During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16- CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B- cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre- B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy- chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

The purposes of this study were to examine the biologic effects of the engagement of the interleukin-7 receptor (IL-7R) with recombinant human interleukin-7 (rhIL-7) in immunophenotypically distinct T-lineage acute lymphoblastic leukemia (ALL) blasts and to elucidate the biochemical nature of the IL-7R-linked transmembrane signal in rhIL-7-responsive T- lineage ALL blast populations. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of freshly isolated leukemic blasts from six to eight T-lineage ALL patients with a mean plating efficiency of 196 +/- 53 (background subtracted) colonies/10(5) blasts plated. Stimulation of T-lineage ALL blasts with rhIL-7 resulted in markedly enhanced tyrosine phosphorylation of six distinct phosphoproteins with molecular weights of 57, 72, 98, 123, 150, and 190 Kd, and induced a rapid increase in the production of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3), which was inhibitable by the tyrosine-specific protein kinase inhibitor genistein, but not by the serine/threonine-specific protein kinase C inhibitor H7. Similarly, rhIL-7 stimulated Ins-1,4,5-P3 production in CEM-1.3 T-lineage ALL cells and this stimulation was inhibitable by the tyrosine-specific protein kinase inhibitors genistein and herbimycin A, but not by H-7. Thus, the transmembrane signal triggered by engagement of the IL-7R is intimately linked to a functional tyrosine-specific protein kinase pathway and stimulates the phosphoinositide (PI) turnover and proliferation of T-lineage ALL blasts. The presented data confirm and extend previous studies on the expression of functional IL-7R on T- lineage ALL blasts and support the hypothesis that IL-7 may play an important regulatory role in the biology of T-lineage ALL.

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B- lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence- activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS- sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.

Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post- BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time- dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor- depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.

Fourteen patients with high-risk T-lineage acute lymphoblastic leukemia (ALL) in complete remission underwent autologous bone marrow transplantation (BMT) in an attempt t o eradicate their residual disease burden. A combined immunochemotherapy protocol using a cocktail of t w o immunotoxins directed against CD5/Tp67 and CD7/Tp41 T-lineage differentiation antigens in combination with the in vitro active cyclophosphamide congener 4hydroperoxycyclophosphamide (4-HC) was used t o purge autografts. Despite high dose pretransplant radiochemotherapy and effective purging of autografts. 9 of 14 patients relapsed at a median of 2.5 months (range. 1.2 t o 16.8 months) post BMT. Two patients remain alive and disease free at 26 and 28 months post BMT. We used a novel quantitative minimal residual disease (MRD) detection assay, which combines fluorescence activated multiparameter flow cytometry and cell sorting with leukemic progenitor cell (LPC) assays, t o analyze remission bone marrow (BM) samples from T-lineage ALL patients for the presence of residual LPCs. ODERN MULTI-AGENT intensive chemotherapyM results in long-term disease-free survival (DFS) in 50% to 80% of patients with acute lymphoblastic leukemia (ALL), and a substantial proportion of these patients are cured of their di~ease.'.~ However, some ALL patients are at high risk for relapse following the currently available firstline chemotherapeutic regimens. Although a prolonged second remission can be attained by intensive salvage chemotherapy in patients who relapse while off treatment, patients who experience a medullary relapse during or shortly after their first-line chemotherapy have a poor outcome.' A number of centers have investigated the role of high-dose radiochemotherapy followed by allogeneic or autologous bone marrow transplantation (BMT) for the treatment of high-risk remission ALL.4-" Recent reports indicate that BMT may be more effective than chemotherapy in a number of high risk ALL patients, especially those who relapse within 18 months after first-line chemotherapy.ls6 More than two thirds of otherwise eligible BMT candidates with high risk remission ALL do not have a matched sibling donor for allogeneic BMT. For these patients autologous BMT has emerged as a potentially curative alternative therapy.'."This report details our preclinical and clinical data in 14 consecutive patients with high-risk remission T-lineage ALL who were prepared for BMT with either high dose cytarabine (ARA-C) in combination with total body irradiation (TBI) or hyperfractionated TBI followed by cyclophosphamide, and who received autografts that were purged ex vivo using the anti-CD5 (Tp67) immunotoxin T101-Ricin plus the anti-CD7 (Tp41) immunotoxin G3.7-Ricin combined with 4-hydroperoxycyclophosphamide (4-HC). Remission bone marrow (BM) samples were analyzed for the presence of residual T-lineage leukemic progenitor cells (LPCs) using a quantitative minimal residual disease (MRD) detection assay system, which combines fluorescence activated multipa-Notably, high numbers ...