Pilar Bravo

Expression of Bcl-2 by human bone marrow Mast Cells and its overexpression in Mast cell leukemia

Bcl‐2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl‐2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl‐2 was studied using quantitative three‐color flow cytometry. We also studied the molecular configuration of the bcl‐2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl‐2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl‐2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl‐2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl‐2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl‐2, which could not be related to molecular rearrangements involving the bcl‐2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl‐2 by BMMC in MCL may help to explain their resistance to chemotherapy‐induced apoptosis. Am. J. Hematol. 60:191–195, 1999.

Overexpression of complement receptors and related antigens on the surface of bone marrow mast cells in patients with systemic mastocytosis

Summary. Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement‐associated antigens. This study analysed the expression of various complement‐related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non‐haematological disorders (control groups). Expression of complement‐associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0·05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0·05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement‐mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease.

We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions.

Immunophenotype of bone marrow mast cells in indolent systemic mast cell disease in adults.

One of the major advances in the histological diagnosis of bone marrow (BM) involvement in mastocytosis has been the specific immunohistochemical detection of tryptase on most cells (MC), which has shown to be of great diagnostic value, especially in cases of malignant mastocytosis. On the other hand, recent studies have clearly shown that bone marrow mast cells can be specifically identified and accurately enumerated using multiparametric flow cytometry, which allow a systematic analysis of the immunophenotypic characteristics of bone marrow mast cells. Once this flow cytometric approach was applied for the analysis of BMMC from mastocytosis patients clear immunophenotypical differences were found between BMMC from normal individuals and adults with mastocytosis. The most characteristic immunophenotypic feature, both in malignant and adult indolent systemic mast cell disease, being the coexpression of CD2 and CD25 antigens, never present in normal bone marrow mast cells and, which constitute an aberrant hallmark of bone marrow mast cells in adult mastocytosis. Furthermore, bone mast cells from mastocytosis display a higher reactivity for CD35, CD63, and CD69 activation-associated antigens. Based on these results it could be concluded that the use of multiparametric flow cytometric immunophenotyping of BMMC in adult patients suffering from cutaneous mastocytosis can be of great utility for the diagnosis of BM involvement; additionally, this might also help to establish the real incidence of BM involvement in cutaneous mastocytosis.

HUMAN BONE MARROW MAST CELLS FROM INDOLENT SYSTEMIC MAST CELL DISEASE CONSTITUTIVELY EXPRESS INCREASED AMOUNTS OF THE CD63 PROTEIN ON THEIR SURFACE

The quantitative measurement of the expression of both cytoplasmic and surface CD63 antigen by human mast cells from both normal and pathological bone marrow samples was studied by use of flow cytometry. Our major goal was to analyze whether in vivo CD63 expression by human bone marrow mast cells could be useful to discriminate bone marrow mast cells from patients with mastocytosis from other conditions. For that purpose, a total of 65 subjects corresponding to 12 healthy volunteers, 25 B-cell chronic lymphoproliferative disorders, 5 reactive bone marrow samples, 4 myelodysplastic syndromes, and 19 mastocytosis were analyzed. The expression of both surface and cytoplasmic CD63 by human bone marrow mast cells is clearly demonstrated. Our results show that high amounts of CD63 are present in human bone marrow mast cells most of it corresponding to an intracellular localization. No significant differences in CD63 expression were observed as regards both total and cytoplasmic CD63, except for higher CD63 levels in adult patients with mastocytosis (P = 0.05). By contrast, the mean level of surface CD63 significantly varied between the different groups of individuals. Accordingly, patients with monoclonal gammopathies displayed a slight decrease (P = 0.1) in surface CD63 expression, whereas bone marrow mast cells from adults with indolent systemic mast cell disease showed significantly (P = 0.0005) higher levels of surface CD63 as compared to healthy controls.

