Quantitative multiparametric immunophenotyping in acute lymphoblastic leukemia: correlation with specific genotype. I. ETV6/AML1 ALLs identification.

The t(12;21)(p13;q22) fusion gene is the most frequent genetic lesion described in precursor B cell acute lymphoblastic leukemia (ALL) of childhood occurring in a quarter of cases. This gene rearrangement is associated with a good outcome presenting a high response rate to chemotherapy. In spite of its potential clinical relevance, the t(12;21) translocation usually goes undetected with conventional cytogenetic procedures. In the present study we utilized an objective flow cytometric approach (multiparametric quantitative analysis) for the phenotypic characterization of this type of ALL. We studied a total of 74 precursor B-ALL children, including 21 t(12;21)+ and 53 t(12;21)- cases. Our results show that the t(12;21)(p13;q22)+ ALLs display a higher intensity of CD10 (P = 0.0016) and HLADR (P = 0.005) expression together with lower levels of the CD20 (P = 0.01), CD45 (P = 0.01), CD135 (P = 0.003) and CD34 (P = 0.03) antigens as compared to the t(12;21) cases. Moreover, as regards CD34 expression, we observed a more heterogeneous antigen expression within individual patients with higher coefficients of variation (median of 202 vs 88, P = 0.0001). A multi-variate analysis disclosed that with the immunophenotypic approach used identification of t(12;21)+ cases can be achieved with a sensitivity of 86% and a specificity of 100%. We conclude that childhood precursor B-ALL carrying the t(12;21) translocation display characteristic phenotypic features which could provide a rapid, simple, sensitive and specific screening method to select for those cases that should undergo confirmatory molecular analysis.

The c-kit receptor and its ligand stem cell factor in childhood malignant lymphoid precursors.

The c-kit receptor (CD117) and its ligand stem cell factor (SCF) play an important role in the development, differentiation, and survival of normal and malignant hematopoietic cells. The aim of this work is to review the cellular distribution of this receptor and the effect of SCF on the hematopoietic system, particularly among lymphoid lineage, either in normal or malignant cell progenitors. We examined reports and results in the field and articles or abstracts published in journals covered by MEDLINE. Additionally, we evaluated CD117 expression on fresh blast cells of 376 newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL) that were referred to centers affiliated with the Italian Association for Pediatric Hematology and Oncology (AIEOP). In view of our data, approximately 11% of ALL are CD117 positive. In particular, this receptor can be expressed in 10% and 11.5% of T-lineage and B-lineage ALL, respectively. Its expression is associated with an intermediate/mature phenotype in T-lineage ALL, whereas in B-lineage ALL, the majority of the positive cases are classified as early B ALL. The effect of SCF on malignant hematopoiesis and its potential clinical uses are reviewed.

Clinical relevance of CD10 expression in childhood ALL. The Italian Association for Pediatric Hematology and Oncology (AIEOP).

BACKGROUND AND OBJECTIVE: Previous studies have considered the prognostic significance of CD10 expression in childhood acute lymphoblastic leukemia (ALL) and showed its linkage to a more favorable prognosis. The aim of this study was to assess the independent significance of CD10 expression in a large population of ALL patients. DESIGN AND METHODS: We revised the independent clinical relevance of CD10 expression in 2038 children with acute lymphoblastic leukemia (ALL), who were consecutively entered in 4 sequential trials of the Italian Association for Pediatric Hematology and Oncology (i.e. AIEOP studies 82, 87, 88, 91); 1142 were males and 896 females, age ranged between 1 and 14 years (yrs) at diagnosis. Of the whole group, 1471 children (72.2%) were defined as having standard risk, 567 (27.8%) as having a high risk. RESULTS: CD10 was detected in blast cells from 1706 of 1784 (95.6%) patients with B-lineage ALL and 46 of 254 (18.1%) with T-cell ALL. In the B-lineage subgroup CD10 expression was associated with presenting features such as age < 9 yrs and inclusion in the standard risk category. No significant differences were found between CD10+ and CD10- cases in T-lineage ALL, concerning presenting features, except for FAB L2 in the former group. We compared the event-free survival (EFS) rates for patients with T-ALL or B-lineage ALL, regarding CD10 positivity, overall and by individual study. Patients with T-ALL fared worse than those with B-lineage ALL (5 and 10 yrs EFS: 46.8% vs. 68.5% and 44.5% vs. 63.7% respectively, p = 0.0001). In multivariate analysis of B-lineage subgroup poorer EFS was associated with male sex, higher WBC (> or = 20 x 10(9)/L), age > 9 yrs. Only WBC > or = 20 x 10(9)/L and age > 9 yrs were parameters linked to poorer EFS in the T-lineage subgroup. Finally, we compared EFS rates for four groups of patients categorized as having high or standard risk, and according to CD10+ and CD10- expression. High-risk patients fared statistically worse than standard risk patients both in the CD10- and in the CD10+ groups (42% vs. 50.7% and 63.6% vs. 66.8%, respectively). INTERPRETATION AND CONCLUSIONS: CD10 expression does not have independent prognostic significance in either the larger subgroup of B-ALL patients or in T-cell ALL.

