Anand Raghavachar

Severe combined immunodeficiency: treatment by bone marrow transplantation in 15 infants using HLA-haploidentical donors

In 15 infants with severe combined immunodeficiency (SCID), immunological reconstitution was attempted by bone marrow transplantation (BMT) from HLA-haploidentical parents. To prevent graft versus host disease (GvHD), marrow grafts were depleted of contaminating T-lymphocytes using lectin agglutination and rosette formation with sheep red blood cells. Thirteen patients received transplants without undergoing prior cytoreductive conditioning. Eleven of these developed donor-dependent T-cell functions, two failed to do this. One of these two as well as two further patients received cytoreductive treatment prior to repeat and to first transplants and in two, complete lymphohemopoietic reconstitution was observed. Of the 15 patients who received transplants, 11 are currently alive. Two recently treated patients remain in the hospital, nine are at home with stable T-cell functions. Normal humoral immune functions have developed upto now in three patients. In the others, gammaglobulins are regularly substituted. Complications of acute or chronic GvHD were not observed with the exception of one case who developed transient GvHD of the skin.These results suggest that in a majority of patients with SCID, T-cell functions can develop without GvHD following haploidentical, T-cell-depleted BMT. Exceptional patients require preconditioning to allow donor cell engraftment, an approach that also appears to facilitate reconstitution of humoral immune functions

2 Immunophenotypic classification of acute lymphoblastic leukaemia

Recent advances in immunophenotyping of acute leukaemias have led to important insights into normal haematopoietic differentiation as well as into the cellular diversity and origins of leukaemic blasts, especially in acute lymphoblastic leukaemia (ALL). These advances include the production and standardization of monoclonal antibodies (mAbs) to a variety of lymphoidand myeloid-lineage-associated antigens, the characterization of reactivity patterns of these reagents with leukaemic blasts, the establishment of an internationally accepted cluster of differentiation (CD) nomenclature and, finally, technical innovations in the evaluation of antigen expression on individual leukaemic cells. Immunophenotyping has become an important element in the modern diagnosis of acute leukaemia for several reasons. First, use of a standardized panel of mAbs to B-cell, T-cell and myeloid as well as non-lineage-restricted antigens permits allocation of more than 98% of acute leukaemias to their respective lineage (Janossy et at, 1989; Campana et al, 1990a). Secondly, in ALL, immunophenotyping has established a solid basis for precise and biologically oriented classification of the disease (Foon and Todd, 1986; Greaves, 1986); and, in acute myeloid leukaemia (AML), immunologic markers are particularly important for identifying acute leukaemia with minimal myeloid or megakaryoblastic differentiation (Bennett et al, 1985; Bennett et al, 1991; Buccheri et al, 1992). Thirdly, based on recent observations that leukaemic blasts frequently disclose aberrant or asynchronous antigen expression compared with normal haematopoietic cell differentiation, leukaemia-associated phenotypic features have been used to detect minimal residual disease (MRD) in both ALL and AML (reviewed in Campana et al, 1991a; van Dongen et al, •992). Finally, immunophenotyping alone or in conjunction with more recently developed cytogenetic and molecular-biological techniques has identified biologically and clinically distinct subsets within the major diagnostic groups and has been essential for

Clonal variation in childhood acute lymphoblastic leukaemia at early and late relapse detected by analyses of phenotype and genotype.

To increase our knowledge of the clonal relationship of leukaemia relapse, the genotypes and phenotypes of ten children with acute lymphoblastic leukaemia (ALL) were examined at initial diagnosis and relapse. Seven patients were phenotyped as common ALL, two as mixed, and one as T-cell ALL (T-ALL). Comparative analyses of immunoglobulin (Ig) heavy and light chain as well as T-cell receptor β-chain (Tβ) sequences revealed clonal variations, i.e. appearance of a novel or an evoluted leukaemic cell clone in five patients coinciding with the loss of common acute lymphoblastic leukaemic antigen (CALLA) in four cases, irrespective of early or late relapse. Conversion of early B- to T-ALL or lymphoblastic to non-lymphoblastic leukaemia was not noted in any of the patients examined. Our results suggest that clonal variation is a frequent event in childhood ALL.

Conversion of acute undifferentiated leukemia phenotypes: analysis of clonal development.

The cellular origin of acute undifferentiated leukemia (AUL) is still a matter of controversy. We report on two cases in which the diagnosis of AUL was established according to restricted criteria. Blast cells of both patients showed phenotypic conversion during the course of disease. In one case, within 24 days from starting treatment, the leukemic phenotype changed from AUL to acute myelomonocytic leukemia (FAB L1, TdT+ to FAB M4, TdT-). The initial phenotype of this acute leukemia was characterized by the co-expression of both B-lymphoid and myeloid markers on the same cell. Moreover, analysis of esterase isoenzyme pattern showed the whole spectrum of isoenzymes typically seen in myelomonocytic leukemias already at diagnosis, yet blast cells additionally contained all three isoenzymes of beta-hexosaminidase typically seen in AUL. However, examination of immunoglobulin (Ig) heavy chain gene rearrangement initially and after conversion revealed an identical monoclonal configuration of Ig heavy chain sequences in both samples. The second AUL patient relapsed after allogeneic bone marrow transplantation with common ALL-antigen (CALLA) positive acute leukemia. Subsequent Southern blot analysis showed a novel rearranged Ig fragment compared to the analysis before transplantation indicating that the leukemic clones prior to and after transplantation were not identical. No chromosomal abnormalities were observed in both cases. These data support the view that AUL cells originate from a pluripotent stem cell that is capable to differentiate in the myelomonocytic lineage (patient 1), and confirm the value of Ig gene analysis as marker for cellular clonality.

