Monoclonal antibody characterization of surface antigens in childhood T-cell lymphoid malignancies

Although childhood T-cell acute lymphocytic leukemia (T-ALL) and T-cell non-Hodgkin's lymphoma (T-NHL) have certain clinical features in common, T-ALL carries a notably poorer prognosis than does T-NHL. To determine whether the malignant cells from patients with these disorders are distinguishable, we examined bone marrow and/or blood from 51 children with T-ALL and tumor biopsy specimens from 17 with T-NHL, using a panel of monoclonal antibodies directed against T-cell differentiation antigens. We found considerable phenotypic heterogeneity in both T-ALL and T-NHL. Of the T-ALL (defined by greater than 25% blasts in the bone marrow) patients, 33% demonstrated a surface antigen pattern consistent with the earliest thymocyte stage of T-cell development (T9+ and/or T10+, or T6-/T4-/T8-/T3-), 37% were of a midthymocyte stage (T6+, and/or simultaneous expression of T4-helper and T8-suppressor antigens), and 30% expressed surface antigen patterns found on mature thymocytes (T3+, variable expression of other antigens). In contrast, tumor cell phenotypes in the 17 T-NHL patients were approximately equally distributed between mid- and mature thymocyte phenotypes. No NHL samples were classified as the early thymocyte phenotype. Clinical features as related to specific T-ALL immunophenotypes are presented, and the implications of these findings in regard to the current understanding of the differences in tumor biology between T-ALL and T-NHL are discussed.     

Expression of the candidate MCT-1 oncogene in B- and T-cell lymphoid malignancies

Our laboratory has recently discovered a novel candidate oncogene, MCT-1, amplified in human T-cell lymphoma and mapped to chromosome Xq22-24. This region is amplified in a subset of primary B-cell non-Hodgkin lymphoma (NHL), suggesting that increased copy number of a gene(s) located in this region confers a growth advantage to some primary human lymphomas. We examined a diverse panel of lymphoid malignancies for the expression of MCT-1. We demonstrated that there are significantly increased levels of MCT-1 protein in a panel of T-cell lymphoid cell lines and in non-Hodgkin lymphoma cell lines. Furthermore, we identified a subset of primary diffuse large B-cell lymphomas that exhibited elevated levels of MCT-1 protein. Interestingly, all transformed follicular lymphomas in our study demonstrated elevated protein levels of MCT-1. There was no detectable MCT-1 protein in leukemic cells from patients with chronic lymphocytic leukemia or in any healthy lymphoid tissue examined. Lymphoid cell lines overexpressing MCT-1 exhibited increased growth rates and displayed increased protection against apoptosis induced by serum starvation when compared with matched controls. We found that MCT-1-overexpressing cells show constitutively higher levels of phosphorylated PKB/Akt protein, especially under serum starvation. Activation of survival pathways may be an additional function of the MCT-1 gene. Our data suggest that high levels of MCT-1 protein may be associated with a high-risk subset of lymphoid neoplasms and may further support the potential role of MCT-1 in promoting human lymphoid tumor development.     

A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors

A murine monoclonal antibody (MoAb) designated FA6-152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6-152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6-152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.     

A monoclonal antibody which identifies an antigen in endothelial cell and epithelial basement membrane.

We isolated a monoclonal antibody which decorates the endothelial cells of normal and lymphatic vessels in formalin-fixed, paraffin-embedded tissue. In addition, the antibody recognizes a previously undescribed substance found in the basement membrane zone and subbasement membrane zone of a variety of epithelial. By ELISA assay, the antigen recognized by this monoclonal antibody is not laminin, type IV collagen or fibronectin. This antibody may be used as a diagnostic tool because it identifies an epitope in neoplasms differentiating towards endothelial cell such as angiosarcoma and Kaposi's sarcoma.     

T-cell replete fludarabine/cyclophosphamide reduced intensity allogeneic stem cell transplantation for lymphoid malignancies

The relative merits of reduced-intensity allogeneic stem cell transplantation (RISCT) for high-risk indolent lymphoid malignancies are emerging, although the preferred conditioning regimen to manage the risks of graft-versus-host disease (GVHD) is not clearly defined. Here we report the outcome of 73 patients with lymphoid malignancies who received RISCT with a fludarabine/cyclophosphosphamide conditioning regimen and a median follow-up of 3 years. Median age was 54 years. Forty-eight per cent of patients had previously undergone autologous stem cell transplantation with a median of three prior therapies. Non-relapse mortality at 3 years was 19% but only 5% for patients with multiple myeloma (MM). Three-year overall survival and current progression-free survival was 67% and 63% respectively. Grade 2-4 acute GVHD occurred in 14% of patients while 49% had chronic GVHD requiring systemic immunosuppression. The preparatory regimen in this study has the advantage of reduced acute GVHD and low mortality, notably in patients with MM. In addition, this strategy provides long-term disease control in a significant proportion of patients with particular benefit in those with high-risk follicular lymphoma.     

