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The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus β-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherpay was not as good as intensive chemotherapy for maintenance.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

Alkyl-lysophospholipids (ALP) are analogues of 2-lysophos-phatidylcholine that have been reported to have selective antitumor activity. These compounds could potentially be useful in purging bone marrow of leukemic cells in autologous marrow transplantation in acute leukemia. To determine the efficacy of pharmacological purging by ALP, we have designed a human assay system to mimic the conditions expected in the clinical setting of autotransplantation using remission marrow. A simulated remission marrow (SRM) was prepared by mixing normal marrow cells and HL60 cells in a ratio of 1,000:1. The effect of cryopreserva-tion on ALP-treated normal, HL60, and SRM cells was examined. In separate experiments, ALP significantly reduced the number of clonogenic HL60 cells with no effect on normal marrow progenitors. The effect of ALP was more apparent after cryopreservation. Incubation of HL60 cells with 50 μ/mL ALP for four hours followed by cryopreservation resulted approximately in a 3 log reduction of clonogenic HL60 cells. ALP also selectively purged the small number of leukemic cells from SRM. In SRM, the data suggested that ALP had indirect cytotoxic activity on leukemic cells by enhancing the cytotoxic activity of monocytes in addition to its direct effect. We found no evidence that clonogenic HL60 cells decreased because of induction of differentiation by ALP. These data indicated that treatment of marrow cells with ALP offers an efficient means to eliminate leukemic cells from the graft.

Two major types of lymphocytosis of large granular lymphocytes (LGLs) were observed. The proliferating LGLs in each type had distinct immunophenotypes, functional characteristics, and probably belonged to different cell lineages. The more common form (Type A) consisted of cells derived from the T cell lineage and had the T suppressor/cytotoxic phenotype (T11+, T3+, T8+). The expression of the Leu 7 and HLA-DR antigen was variable. These cells did not have natural killer (NK) function but showed a variable degree of antibody-dependent cell-mediated cytotoxic (ADCC) activity. Neutropenia was invariably present and rheumatoid arthritis and autoantibodies were frequent associations. These lymphocytes had many similarities to the major type of LGLs present in normal adult bone marrow. The other type of LGL lymphocytosis (Type B) consisted of cells lacking the antigens T3 and T8 but expressing M1 and NKH1. These cells possessed strong NK and ADCC activity but their cell lineage was not clear. Neutropenia and autoimmune phenomena were not detected. The cytochemical characteristics of the LGL granules from both types of patients were similar but differences in ultrastructure were observed. LGLs from Type B patients proliferated in the presence of Interleukin 2 (IL-2) and 12-O-tetradecanoyl-phorbol-13 acetate (TPA). Significant changes in their basic T11+, T3–, T8– phenotype were not observed. IL-2 and TPA, however, had profound influence on the NK function of the cells with enhancement in the case of IL-2 and marked suppression when stimulated by TPA.

We have studied the effect of recombinant human hematopoietic growth factors (interleukin-3 [rhlL-3], granulocytemacrophage colony-stimulating factor [rhGM-CSF], and granulocyte CSF [rhG-CSF]) on the clonal growth of human colon adenocarcinoma cell lines HTB-38, CCL 187, and WiDr (CCL 218). The factors stimulated clonal growth of HTB-38 and CCL 187 in a capillary modification of the human tumor clonogenic assay in agar up to twofold. There were dose-response correlations over a range of 1 to 10,000 U/mL for rhlL-3, rhGM-CSF, and rhG-CSF. Incubation with neutralizing monoclonal antibodies abolished the stimulation of clonal growth by rhGM-CSF. The WiDr cell line was nonresponsive to rhlL-3 and rhGM-CSF. These results represent the first evidence that a variety of hematopoietic growth factors can stimulate the growth of clonogenic cells of some nonhematopoietic malignant cell lines in vitro. © 1989 by Grune & Stratton, Inc.

