Use of specific antisera against leukemia-associated antigens in the diagnosis of childhood acute lymphoblastic leukemia.

A series of 66 children with acute lymphoblastic leukemia (ALL) at diagnosis were investigated (simultaneously) for various surface markers. For this purpose the reaction of specific antisera against ALL antigens and T cell antigens was analysed in every case by several test systems, namely immunofluorescence, microcytotoxicity and complement fixation. A clearly defined classification in 6 subgroups of ALL emerged. The clinical data at presentation and possible correlations with the immunological subgroups were demonstrated.     

Expression of common acute lymphoblastic leukemia antigen (CALLA) by lymphomas of B-cell and T-cell lineage

Previous studies have demonstrated that the common acute lymphoblastic leukemia antigen (CALLA) is expressed by leukemic cells from approximately 80% of patients with non-T-cell ALL and 30%-50% of patients with chronic myelocytic leukemia in blast crisis. A small number of normal bone marrow and fetal liver cells also express CALLA, but the functional role of this molecule is unknown. In the present study, we have used a monoclonal antibody (J5) specific for CALLA to study the expression of this antigen in non-Hodgkin's lymphomas. Within the B-cell lymphomas, it was found the CALLA was expressed by almost all Burkitt's and nodular poorly differentiated lymphocytic lymphomas. Within the T-cell lymphomas, CALLA was expressed in 40% of patients with lymphoblastic lymphoma. Three of 3 Burkitt's lymphoma cell lines and three of eight T-lymphoblast cell lines were also found to express CALLA. Normal spleen, lymph node, and thymus cells were not reactive with J5 antibody. These findings indicate that expression of CALLA is not limited to relatively undifferentiated leukemic lymphoblasts but also occurs in more differentiated lymphoid malignancies. However, normal differentiated lymphoid cells in lymph node, spleen, and thymus, which have a phenotype similar to that of lymphoma cells, do not appear to express CALLA.     

Common acute lymphoplastic leukemia antigen (CALLA) and intestinal metaplasia

Intestinal metaplasia (IM) foci in 19 antral and 14 fundal gastric biopsies from patients with chronic atrophic gastritis were studied immunohistochemically for the presence of CALLA antigen. In only 2 cases were metaplastic glands completely negative, in 14 cases they were all positive, and in 17 cases variable proportions of CALLA positive and negative metaplastic glands were present. Complete IM seems to be less frequent than the incomplete type when the presence of CALLA is taken in consideration. CALLA is obviously a much better marker for complete or incomplete small IM. The possible importance of the presence of CALLA in IM foci for the future development of gastric cancer is discussed.     

Clinical features of common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma: Report of four cases

Summary Four patients with common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma are presented. The subclasses of monoclonal protein were IgD (1 case), IgA (1 case), and IgA (2 cases). Bence Jones proteinuria was seen in all cases. The clinical stages were determined as IIA (2 cases) and IIIA (2 cases). All patients died with a median survival time after diagnosis of 62 days due to rapid development of renal failure (3 cases), and renal insufficiency and pneumonia (1 case). According to light microscopic evaluation, these myelomas corresponded to plasmablastic (1 case), immature (2 cases), and intermediate (1 case) types. Both CALLA and a cytoplasmic immunoglobulin identical with the serum monoclonal protein were simultaneously detected in single cells from all cases using immunofluorescent double labeling. These findings suggest that CALLA-positive and plasmablastic myelomas constitute clinically a subgroup characterized by extremely poor survival but they represent cytologically different subcategories.     

Characterization of murine monoclonal antibodies against the common acute leukemia antigen (CALLA)

This study presents two murine monoclonal antibodies which react with the Common Acute Lymphoblastic Leukemia Antigen (CALLA). Both antibodies can be used for the diagnosis of common ALL (cALL). Indirect immunofluorescence studies (FACS-analysis) showed that the antibodies react with granulocytes and different human cell lines (Nalm-6, Reh, Raji, CCRF-CEM). The monoclonal antibodies BL-CALLA/1 and BL-CALLA/2 identify a single polypeptide chain of 95 kD. Both antibodies recognize the same or closely related epitope of the CALLA-molecule and are able to modulate in vitro the antigen on the CALLA-positive cell line Reh.     