Shifts in expression of cell membrane phenotypes in childhood lymphoid malignancies at relapse.

To determine if cell membrane phenotypes change under the selective pressures of therapy we have conducted a prospective study of 54 children with lymphoid malignancies of T-like, B-like, common, and null cell types. Membrane phenotypes were determined at diagnosis in all patients and again 1-24 mo later in 18 children who either failed induction therapy or had one or more relapses. In 7 patients the cells tested were from relapse sites different than those of the original diagnoses. The data indicate that at relapse most children with lymphoid neoplasias had the same cell membrane phenotype as established at diagnosis, and suggest that the site of relapse did not affect the expression of cell surface markers. However, there were three exceptions: (1) a child initially diagnosed as having null cell acute lymphocytic leukemia had 90% T-antigen-positive blasts in her second-relapse bone marrow; (2) only membrane IgM was present on relapse blasts from a B-cell lymphoma that had both membrane IgM and IgD before initiation of treatment; (3) at diagnosis, bone marrow blasts from a child with T-like leukemia expressed both T antigen and E receptors, but at relapse, bone marrow and pleural fluid cells expressed only T antigens. We postulate that these phenotype shifts may be due to selective effects of therapy on cells at different stages of differentiattion.     

Reovirus therapy of lymphoid malignancies

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.     

Treatment of cutaneous T-cell lymphoma with chimeric anti-CD4 monoclonal antibody

Chimeric anti-CD4 monoclonal antibody was administered intravenously as a single dose to eight patients with mycosis fungoides. The dose was escalated throughout the study between patients groups, and individual patients received 50, 100, or 200 mg per dose. Seven of eight patients responded to treatment with an average freedom from progression of 25 weeks (range, 6 to 52 weeks). The treatment was well tolerated, and there was no clinical evidence of immunosuppression. Following treatment, there was significant suppression of peripheral blood CD4 counts in all patients for 1 to 22+ weeks. Only one patient made a very low titer human antichimeric antibody response. All but two patients made primary antibody and T-cell proliferative responses to a foreign antigen administered 24 hours after antibody infusion. However, there was generally marked, but temporary suppression of T-cell proliferative responses in vitro to phytohemagglutinin (PHA), tetanus toxoid, and normal donor lymphocytes. We conclude that at the dose levels studied, this antibody (1) had clinical efficacy against mycosis fungoides; (2) was well tolerated; (3) had a low level of immunogenicity; (4) decreased T-cell proliferative responses in vitro, and (5) did not induce tolerance to a foreign antigen.     

Functional characteristics of human T-cell subpopulations distinguished by a monoclonal antibody

In animals and in man, diverse immunologic functions are mediated by specialized T-cell (thymus-derived lymphocyte) subsets that are distinguishable from one another on the basis of differences in cell surface determinants. Unfortunately, in humans, subset-specific antibodies have been difficult to generate. In this study, production of a murine monoclonal antibody specific for a subset of human T cells was achieved by fusing a sensitized B cell (bone marrow-derived cell) with a myeloma cell and isolating the antibody secreted by the resultant hybrid clone. This antibody binds 30-35% of peripheral T lymphocytes (T_a⁺ cells) but fails to bind remaining T lymphocytes (T_a^- cells), B lymphocytes, or monocytes. T_a⁺ and T_a^- subpopulations were separated with a fluorescence-activated cell sorter and their in vitro responses to various stimuli were assessed. T_a⁺ and T_a^- cells respond equally well to soluble antigens, allogeneic B cells, and autologous B cells, but only T_a⁺ cells respond to concanavalin A. T_a⁺ cells cultured in the presence of concanavalin A gradually lose the T_a marker, an effect not observed after stimulation with phytohemagglutinin, soluble antigens, or alloantigens. These results suggest that the functional subpopulation of T cells defined by T_a does not correspond to any previously described human T cell subset. Furthermore, somatic cell hybridization has been shown to be a feasible method for production of monoclonal antibodies specific for subpopulations of human lymphocytes.     