A 43-year-old male with a phenotypically homogeneous, expanded subset of T cells presented in 1981 with anemia and neutropenia. The surface antigen phenotype of 99% of the peripheral blood lymphocytes was T3+, T8+, T4-, and they were morphologically large granular lymphocytes (LGL). The same cells comprised 37% of the marrow nucleated cells. Eight months after he presented, the peripheral blood T8+, LGL diminished spontaneously, and the anemia and neutropenia completely resolved. The patient remains hematologically normal as of October 1984. To determine if the T8+, LGL represented a clonal expansion, DNA from peripheral blood lymphocytes collected and cryopreserved when the patient was neutropenic and anemic, and when he was hematologically normal, was analyzed for clonal T-cell antigen receptor gene rearrangements. Using Southern blot analysis, a clonal DNA rearrangement was demonstrated, and this clone diminished but was still demonstrable in peripheral blood lymphocytes collected in 1984. The above observations implicate the expanded T8+, LGL in the pathogenesis of the neutropenia and anemia, yet the exact mechanism remains to be elucidated.

T-γ lymphoproliferative disease (T-γ LPD) is a chronic disorder of mature T cells that is associated with neutropenia and autoimmune phenomena. Although the progression of the lymphoproliferation is indolent, it is often associated with a monoclonal proliferation of T-cell-type large granular lymphocytes (LGL) that manifest multiple in vitro suppressor and cytotoxic activities. We considered the possibility that the granulocytopenia or anemia might represent an autoimmune disorder mediated by the monoclonal LGL via T-cell receptor (TCR) recognition of an antigen involved in hematopoiesis. Therefore, in an effort to characterize the usage of the TCR α-and β-chain genes in patients with T-7 LPD, we cloned and sequenced TCR α-and β-chain mRNAs derived from the T-cell type LGL of five patients. The five patients studied did not use a common Vαnor a common Jαsegment. However, an unusual finding was observed in one of the patients where the occurrence of a single variable-diversity-junctional (VDJ) rearrangement of the βchain confirmed the monoclonal origin of the LGL proliferation. In accord with this evidence for monoclonality, many of the cells studied used a common Vα(Vα19.1). In contrast to this common Vαusage, there was a marked diversity of the Jαsegments and N-region addition that were associated with the Vα19.1 segment. This pattern of common Vαusage associated with different N and Jαsegments suggests an immune-mediated selection process affecting the TCR αchain occurring after the transformation event that established the clone. We suggest that the T-cell-type LGL malignant clone might have developed autoreac-tivity conferred by the selected TCR αchain and that this autoreactivity might be implicated in this patient’s anemia. This is a US government work. There are no restrictions on its use.

Human lactoferrin (LF) has been reported to inhibit in vitro granulopoiesis by means of decreasing colony-stimulating activity production by monocytes. We performed a series of experiments to determine if the reported experimental results could be replicated using highly purified murine LF and murine target cells. Three different types of experiments were performed. (1) Medium was conditioned by lung, femoral shaft, and adherent peritoneal cells in the presence and absence of LF, and the granulopoietic stimulating activity in the conditioned media was assayed by means of a 7-day agar colony assay and a 3-day liquid slide chamber assay, which quantitates 3H-TdR incorporation into DNA. (2) In cultures stimulated by an underlayer of adherent peritoneal cells, marrow cell colony formation in agar was determined after 7 days of culture in the presence or absence of LF. (3) LF was added to 3-day liquid marrow cell cultures that had been stimulated by lung or femoral shaft conditioned media. In all experimental situations, highly purified, iron-saturated LF in concentrations up to 107Mhad no effect on in vitro granulopoiesis. These results do not support LF’s reputed regulatory role in granulopoiesis.

An unusual case of an aggressive leukemia of natural killer (NK) cells occurred in a 65-year-old male. Clinical characteristics of this case included hepatosplenomegaly, ascites, marrow infiltrate with leukemic cells, and a WBC up to 82.8 × 109before therapy. One year before his presentation he had been noted to have a WBC of 12.1 × 109with 78% lymphocytes, and 6 months before had noted intermittent fever and weight loss. He and his brother had well documented hereditary cold urticaria. The patient was treated with a modification of ProMACE CYTABOM regimen and had prompt regression of the leukemia with associated acute tumor lysis. Renal, hepatic, and marrow failure predominated during a terminal course that ended 22 days after therapy was commenced, and at autopsy there was no evidence for leukemic cell infiltrate in the liver, spleen or marrow. The leukemic cells were large granular lymphocytes by light and electron microscopic criteria, and had the following immunophenotype: CD2 +, DR +, Leu7 +, NKH1 +, CD11+, CD3-, CD5-, CD4-, CD8-, CD16-. The cells displayed high antibody-dependent cell-mediated cytotoxicity (ADCC) and NK activity, and had a high rate of spontaneous proliferation in vitro that was not augmented by phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). Southern analysis of DNA from leukemic cells revealed normal germline arrangements for the βand γchains of the T cell antigen receptor and immunoglobulin heavy chain genes. The majority of metaphases were clonally abnormal revealing consistent rearrangements involving extra material attached to the long arms of chromosomes 5 and 11.

