients in hematologic remission are not jeopardized by the long-term administration of Imatinib.

An accurate and rapid flow cytometric diagnosis of BCR-ABL positive acute lymphoblastic leukemia.

Tyrosine kinase inhibitors have profoundly modified the treatment and prognosis of chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia. A rapid and accurate detection of the BCR-ABL fusion protein is paramount today for an optimal management of Ph(+) acute lymphoblastic leukemia. We have utilized a recently described and commercialized immunoassay that identifies qualitatively the presence of the BCR-ABL protein in leukemic cell lysates. The BCR-ABL fusion protein is captured and detected by a cytometric bead assay and analyzed by flow cytometry. The assay was applied to 101 primary patient samples (94 acute leukemias and 7 chronic myeloid leukemia blast crisis) and the results of the immunoassay were concordant with those obtained by conventional molecular techniques. The method proved reliable, reproducible, of simple execution and it was successfully completed within four hours. This flow cytometric immunoassay has important implications for perfecting the management of Ph(+) acute lymphoblastic leukemia patients worldwide.

B-cell concentration in the apheretic product predicts acute graft-versus-host disease and treatment-related mortality of allogeneic peripheral blood stem cell transplantation.

BACKGROUND: The influence of the graft composition on the clinical outcome after allogeneic peripheral blood stem cell (PBSC) transplantation is not well established. METHODS: The cellular composition of the apheretic products obtained from 63 human leukocyte antigen-identical siblings was prospectively correlated with the outcome of patients with hematological malignancies undergoing an allogeneic PBSC transplant after myeloablative conditioning. The concentration of nuclear, mononuclear, CD34+, T-cell subsets, B cells, and natural killer cells in the graft has been analyzed. RESULTS: In univariate analysis, acute graft-versus-host disease (GVHD) correlated with the disease (P=0.002), with the phase of disease at transplant (P=0.01), and with the number of CD20+ cells infused (P=0.05). In multivariate analysis, a dose of CD20+ cells in the graft higher than the median dose remained the only factor negatively affecting the incidence of acute GVHD (P=0.01; 95% confidence interval [CI]: 0.12-0.78). In univariate analysis, treatment-related mortality (TRM) correlated with the disease (P=0.04) and was negatively affected by a dose of infused B cells greater than the median value (28% versus 50%; P=0.02). In multivariate analysis, TRM was close to statistical correlation with the dose of CD20+ cells (P=0.06; 95% CI: 0.02-1.05). No other clinical parameter was influenced by the composition of the graft. CONCLUSIONS: Our results suggest that the concentration of B cells in the apheretic product may predict the incidence of acute GVHD and TRM in patients undergoing an allogeneic PBSC transplantation and open the way to the new preventive and therapeutic strategies for the management of GVHD.

Generation of functional dendritic cells (DC) in adult acute lymphoblastic leukemia: rationale for a DC-based vaccination program for patients in complete hematological remission.

The capacity to generate effective dendritic cells (DC) from adult acute lymphoblastic leukemia (ALL) patients in complete remission (CR) and off-therapy was investigated. Monocyte-derived DC cultured in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF)-alpha expressed maturation markers, produced IL-12 and loaded apoptotic bodies to a similar extent to normal DC. Patients' circulating T and NK lymphocytes were normally represented and, after stimulation, were capable of producing TNF-alpha and interferon-gamma to a similar extent to control lymphocytes. DC loaded with leukemia-derived apoptotic bodies increased their ability to stimulate both allogeneic and autologous lymphocytes, and to generate specific anti-leukemic CD3 + cells. These findings offer a rationale for the design of DC-based vaccine programs for adult ALL patients in CR with the aim of controlling/eradicating the disease.

ZAP-70 expression in acute lymphoblastic leukemia: association with the E2A/PBX1 rearrangement and the pre-B stage of differentiation and prognostic implications.

We evaluated the expression of 2 members of the Syk family, ZAP-70 and Syk, in acute lymphoblastic leukemia (ALL) samples, using data derived from a series of 33 T-ALL and 95 B-lineage adult ALL patients analyzed by oligonucleotide arrays. Of the B-lineage ALL cases, 37 were BCR/ABL+, 10 were ALL1/AF4+, 5 were E2A/PBX1+, and 43 carried no known molecular abnormality. ZAP-70 was highly expressed in T-ALL. A high ZAP-70 expression was also found in a proportion of B-lineage ALL, the highest levels being associated with the E2A/PBX1+ group and the lowest with ALL1/AF4+ cases (P < .001). A higher ZAP-70 expression was also observed in the pre-B group (P < .001). Remarkably, Syk expression was always preserved, suggesting that ZAP-70 expression is not substitutive of Syk. At the protein level, ZAP-70 was evaluated on 39 newly diagnosed ALL patients (25 adults, 14 children) and was detected in 23 cases (59%). ZAP-70 expression was consistently found in Ig mu+ cases. Evaluation of long-term outcome in cases without molecular abnormalities showed that the higher levels of ZAP-70 were coupled to a higher relapse rate. In ALL, ZAP-70 expression is associated with the E2A/PBX1 rearrangement and pre-B stage and may have a prognostic role and be a candidate molecule for targeted therapies.