Development of an anti-porcine CD34 monoclonal antibody that identifies hematopoietic stem cells

OBJECTIVES: The isolation of porcine hematopoietic stem cells (HSC) would be an important step toward development of porcine-to-human chimerism for induction of tolerance in clinical xenotransplantation. CD34 is a common marker of HSC and has not been developed as a marker in pigs. In this study we have generated and characterized a monoclonal antibody (mAb) that identifies porcine CD34 on a subset of porcine bone marrow (BM) stem/progenitor cells. METHODS: The porcine CD34 gene was cloned and a recombinant protein produced. An anti-porcine CD34 mAb was produced that could detect both the recombinant protein and a subset of porcine BM cells. The CD34(+) cells were phenotyped by lineage and HSC associated markers. Furthermore, the CD34(+) cells were analyzed by colony-forming unit (CFU) assay. RESULTS: Two splice variants of the porcine CD34 gene were cloned and a recombinant protein produced for mAb production. The mAb developed can detect both the recombinant protein and the native CD34 protein on a range of pig tissues, including BM. This subset of BM cells was negative for hematopoietic lineage makers, including CD3, CD14, and CD21 and positive for other known porcine HSC markers, including CD90, CD172a, histocompatibility complex (MHC) class I, and MHC class II. Moreover, the CD34(+) BM cells were enriched for multilineage progenitor cells as determined by CFU assay. CONCLUSIONS: Similar to human and mouse CD34, pig CD34 detects a subset of BM progenitor cells. This mAb will now provide a means for isolating porcine CD34(+) cells to be further analyzed for HSC activity and to assess their potential to develop pig-to-human chimeras to induce xenograft tolerance.     

Phenotypic heterogeneity of human T-cell malignancies: demonstration by monoclonal antibodies and cytochemical markers

The present study sought to delineate the phenotypic heterogeneity of the human T-cell malignancies. Twenty T-cell neoplasms were investigated for reactivity with the OKT hybridoma monoclonal antibodies and expression of acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (BG), and acid phosphatase (AP) activity. Twelve cases (Mycosis fungoides, Sezary syndrome, cutaneous T-cell lymphoma, chronic lymphocytic leukemia) were OKT3'T4', ie, expressed the phenotype commonly associated with mature T-helper cells. These cases were further divisible into ANAE+BG+ (6 cases), ANAE-BG+ (5 cases), and ANAE-BG- (1 case) phenotypes. In contrast to the 12 OKT3+T4+ cases, the remaining 8 cases showed considerable inter- and intratumor heterogeneity with respect to reactivity with the OKT antibodies. Six of these cases (acute lymphoblastic leukemia, lymphoblastic lymphoma) expressed phenotypes consistent with various intrathymic stages of T-cell differentiation. Five of the latter 6 cases were AP+BG+ANAE-, analogous to the majority of normal cortical thymocytes; an OKT3+T4-T8+T10+ neoplasm was ANAE+, analogous to normal medullary thymocytes. Two cases expressed the previously undescribed OKT3+T4-T8-T10+ phenotype. These studies demonstrate that the T-cell malignancies are divisible into phenotypes which correspond to normal maturational stages of T-cell differentiation and functionally distinct T-cell subsets. Phenotypic analysis of the human T-cell malignancies may provide a basis for understanding their biological heterogeneity and may aid in the identification of transitional stages of T-cell differentiation and minor T-cell subsets.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Detection of immunoglobulin gene rearrangement in lymphoid malignancies of B-cell lineage by seminested polymerase chain reaction gene amplification

Seminested polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity-determining region 3 of the immunoglobulin (Ig) gene heavy chain from the malignant cell specimens of patients with leukemias and lymphomas of B-cell lineage. Two different pairs of primers were used sequentially. Twenty of the 27 (74%) acute lymphoblastic leukemia (ALL) patients, 14 of 19 (74%) chronic lymphocytic leukemia (CLL) patients and eight of 20 (40%) non-Hodgkin's lymphoma (NHL) patients, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by the seminested PCR. False-negative results appeared to occur more commonly in cases of lymphoma. The PCR analysis was also less likely to be positive if one-stage PCR studies with either pair of primers were both negative. The seminested PCR technique was found to have a high sensitivity of detecting malignant cells at the level of 0.02%. The clinical application of this assay needs to be investigated further.     