The status of the T cell receptor β and  γ genes in natural killer (NK) cells was investigated in two patients with a marked expansion of CD2 +, CD3- NK cells. Both genes were found to be in the germline state. The Tαand complete Tβgene transcripts were not detected, but a 1.0-kilobase Tβgene transcript could be demonstrated at low levels in freshly isolated cells and at a much higher level in interleukin-2 (IL-2)-cultured cells. The transcript coding for the δ chain of the CD3 complex was also absent. These cells were cultured in IL-2 with or without the addition of the differentiation-inducing agents: retinoic acid, N,N-hexamethylene bisacetamide, and sodium butyrate. The cultured cells retained their NK activity except in culture with sodium butyrate at ≥1 mmol/L. Expression of CD3 or other T cell surface markers by the NK cells was not observed in these cultures. Either CD2 +, CD3- NK cells are derived from a non-T lineage, or they have diverged from the T cell lineage earlier than the stage of Tγ gene rearrangement and CD3 δ chain expression; they are refractory to many induction signals in undergoing further T cell differentiation.

A liquid culture system employing slide chambers was developed to facilitate the study of proliferation and differentiation of mouse neutrophilic granulocytes and cells of the monocyte-macrophage series. Cultures were initiated with 1-2.5 × 104light density marrow cells which had been fractionated on Ficoll-Hypaque. In the presence of colony-stimulating activity (CSAHU), two types of clusters were observed. One was tight, spheroidal, and composed of neutrophilic granulocytes, while the other was a loose grouping of flattened cells of the monocyte-macrophage series. The slide chamber culture is adaptable to microscopic assay techniques (e.g., cluster size, autoradiography, immunofluorescence) as well as quantitative biochemical methods (e.g., rate of 3H-thymidine incorporation, DNA quantitation). We have demonstrated both a shortening of the generation time of granulocytes in tight clusters and increasing rates of 3H-thymidine incorporation into DNA, with a corresponding increase in total culture DNA as a function of CSAHUconcentration. Granulocytic differentiation in the tight spheroidal clusters has been demonstrated by histochemical stains and immunofluorescence of an antibody to a marker protein specific for the secondary granule of the neutrophil (lactoferrin).

A randomized trial was conducted comparing ketoconazole and nystatin in the prevention of oral candidiasis and appearance of invasive fungal infections in 51 neutropenic leukemic patients undergoing induction chemotherapy. Ketoconazole was administered in a 200 mg dose twice daily. Nystatin oral suspension was given in doses of 500,000 units four times daily. Surveillance cultures of the throat and urine were obtained prior to treatment and conducted weekly. Patients were enrolled in the study if the absolute granulocyte count was less than 1500μl, if physical examination revealed no evidence of oral candidiasis, no evidence of urinary tract infection, and there was no pulmonary infiltrate on chest x-ray. Patients were continued on study until the absolute granulocyte count reached 1500μl, evidence of oral candidiasis appeared, or presumed or proven invasive fungal infections appeared. Of the 46 evaluable patients, 22 received ketoconazole, 3 (14) developed oral candidiasis, and 5 developed suspected systemic fungal infections (23). Of 24 patients who received nystatin, 4 (17) developed oral candidiasis and 8 (33) developed systemic fungal infections, 4 proven and 4 suspected. Significantly more patients on the nystatin arm progressed to invasive fungal infections. Ketoconazole was not superior to nystatin in reducing the frequency of oral candidiasis but possibly reduced the frequency of invasive fungal infections.