Cultured marrow stromal cells express common acute lymphoblastic leukaemia antigen (CALLA): implications for marrow transplantation

This study demonstrates the presence of an antigenic determinant associated with the common acute lymphoblastic leukaemia antigen (CALLA), and presumably CALLA itself, on stromal cells in normal human long-term marrow cultures by using two monoclonal anti-CALLA antibodies, J-5 and 24.1. Treatment of cultured stromal cells with antibody and complement resulted in the loss of most flat angulated cells and many of the fat-containing cells. However, long-term cultures were generated with cytotoxic antibody-treated marrow buffy coat cells, and the stromal cells in these cultures were also CALLA-positive. We conclude that CALLA-bearing stromal cells arise from CALLA-negative progenitors. CALLA therefore could be either a differentiation antigen acquired on mature marrow stromal cells or may arise as a proliferation-dependent antigen. These studies suggest that the generation of long-term cultures from cytotoxic antibody-treated marrow may be an appropriate in vitro model for the functional assessment of such marrow prior to its use in autologous transplantation.     

Analysis of two new leukemia-associated antigens detected on human T- cell acute lymphoblastic leukemia using monoclonal antibodies

Two monoclonal antibodies (anti-3-3 and anti-3-40) were produced, which identify two new leukemia-associated antigens. Both antibodies reacted with most cell lines derived from patients with T lymphoblastic leukemia (T-ALL), but were not detected on suspensions of normal hematopoietic cells (including thymocytes) by cytotoxicity, absorption, or indirect immunofluorescence assays. Analysis of fresh leukemic cells indicated that anti-3-3 only reacted with T-ALL cells, while anti-3-40 also reacted with some non-T, non-B ALL cells and a few acute myelocytic leukemia (AML) cells. The 3-40 antigen was also found histopathologically in frozen sections of several normal tissues, including the epithelial cells and a few lymphoid cells of the thymus, and some malignant tissues. The 3-3 antigen was not found in any tissue studied. A "double absorption"assay provided additional serologic evidence that the two antibodies identify different antigenic determinants. Biochemical analysis indicated that the molecules immunoprecipitated by anti-3-3 and anti-3-40 have molecular weights of 35,000-40,000 daltons. This study demonstrated that the 3-3 and 3-40 antigens are markers for human T-ALL and can be used along with the normal T-lymphocyte antigen, 3A1, to discriminate T-ALL from cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia (ATL), and T-cell chronic lymphocytic leukemia (T-CLL).     

Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells

The phosphatidyl-inositol 3 kinase (PI3k)/Akt pathway has been implicated in childhood acute lymphoblastic leukemia (ALL). Because rapamycin suppresses the oncogenic processes sustained by PI3k/Akt, we investigated whether rapamycin affects blast survival. We found that rapamycin induces apoptosis of blasts in 56% of the bone marrow samples analyzed. Using the PI3k inhibitor wortmannin, we show that the PI3k/Akt pathway is involved in blast survival. Moreover, rapamycin increased doxorubicin-induced apoptosis even in nonresponder samples. Anthracyclines activate nuclear factor kappaB (NF-kappaB), and disruption of this signaling pathway increases the efficacy of apoptogenic stimuli. Rapamycin inhibited doxorubicin-induced NF-kappaB in ALL samples. Using a short interfering (si) RNA approach, we demonstrate that FKBP51, a large immunophilin inhibited by rapamycin, is essential for drug-induced NF-kappaB activation in human leukemia. Furthermore, rapamycin did not increase doxorubicin-induced apoptosis when NF-kappaB was overexpressed. In conclusion, rapamycin targets 2 pathways that are crucial for cell survival and chemoresistance of malignant lymphoblasts--PI3k/Akt through the mammalian target of rapamycin and NF-kappaB through FKBP51--suggesting that the drug could be beneficial in the treatment of childhood ALL.     

