Tom Grunberger

Detection of Residual Acute Lymphoblastic Leukemia Cells in Cultures of Bone Marrow Obtained during Remission

We used a semisolid culture assay to quantitate leukemia cells in the bone marrow of patients with childhood acute lymphoblastic leukemia (ALL). In bone marrow cultures from 40 patients with newly diagnosed disease, the colonies that developed in vitro consisted of lymphoblasts with the same surface markers and abnormal karyotype as the original diagnostic marrow specimens. We also studied marrow cultures from 13 patients in chemotherapy-induced remission; 6 of these, including 1 obtained from a patient during successful engraftment after marrow transplantation, also yielded lymphoblast colonies in culture, with the same immunologic phenotype or abnormal karyotype as the original leukemic marrow. Four of these patients, including the one who underwent marrow transplantation, relapsed within 2 to 30 months of the abnormal cultures; the other two are still in remission, one of them 30 months after diagnosis. Bone marrow cultures from eight normal controls and from the other seven patients in remission did not yield lymphoblast colonies; all seven of the latter are still in remission. This assay appears to allow detection of small numbers of residual leukemic cells. We conclude that the technique will be valuable in monitoring the efficacy of chemotherapy and allogeneic bone marrow transplantation in acute lymphoblastic leukemia, as well as in evaluating the quality of purged marrow for autologous marrow transplantation.

Central role of tumour necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia

Summary. In previous studies on patients with juvenile chronic myelogenous leukaemia (JCML), we found excessive proliferation of malignant monocyte‐macrophage elements in the absence of exogenous growth factor, and impaired growth of normal haematopoietic progenitors. In the current study, six newly‐diagnosed JCML patients were investigated to characterize the disease further. In co‐cultures, JCML cell culture supernatant as well as patient plasma obtained at diagnosis produced a striking reduction in numbers of control marrow BFU‐E, CFU‐GM, CFU‐Meg and CFU‐GEMM colonies. Monoclonal anti‐tumour necrosis factor alpha neutralizing antibodies (anti‐TNF‐α Ab) abolished these inhibitory properties. In sharp contrast, JCML supernatants exerted a marked growth‐promoting effect on autologous JCML cells cultured in clonogenic assays. Anti‐TNF‐α Ab and anti‐granulocyte‐macrophage colony‐stimulating factor neutralizing antibodies (anti‐GM‐CSF Ab) both reversed the stimulating effect. Recombinant GM‐CSF and recombinant TNF‐α produced a profound increase in JCML colonies when tested individually and anti‐GM‐CSF Ab reversed the TNF‐α effect. Expression studies of TNF‐α and TNF‐α receptor genes of cultured JCML cells demonstrated mRNAs for both. Further, TNF‐α activity was assayed in a wide variety of cell culture supernatants and in normal and patients’plasma, and only the JCML specimens showed increased TNF‐α values. Recombinant interleukin‐1 alpha (IL‐1α) also stimulated JCML colony growth, but polyclonal anti‐IL‐1 neutralizing antibodies did not suppress JCML colony numbers nor did it reverse the effects of TNF‐α or GM‐CSF. The evidence indicated that the JCML monokine which inhibits normal haematopoiesis is TNF‐α and that the endogenously‐produced TNF‐α and GM‐CSF from JCML cells play an important role in the pathogenesis of the disease by acting as autocrine growth factors. IL‐1α also stimulates JCML cell proliferation as an accessory factor and augments the effect of GM‐CSF, TNF‐α or both.

Interleukin 6 induces myeloid differentiation of a human biphenotypic leukemic cell line.