Possible Mechanism of Interferon Action in Hairy Cell Leukemia

Recently, we described that peripheral blood mononuclear cells (MNC) from patients with hairy cell leukemia (HCL) can be induced in vitro to produce higher levels of interferon-gamma (IFN-γ) but lower levels of IFN-α than in healthy controls [7]. The reduced IFN-α production has been explained by the lack of monocytes in this disease. The increased production of IFN-γ suggests an increased release of cytokines by T cells. This assumption has been confirmed by Ford et al. [2], who described that a conditioned medium of T4-lymphocytes from patients with HCL contains a cytokine that stimulates the growth of activated, normal B cells. Kehrl et al. [5] demonstrated tumor necrosis factor alpha (TNF-α) to be a stimulator of activated normal B cells, and we (Digel et al. 1988) have shown recently that recombinant TNF-α stimulates the growth of purified B cells from patients with B-chronic lymphocytic leukemia (B-CLL). Based on these findings, we hypothesize that TNF is produced in B-cell disorders like HCL and that TNF may stimulate the growth of hairy cells (HC).

Immunoglobulin and T-Cell Receptor Gene Rearrangements in Human Acute Leukemias

Development of immunological methods has made it possible to examine the cellular origin of leukemia cells in relation to their normal counterparts and acute lymphoblastic leukemia (ALL) is generally classified into the following major subgroups: T-ALL, B-ALL and common ALL (cALL) (1). Not infrequently, however, it is impossible to classify acute leukemias as B or T cell or nonlymphoid in origin despite examining lineage associated surface markers, as some malignancies represent stages of differentiation prior to the expression of lineage restricted surface markers. These limitations can now be overcome by utilizing the DNA rearrangement of immunoglobulin (Ig) and T cell receptor genes to reveal the clonality, cellular lineage, and stage of differentiation of such acute undifferentiated leukemias (AUL) (2).

T Cell Depletion of Marrow for the Prevention of Graft Versus Host Disease

Selective depletion of T cells from marrow grafts is an attractive approach to reduce the incidence and severity of GvHD. Recent studies in man clearly indicate that this approach is valid, opening the way to perform BMT across HLA barriers (1,2,3). However, this initial experience also reveals an increased risk of graft failures after transplantation of T cell depleted marrow, in particular when HLA nonidentical donors are used (4). We report similar experience obtained in 24 patients transplanted with in vitro processed marrow from HLA matched and mismatched donors, using lectin agglutination and/or rosetting by sheep red cells for T cell depletion.

Immunoglobulin and T Cell Receptor Gene Rearrangements in Acute Leukemias

Acute leukemias are commonly classified on the basis of morphologic, cytochemical and immunologic criteria. Immunologic techniques, particularly surface antigen analysis using monoclonal antibodies, have related leukemic cells to normal cell counterparts [1]. In general, lineage fidelity is maintained in leukemic blast cells. However, there are exceptions [2, 3]. Recently, molecular analysis using specific Ig and T cell receptor gene probes has become a valuable addition to morphologic, cytochemical and surface marker studies in determining lineage, clonality, and differentiation of leukemic cell populations [4, 5]. We therefore investigated the diagnostic potential of molecular analyses (a) to develop an understanding of the cellular origin of leukemias which cannot be allocated to one of the defined myeloid or lymphoid lineages by cellular phenotypes, and (b) to reveal the clonal development of phenotypic conversion in acute leukemia.

Use of Two Monoclonal Anti-Human B Cell Antibodies in the Study of Early B Cell Differentiation

UL-38 and UL-90 were raised against fresh CALLA+, cµ - leukemia cells. Details of the fusion protocol and properties of the antibodies are described elsewhere (1). Briefly somatic cell hybridization and growth of hybridomas UL-38 and UL-90 was performed essentially as described by Kohler and Milstein (2). As fusion partners PAI-O BALB/c myeloma cells (3), a nonproducing subclone of the mouse myeloma cell P3x63Ag8, were used (obtained from Dr. J.W. Stocker, Basel). mAbs BA-1 (4), BA-2 (5), and BA-3 (5) were obtained from Hybritech Inc., San Diego, CA, B1 (6) and J5 (7) from Coulter, Hialeah, FL, and VIL-A1 (8) was a kind gift of Dr. W. Knapp, Vienna. All antibodies were used at a concentration of 1 µg/ml.