High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies

Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.     

The future role of monoclonal antibody therapy in childhood acute leukaemias

Leukaemia is the single most common childhood malignancy. With modern treatment regimens, survival in acute lymphoblastic leukaemia (ALL) approaches 90%. Only about 70% of children with acute myeloid leukaemia (AML) achieve long term survival. Patients who relapse have a dismal prognosis. Novel therapeutic approaches are needed to improve treatment outcomes in newly-diagnosed patients with a poor prognosis and for patients with relapsed/refractory disease that have limited treatment options. One promising approach in treating haematological malignancies has been the use of monoclonal antibodies to target cell surface antigens expressed on malignant cells. Most success with monoclonal antibody therapy in the treatment of haematological malignancies has come in the setting of adult B-cell non-Hodgkin lymphoma with the addition of the anti-CD20 monoclonal antibody rituximab to standard treatment regimens. In order to further advance treatment of haematological malignancies, novel monoclonal antibodies continue to be developed that target a variety of cell surface antigens. Several antibodies continue to be investigated in childhood leukaemias. This review will discuss the development of monoclonal antibodies that target a variety of cell surface antigens for the treatment of childhood ALL and the use of the anti-CD33 antibody gemtuzumab ozogamicin in the treatment of childhood AML.     

Anti-K562 cell monoclonal antibody RTF8X recognizes tumor-associated antigen of erythroid lineage

A new anti-K562 cell monoclonal antibody, RTF8X, a cytotoxic IgM, recognized a surface antigen on erythroblasts from patients with erythroleukemia and polycythemia vera. RTF8X, which is highly specific to K562 cells, did not react with the other 14 hematopoietic cell lines and the seven nonhematopoietic cell lines. RTF8X antigen was not detected in normal peripheral blood, but was found in less than 1% of normal marrow cells. RTF8X did not inhibit in vitro colony formation of CFU-E and BFU-E in a complement-dependent cytotoxicity assay. Cell- sorting analysis showed that, morphologically, the RTF8X-positive marrow cells from the patients and normal volunteers contained more than 60% erythroblasts and that CFU-E and BFU-E were not demonstrated in cells with RTF8X antigen. Enzyme treatment suggested that RTF8X antigen was a sialoglycolipid. These results indicate that RTF8X may recognize the surface antigen found increasingly in association with tumors of erythroid lineage. RTF8X should be useful for studies of erythroid differentiation and proliferation in patients.     

Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage

A monoclonal antibody (MoAb), Ki-M8, that reacts specifically with cells of the monocyte/macrophage system is described. On light and electron microscopic immunohistochemistry, Ki-M8 recognizes intracytoplasmatically localized antigens of mol wt 30,000 and 32,000, increasingly expressed during differentiation of monocytes into macrophages. Ki-M8 antigen is detectable on almost all known tissue macrophages and monocyte/macrophage-related cell lines after appropriate stimulation. In functional terms Ki-M8 significantly impairs the generation of oxygen radicals during an induced respiratory burst. Applied to acute nonlymphoblastic leukemias, a clear-cut differentiation of the monocytic phenotype and differentiation is possible on the basis of Ki-M8 immunoreactivity. Ki-M8 represents a reagent specific for the monocyte/macrophage system with regard to antigen distribution in normal and neoplastic cells as well as with regard to its influence on a typical monocyte/macrophage-related function.     

A monoclonal antibody (WT1) for detecting leukemias of T-cell precursors (T-ALL)

The selectivity of a monoclonal anti-T antibody, designated WT1, has been assessed in a series of 906 leukemias and lymphomas. In acute lymphoblastic leukemias, WT1 reacts comprehensively and selectively with thymic acute lymphoblastic leukemia (ALL) cells in untreated or relapsed patients, thus overriding the extensive antigenic diversity of this cancer and the immaturity of the cell type involved. All 80 cases of thymic ALL examined were WT1-positive. In addition, 18 cases of presumptive prethymic ALL were also WT1-positive, but were unreactive with other maturation-linked T-cell markers. The phenotype WT1+ HLA-DR TdT+ appears to be unique to T-ALL and can therefore be used systematically for the differential diagnosis of this poor prognosis subtype of ALL. Virtually all ALL cases can now be placed into one of two major subgroups representing transformed precursors of either the T- or B-cell lineage. WT1 identifies a single polypeptide of approximately 40,000 mol wt and is similar to two previously described monoclonal antibodies.