Treatment of young patients with Philadelphia chromosome‐positive acute lymphoblastic leukaemia using increased dose of imatinib and deintensified chemotherapy before allogeneic stem cell transplantation

The main outcomes of the Programa Español para Tratamiento de Hemopatías (PETHEMA)‐acute lymphoblastic leukaemia (ALL)‐Ph‐08 trial were described and compared with those of the historical PETHEMA‐CSTIBES02 trial. The trials differed in imatinib dose (600 vs. 400 mg/d) and amount of chemotherapy (one vs. two consolidation cycles) before stem cell transplantation (SCT). All patients (n = 29) enrolled in the ALL‐Ph‐08 trial achieved complete remission (CR) (vs. 90% in CSTIBES02), and SCT was performed in CR in 90% (vs. 78%). The reduction in early death, relapse before SCT and transplant‐related mortality observed in the ALL‐Ph‐08 trial resulted in an improved 2‐year event‐free survival (63% vs. 37%, P = 0·009).

Expression of high amounts of the CD117 molecule in a case of B‐cell non‐Hodgkin's lymphoma carrying the t(14:18) translocation

The c‐kit proto‐oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK‐cells, and to early T‐cell precursors and it is not expressed by normal B‐cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkins disease and anaplastic large‐cell lymphoma. In the present paper we describe a case of a B‐cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B‐cell population. Further studies are necessary to explore both the functional role of c‐kit expression in the neoplastic B‐cells from this patient and its potential utility for the diagnosis and follow‐up of patients with B‐cell non‐Hodgkins lymphoma. Am. J. Hematol. 63:226–229, 2000.

Blastic plasmacytoid dendritic cell neoplasm frequently shows occult central nervous system involvement at diagnosis and benefits from intrathecal therapy

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive myeloid neoplasm which shows a high rate of central nervous system (CNS) recurrence and overall survival (OS) of <1 year. Despite this, screening for CNS involvement is not routinely performed at diagnosis and intrathecal (IT) prophylaxis is not regularly administered in BPDCN. Here, we prospectively evaluated 13 consecutive BPDCN patients for the presence of CNS involvement by flow cytometry. Despite none of the patients presented with neurological symptoms, occult CNS involvement was detected in 6/10 cases evaluated at diagnosis and 3/3 studied at relapse/progression. BPDCN patients evaluated at diagnosis received IT treatment -either CNS prophylaxis (n = 4) or active therapy (n = 6)- and all but one remain alive (median follow-up of 20 months). In contrast, all three patients assessed at relapse/progression died. The potential benefit of IT treatment administered early at diagnosis on OS and CNS recurrence-free survival of BPDCN was further confirmed in a retrospective cohort of another 23 BPDCN patients. Our results show that BPDCN patients studied at diagnosis frequently display occult CNS involvement; moreover, they also indicate that treatment of occult CNS disease might lead to a dramatically improved outcome of BPDCN.

Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry

The immunophenotypic identification and enumeration of human bone marrow mast cells represents a clear demonstration of the utility of flow cytometry for rare‐event analysis. The basic approach can be applied to a variety of specimens, including bone marrow, peripheral blood, ascitic fluid, and lymphoid tissue such as adenoids. Special emphasis is placed on markers with potential utility for distinguishing between normal, reactive, and pathological mast cells. From the clinical aspect, immunophenotypic analysis of mast cells may have great utility in supporting the diagnosis of tissue involvement in mastocytosis.

Clinical characteristics of patients with central nervous system relapse in BCR-ABL1-positive acute lymphoblastic leukemia: the importance of characterizing ABL1 mutations in cerebrospinal fluid

We investigated the frequency, predictors, and evolution of acute lymphoblastic leukemia (ALL) in patients with CNS relapse and introduced a novel method for studying BCR-ABL1 protein variants in cDNA from bone marrow (BM) and cerebrospinal fluid (CSF) blast cells. A total of 128 patients were analyzed in two PETHEMA clinical trials. All achieved complete remission after imatinib treatment. Of these, 30 (23%) experienced a relapse after achieving complete remission, and 13 (10%) had an isolated CNS relapse or combined CNS and BM relapses. We compared the characteristics of patients with and without CNS relapse and further analyzed CSF and BM samples from two of the 13 patients with CNS relapse. In both patients, classical sequencing analysis of the kinase domain of BCR-ABL1 from the cDNA of CSF blasts revealed the pathogenic variant p.L387M. We also performed ultra-deep next-generation sequencing (NGS) in three samples from one of the relapsed patients. We did not find the mutation in the BM sample, but we did find it in CSF blasts with 45% of reads at the time of relapse. These data demonstrate the feasibility of detecting BCR-ABL1 mutations in CSF blasts by NGS and highlight the importance of monitoring clonal evolution over time.