Long-term survival following acute lymphoblastic leukemia (ALL) in childhood

48 patients with ALL (3%) treated according to two different protocols survived 5 years or longer. The mean survival time was 13.0 +/- 5.0 years, but 29 patients lived 16.5 +/- 3.0 years. Only 18 patients (38%) were 16.9 +/- 3.0 years in them 1. CCR, the longest remission time amounted to more than 25 years. Of the patients relapsed 9 are 8.4 +/- 2.8 years in 2. CR. Late relapses after 5 years were observed in seven cases the last in the 9th year of disease. After 5 years the survival rate in both protocols was not different. As a late sequelae, there was one patient with intrahepatic block and portal hypertension and one with encephalopathy and imbecility. All patients were able to leave their professional examination, 5 patients married and 6 healthy children were born.     

Expression of CD20 on acute lymphoblastic leukemia cells in children

CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells. The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II. However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated. The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients). The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy. This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%). The expression of CD20 showed no association with the expression of CD34, CD22 and MDR. The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells. The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20. There was no association of CD20 expression with the time of reaching the hematological remission. The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.     

Monocyte markers and the common acute lymphoblastic leukemia antigen on chronic lymphocytic leukemia cells

The mononuclear cells from 42 patients with chronic lymphocytic leukemia (CLL) were analyzed for surface markers using monoclonal antibodies and flow cytofluorimetry. All 42 CLLs were Ig+HLA.DR+OKT3-OKT4-OKT6-OKT8- and monoclonal with respect to light chain type. Thirty of the 42 tumors expressed the common acute lymphocytic leukemia antigen, and cells from 18 leukemias stained with the antibodies OKM1 and 63D3, which are specific for different antigens characteristic of myeloid cells. Eight of the leukemias failed to stain with PI153, which has been reported to recognize all B-cell CLLs.     

Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopeptidase, which has been shown to inactivate a variety of peptide hormones including enkephalin, chemotactic peptide, substance P, neurotensin, oxytocin, bradykinin, and angiotensins I and II, had not been identified in lymphoid cells. To determine whether CALLA cDNA derived from human acute lymphoblastic leukemia cells (Nalm-6 cell line) encodes functional neutral endopeptidase activity, we generated CALLA+ stable transfectants in the CALLA- murine myeloma cell line J558 and analyzed them for enzymatic activity in a fluorometric assay based upon cleavage of the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total lysates as well as whole-cell suspensions of the Nalm-6 line and of the CALLA+ transfectants, but not of the CALLA- J558 cells, possessed neutral endopeptidase activity. This enzymatic activity was associated with the cellular membrane fraction and was abrogated by the specific neutral endopeptidase inhibitor phosphoramidon. The unequivocal identification of CALLA as a functional neutral endopeptidase provides insight into its potential role in both normal and malignant lymphoid function.     

Near-triploid and near-tetraploid acute lymphoblastic leukemia of childhood

Cytogenetic and DNA flow cytometric analyses of leukemic cells from 1,971 children with newly diagnosed acute lymphoblastic leukemia (ALL) identified stem lines with modal chromosome numbers greater than 65 in 26 patients (1.3%). Near-triploidy (66 to 73 chromosomes) was found in six cases and near-tetraploidy (82 to 94 chromosomes) in 20. A striking morphologic finding was the presence of clumped chromatin with grooved nuclei or Rieder cell formation in 20 cases. Other than a slight excess of the pre-B immunophenotype, the near-triploid cases did not appear to differ substantially from the general ALL population in clinical features. In contrast, near-tetraploid cases were more often associated with a T-cell immunophenotype (47% v 14%, P less than .001) and L2 morphology (30% v 22%, P less than .01), and were older at diagnosis (median age, 8.6 v 4.8 years, P = .01) than cases with other ploidies. Moreover, an unusually high proportion of near-tetraploid cases tested (6 of 15) expressed one or more of the myeloid-associated antigens CD13, CD15, and CD33. Despite the use of contemporary intensive chemotherapy and short follow-up for most patients, 6 of the 20 near- tetraploid cases have relapsed or died. This study suggests that the near-tetraploid subtype differs from other cases of hyperdiploid greater than 50 ALL, which have been associated with a favorable prognosis.     