The human leukemic cell line B1, is characterized by a specific 4;11 chromosomal translocation, immature myeloid/pre-B biphenotypic features, expression of multiple cytokine receptors and IL-1-dependent autocrine growth regulation [Cohen et al. (1991) Blood 78, 94]. Exposure of B1 cells to low concentrations of IL-6 abolished the leukemic cells ability to form colonies in semi-solid medium and slowed down their proliferation rate in suspension. Associated with these changes in growth characteristics, the B1 cells differentiated along the myeloid lineage as judged by the induction of the myeloid-specific surface antigens CD33, CD13 and CD11b, as well as histochemical and morphological changes characteristic of myeloid cells. The induction of differentiation was specific to IL-6 since none of the other cytokines which inhibited B1 cell growth (IL-7, gamma IFN and TNF alpha) were able to induce myeloid or lymphoid differentiation in these cells. The IL-6-induced differentiation was completed over a two week period and was essentially irreversible. Together with the phenotypic changes, IL-6 induced the expression of the protein tyrosine phosphatase (CD45) which may be associated with altered growth observed in IL-6-treated cells. Induction of terminal differentiation of leukemic cells by recombinant bioregulators has therapeutic implications and merits further study.

809 DETECTION AND CHARACTERIZATION OF ERYTHROPOIETIC INHIBITORS IN ANEMIA OF CHRONIC RENAL FAILURE

The mechanism of anemia in pts with end-stage renal disease was studied by assessing erythroid colony growth in methylcellulose cultures. Peripheral blood BFU-E from 10 anemic pts were normal when cultured in control serum (mean 22 BFU-E/105, range 14-41 vs control mean 18±10/105), but declined a mean of 67% when autologous uremic serum was substituted. Sera from 53 of 60 pts cultured with control marrow produced a mean decrease in BFU-E of 74% and in CFU-E of 79%. The serum inhibition was confirmed by reproducing the effect on control marrow colony growth with specific fractions of pts serum separated by sephacryl gel chromatography. Neither peritoneal dialysis nor hemodialysis reduced the inhibitory activity, but it disappeared with successful renal transplantation. Analysis of uremic serum revealed a striking increase in a ribonuclease of M.W. 33,000 in all pts (9,500-40,000u/ml vs control mean 1,047±247u/ml) that was not eliminated by therapeutic dialysis. Purified ribonuclease produced dose-dependent inhibition of control marrow CFU-E but had unpredictable effects on BFU-E. We conclude that in anemic uremic pts: erythroid progenitors are adequate; the serum contains erythropoietic inhibitors; a ribonuclease is increased and appears to have a role in the erythropoietic suppression.

903 JUVENILE CHRONIC MYELOGENOUS LEUKEMIA (JCML) CHARACTERIZATION OF THE DISEASE AND PATHOGENESIS USING CELL CULTURES

The pathogenesis of JCML was studied in 9 pts. Using cell cultures and chromosome markers, marrow and peripheral blood consistently showed 2 features: impaired expression of normal hematopoietic progenitors (CFU-E, BFU-E, CFU-GEMM), and excessive clonal proliferation of monocyte-macrophage elements whose growth was independent of added CSA or an adherent cell fraction. In contrast, 5 adult CMLs (Phl+) showed normal hematopolesis in vitro and CSA-dependent CFU-C growth similar to controls. Using monoclonal antibodies, cloned JCML cells were positive for surface antigens Ia, Mo2, LeuM1, LeuM3, and OKM1, thus confirming monocytic lineage. Characterization studies on cloned cell populations revealed a wide spectrum of features, some mature (latex ingestion, non-specific esterase positive, sensitivity to growth inhibition by PGE2 and to gene-cloned interferon), and some primitive (negative for lysozyme, β-glucuronidase, procoagulant activity, and plasminogen activator). Functionally, JCML cells markedly impaired hematopoiesis in vitro from normal marrow; thus, co-cultures of normal marrow with JCML adherent cells, or fresh JCML marrow, or JCML plasma, resulted in suppressed normal colony formation. We conclude that JCML is a malignant clonal disorder of monocytic lineage, and that cell cultures provide a reliable and specific diagnostic test. The mechanism of hematopoietic failure in JCML is likely mediated by an inhibitory monokine secreted by JCML cells.