Immunophenotype of adult and childhood acute promyelocytic leukaemia: correlation with morphology, type of PML gene breakpoint and clinical outcome. A cooperative Italian study on 196 cases.

Acute promyelocytic leukaemia (APL), characterized by a specific PML-RARalpha fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all-trans retinoic acid, presents clinical (varying WBC counts, age and treatment outcome), morphological (hypergranular M3 and hypogranular M3V) and molecular (three isoforms of PML breakpoint) heterogeneity. We correlated leukaemic immunophenotype with these aspects in 196 molecularly confirmed APLs (63 children and 133 adults) in Italy. The bcr3 isoform (P = 0.05) and FAB M3V (P = 0.05) were more frequent in children. We confirmed in APL an immunophenotype characterized by frequent expression of CD13, CD33 and CD9 and rare expression of HLA-DR, CD10, CD7 and CD11b. However, we recognized CD2 in 28%, CD34 in 23% and CD19 in 11% of cases and demonstrated by double labelling that CD34 and CD2 may be co-expressed. CD2, CD34 and CD19 were significantly intercorrelated, and variably associated to other features: CD2 and CD34 with PML bcr3 (P < 0.001 and P < 0.001, respectively) and with M3V (P < 0.001 and P = 0.002), whereas only CD19 was directly correlated with WBC counts and only CD2 positively influenced CR rate (logistic model) and event-free survival (Cox model). We conclude that immunophenotype plays a role in the determination of the biological and clinical heterogeneity of childhood and adult APL.

Expression of myeloid markers lacks prognostic impact in children treated for acute lymphoblastic leukemia: Italian experience in AIEOP-ALL 88-91 studies.

The importance of coexpression of myeloid antigens in childhood acute lymphoblastic leukemia (ALL) has long been debated; results are conflicting. We studied children with ALL treated at Italian Association for Pediatric Hematology-Oncology (AIEOP) institutions over 6 years with Berlin-Frankfurt-Muenster (BFM)-based protocols and have analyzed the incidence of coexpression of six MyAg (CD11b, CD13, CD14, CD15, CD33, CD65w) to determine its prognostic impact. Criteria for MyAg coexpression (MyAg+ALL) included positivity to one or more MyAg on at least 20% of blasts and confirmation of coexpression at double-fluorescence analysis. A total of 291 of 908 cases were MyAg+ALL (32%). Incidence was similar in B-ALL and T-ALL; among common, pre-B, and pre-pre-B-ALL. CD13 and CD33 were most common. Patients with MyAg+ALL had presenting features similar to MyAg-ALL. They entered standard or intermediate risk protocols more frequently and had better prednisone response, but similar complete remission rates. Six-year event-free survival (EFS) was 69.0% in 291 MyAg+ALL cases and 65.3% in 617 MyAg-ALL cases, without significant difference. Cases expressing two or more MyAg presented similar clinical features and treatment response. MyAg+ALL had worse EFS only in infants (0% v 47%) (P = .01). Therefore, in this series of homogeneously diagnosed and treated ALL, coexpression of MyAg was not associated with prognostic significance, without relevance for clinical purposes or for patient stratification, except for infants.

Transient acute monoblastic leukemia with reciprocal (8;16)(p11;p13) translocation.

The translocation of t(8;16)(p11;p13) has been demonstrated in the blasts of a phenotypically normal newborn baby with acute monoblastic leukemia. No antileukemic therapy was administered and spontaneous, complete remission was observed at 2 months of age. The patient remains well 18 months after the diagnosis and continues to have a normal hemogram.

Immunocytochemical evidence of common-ALL antigen in null-ALL.

4 cases of acute lymphoblastic leukaemia (ALL), diagnosed as null-ALL by indirect immunofluorescence using monoclonal antibodies, were similarly investigated using a sensitive immunoperoxidase method. The Avidin-Biotin system was employed. The immunoenzymatic results were in agreement with those obtained with immunofluorescence techniques for all antigens except common-ALL (C-ALL). The C-ALL antigen, recognized by the J5 antibody, was detected only by the immunoperoxidase method on cell membranes of the 4 ALL. This paper discusses the possibility of false negative results in testing for C-ALL antigen by conventional indirect immunofluorescence as suggested by refined immunocytochemical screening. Moreover, the ability of the immunoperoxidase system to identify antigens on cell membranes, even at very low density, is discussed. The clinical significance of the presence of C-ALL antigen at weak intensity in cases of null-ALL is also considered.