Prognostic significance of CD20 expression in childhood B-cell precursor acute lymphoblastic leukemia

CD20 expression is associated with inferior survival in adults with acute lymphoblastic leukemia (ALL). We analyzed the prognostic impact of CD20 expression in 353 children with B-cell precursor ALL treated in 3 consecutive St Jude Total Therapy studies. CD20 expression (> 20%) was found in 169 patients (48%) and was more frequent in patients between 1 and 10 years of age than in those younger than 1 or older than 10 years (P = .001). None of 14 patients with MLL-AF4 expressed CD20. There was no association between CD20 expression and E2A-PBX, TEL-AML1, ploidy, white blood cell count at diagnosis, or sex. In contrast to the experience in adult ALL, our patients with CD20 expression tended to have a better treatment outcome than those without the expression: 5-year event-free survival 84% +/- 2.9% versus 78% +/- 3.1% (P = .08). These data suggest that CD20 expression is not associated with inferior outcome in pediatric patients treated with contemporary regimens.     

Decreased expression of the common acute lymphoblastic leukaemia antigen (CALLA/CD10) on neutrophils from patients with thermal injury

Summary. The common acute lymphoblastic leukaemia antigen (CALLA/CD10) is a normal component of the circulating neutrophil cell surface membrane. In order to examine the potential functional significance of CALLA/CD10 we analysed the expression of this molecule on neutrophils isolated from thermal injury patients, since these patients have a well-documented constellation of neutrophil defects affecting their microbicidal functions. Expression of neutrophil CALLA/CD10 was monitored by indirect immunofluorescence and flow cytometry. We observed that CALLA/CD10 expression was quantitatively reduced on burn patient neutrophils, compared to healthy donors ( P < 0.001). In contrast, burn patient neutrophils expressed normal levels of class I HLA molecules and the C3bi receptor. Reduced expression of CALLA/CD10 was not associated with neutrophil activation or exposure to plasma 'factor(s)' in vivo . Analysis of normal bone marrow neutrophils by cell sorting indicated that expression of CALLA/CD10 occurs late in neutrophil maturation, since 25% of polymorphonucleated bone marrow neutrophils did not express cell surface CALLA/CD10. Attempts to examine the chemotactic responses of CALLA/CD10 positive and negative neutrophils from burn patients were hampered by previous exposure of these cells to chemoattractants in vivo . Collectively, our findings suggest that burn patient peripheral blood neutrophils may be deficient in CALLA/CD10 due to insufficient maturation time in the bone marrow following thermal injury.     

Selective expression of the common acute lymphoblastic leukemia (gp 100) antigen on immature lymphoid cells and their malignant counterparts

The selectivity of monoclonal antibody J-5 (anti-gp 100, common ALL antigen) for normal and leukemic hemopoietic cells has been investigated. J-5 gave concordant reactions with rabbit anti-cALL, coredistributed on the cell surface, and precipitated a similar if not identical glycoprotein from leukemic and normal tissue. Normal, immature lymphoid cells reactive with J-5 were detected in bone marrow and in fetal thymus. In marrow they were largely coincident with the TdT+ population. J-5 defines a major subgroup of ALL (common ALL) with a favorable prognosis. Of 853 non-ALL acute leukemias investigated, 80 were J-5 positive. These included 14 cases diagnosed as AML, 51 TdT+ blast crises of CGL, and 15 cases diagnosed as "AUL." Of the 14 J-5+ AML, 13 were subsequently rediagnosed either as cALL (10 cases) or mixed lymphoid-myeloid leukemias (3 cases). One-hundred forty-three cases of mature lymphoid cell leukemia (91 B, 52 T) were investigated with J-5; 3 cases only, of disseminated B lymphoma, were positive, albeit weakly. A higher proportion of follicular lymphomas are, however, J-5 positive when studied in sections of biopsy material. A similar pattern of selective reactivity was observed in a series of leukemia/lymphoma cell lines. These studies emphasize the diagnostic value of monoclonal anti-cALL reagents.