Non-T, non-B childhood acute lymphoblastic leukemia. Correlation between cytochemical markers and first complete remission.

The positivity for four cytochemical reactions, acid phosphatase (AcP), alpha-naphtyl acid acetate esterase (ANAE), beta-glucuronidase (BG), and N-acetyl beta-glucosaminidase (NABG) was correlated to first remission duration in 120 children affected with non-T, non-B acute lymphoblastic leukemia (ALL). The percentages of patients remaining in complete remission at 72 months were always higher for children whose blasts lacked these enzymatic reactions; however, a statistical difference was found only between BG+ and BG- ALL. It also appears that more complete enzymatic patterns of leukemic cells are associated with a poorer prognosis. The percentage of patients still in their first remission was 89% for leukemias with no cytochemical markers, 59% when one reaction was present, but less than 39% when two or more enzymes were detected in the blasts. It is noteworthy that the blasts of patients with more severe prognosis demonstrated a simultaneous positivity for AcP-ANAE or BG-NABG cytochemical reactions. The possible usefulness of these cytochemical markers to detect subsets of patients with different prognostic significance among non-T, non-B ALL is discussed.

[Late recurrence of Wilms' tumor]. / Recidiva tardiva di tumore di Wilms.

We present a patient with a Wilms' Tumor recurring after 7 years from the primitive surgical excision. After the relapse, surgery and conventional chemotherapy (with Actinomycin-D, Cyclophosphamide and Vincristine) permitted to achieve a second complete remission. Now the patient is alive without evidence of disease after 4 years from the relapse. The non-complete surgical excision of the tumor and the particular biological activity of the residual cells could explain the late relapse.

Cytochemical study in human lymphocyte subpopulations as defined by Fc receptors.

Three subpopulations of human T lymphocytes (TG, TM, and Tnon-G/non-M cells) were evaluated by means of six different cytochemical reactions. Elevated staining activity was demonstrated in all the populations when stained for acid phosphatase (AcP), beta-glucuronidase (BG) and N-acetyl beta-glucosaminidase (NABG). While the majority of TM cells displayed an intense acid alpha-naphthyl acetate esterase (ANAE) positivity, only a smaller proportion of the Tnon-G/non-M exhibited a positive reaction and a much lower activity was found in the TG group. TM cells were also alpha-naphthyl acetate esterase (NAE)-and alpha-naphthyl butyrate esterase (NBE)-positive, showing a dot-like pattern. These reactions were present in a lower proportion in the Tnon-G/non-M population and nearly completely absent in the TG cells. The present study identifies different cytochemical phenotypes for the different T-lymphocyte subsets and the analysis of these phenotypes may indicate that the TM is the most cytochemically differentiated subpopulation.

Congenital hypoplastic anaemia developed in acute megakarioblastic leukaemia. A case report.

A 14-month-old child with congenital hypoplastic anemia, in temporary remission after steroid therapy, developed an acute megakarioblastic leukemia. After complete remission due to immunosuppressive treatment, the leukemia rapidly relapsed, and the child died at 23 months of age. The rapid development of leukemia puts some doubt on the initial diagnosis.

Cytochemical study of thymocytes and T lymphocytes.

Fetal and postnatal thymocytes and circulating T lymphocytes were evaluated for six cytochemical reactions. Acid phosphatase activity was present in a high percentage of cells in all threee groups. Beta-glucoronidase and alpha-naphthyl acetate acid esterase were negative in the most immature fetal thymocytes, but become increasingly positive with T-cell maturation. Only the circulating lymphocytes presented a high percentage of N-acetyl beta glucosaminidase, alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase positive cells. This study discusses the presence of these enzymes as proportional to different stages in T-cell maturation, and also of certain cytochemical phenotypes characteristic of these stages.

Immunological features in chronic lymphocytic leukaemia (CLL) of T cell origin.

Peripheral blood lymphocytes from a patient with chronic lymphocytic leukaemia of T cell origin were studied. The thymus derived nature of these lymphocytes was confirmed by surface markers, mitogen cultures, mixed lymphocyte reaction, cytotoxicity studies, and cytochemical stains. This case is notable for several clinical and laboratory findings. Among these, the benign clinical course, the reduced rate of serum immunoglobulins, the elevated number of active E rosettes, the increased PHA-induced response to low mitogen doses, the absence of PHA mediated cellular cytotoxicity, and the thy-like positivity to ANAE should be pointed out. Emphasis should be placed, however, on the loss of stimulatory ability in MLR. This last feature supports the hypothesis that these cells proliferate as a clone.