Early recovery of aggressive cytotoxic cells and improved immune resurgence with post-transplant immunotherapy for multiple myeloma.

A phase I/II trial evaluated early administration and dose escalation of interleukin (IL)-2 with granulocyte macrophage colony stimulating factor (GM-CSF) post-transplant. Following melphalan (200 mg/m(2)) and an autologous transplant, IL-2 was initiated (day 0) and continued for 4 weeks. GM-CSF (250 mcg/m(2)/day) began on day 5. Fifteen of 19 patients completed therapy. No treatment-related deaths occurred. IL-2 (1 x 10(6) IU/m(2)/day) was not tolerated in two of six patients due to > or =grade 3 fatigue/diarrhea (n=1) or supraventricular tachycardia (n=1). The maximum tolerated dose of IL-2 was 6 x 10(5) IU/m(2)/day; this dose was well tolerated by 11 of 13 patients. Neutrophil and platelet engraftment occurred on day 13 (median; range 10-17 days) and day 13 (median; range 0-74 days), respectively. When compared to control patients, there was a marked increase in the number of CD3+ T cells (P=0.005), CD4+ T cells (P=0.01), CD8+ T cells (P=0.001) and CD4+CD25+Treg cells (P=0.015) post-transplant. Cytotoxicity directed against myeloma cells was markedly increased when compared to control patients (P=0.017). This unique trial design using early administration of IL-2 with GM-CSF during the period of lymphodepletion, demonstrated a marked increase in the number and function of early cytotoxic effector T cells, without suppression of engraftment.

Differential expression of CD 180 and IgM by B-cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes.

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

Amplification of the MLL region in acute myeloid leukemia.

We report amplification of the MLL gene region (11q23-->11qter) in a 72-year-old woman with myelodysplastic syndrome progressing to acute myelomonocytic leukemia and in a 51-year-old man with a history of hairy cell leukemia and secondary myelodysplasia progressing to acute myelogenous leukemia. The amplicons containing MLL were shown by molecular cytogenetics to extend from chromosomal region 11q23 to the distal long arm of chromosome 11 and to be present in the first patient in five copies on a large ring chromosome and present in the second patient also in five copies on two derived chromosomes. Other karyotypic findings in the first patient included del(5q), +8, and der(21)t(17;21), resulting in the loss of a copy of 17p, whereas deletion 7q was observed in the second patient. Southern-blot analysis for the second patient was consistent with MLL amplification but did not demonstrate rearrangement of the germ-line MLL band. Amplification of MLL and the 11q23 region has been documented in only a few cases and appears to be yet another mechanism by which MLL contributes to the leukemia phenotype.

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma.

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.

Mature polymorphonuclear leukocytes express high-affinity receptors for IgG (Fc gamma RI) after stimulation with granulocyte colony-stimulating factor (G-CSF).

The high-affinity receptor for the constant region of immunoglobulin G IgG (Fc gamma RI; CD64) is virtually undetectable on mature polymorphonuclear neutrophils (PMNs) in healthy individuals but is expressed on PMNs in patients with certain infections and in patients treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). The induction of Fc gamma RI by rhG-CSF has previously been reported to result from effects on immature granulocyte progenitors. To evaluate the G-CSF effect on mature PMNs, we studied the correlation between G-CSF plasma concentration and expression of Fc gamma RI on PMNs in vivo as well as the effect of G-CSF on Fc gamma RI expression on mature PMNs in vitro. Fc gamma RI expression on PMNs correlated (R = 0.79; p < .001) with plasma concentrations of endogenous or recombinant G-CSF in healthy volunteers and in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation. PMNs exhibited a unimodal distribution for elevated Fc gamma RI expression, suggesting that G-CSF induced increased expression of Fc gamma RI on mature as well as on immature PMNs. In vitro, incubation of mature PMNs with G-CSF induced mRNA for Fc gamma RI. Significant Fc gamma RI surface expression was induced in a time- and dose-dependent manner. Thus, G-CSF can act on mature PMNs to increase Fc gamma RI expression and may be useful for stimulating antibody mediated immune functions of PMNs in vivo.

A new in situ hybridization technique for spliced RNA species documents the bone marrow origin of pulmonary macrophages in chronic myelogenous leukemia.

Tissue macrophages derive from monocytes of bone marrow origin. Because monocytes from patients with chronic myelogenous leukemia (CML) contain the Philadelphia chromosome (Ph), it seemed probable that tissue macrophages in CML would originate from the malignant clone. Using powerful molecular techniques, we studied pulmonary alveolar macrophages (PAM) from two patients with CML. PAM from Patient 1, a patient in chronic phase studied before bone marrow transplantation (BMT), contained the Ph by Southern blot analysis. Patient 2, an accelerated phase patient, was studied after post-BMT relapse. PAM from this patient not only contained the Ph, but also expressed the BCR/ABL message documented by a new splice junction in situ hybridization technique. This new technique allows detection of BCR/ABL mRNA and determination of splice useage in individual cells. These data confirm the continued replenishing of PAM from peripheral blood monocytes in non-BMT settings and represent the first direct evidence that tissue macrophages are derived from the malignant clone in patients with CML.

bcr/abl mRNA detection following bone marrow transplantation for chronic myelogenous leukemia.

We have utilized the polymerase chain reaction (PCR) to sensitively detect persistence of the chronic myelogenous leukemia (CML) malignant clone and to study bcr/abl mRNA splicing patterns following bone marrow transplantation. Thirteen of sixteen patients displayed persistent malignant cells during post-BMT clinical remission. In two patients bcr/abl mRNA was detected 4 and 9 months prior to clinical relapse. In eleven of fourteen patients in continued clinical remission malignant cells were detected post-BMT. Ten of these eleven patients were also cytogenetically normal. Seven patients have lost all evidence of bcr/abl transcript, but only at 1-2 years posttransplant, while four have shown persistence of the bcr/abl transcript from 28 days to 3 years post-BMT and one has converted from an initially negative result at 1 year post-BMT to detectable levels of chimeric mRNA at 2 years. Thus, 8/9 patients tested at or before 6 months, 7/12 at 1 year, and 3/10 at 2 years showed persistent detectable CML cells. Intriguingly, mRNA splicing patterns changed in 5 patients following BMT, with complete loss of mRNA containing bcr exon 3 (n = 2) or new appearance of mRNA not containing bcr exon 3 (n = 2). A single patient transiently lost evidence of bcr exon 3 expression while persistently expressing the bcr exon 2/abl exon 2 splice. Our data suggest that the majority of patients harbor small numbers of malignant cells following transplantation, and that such persistence may not inevitably predict clinical relapse. Complete elimination of the malignant CML clone post-BMT may rely on immunological mechanisms (e.g., graft-vs-leukemia).

Use of a somatostatin analogue, octreotide acetate, in the management of acute gastrointestinal graft-versus-host disease.

PURPOSE: Because the secretory diarrhea of acute graft-versus-host disease (GvHD) of the gut induces serious metabolic and nutritional disturbances, this study was initiated to assess the use of a somatostatin analogue, octreotide acetate, as adjunctive therapy for severe GvHD of the gut with massive diarrhea. PATIENTS AND METHODS: In a pilot study, six patients with biopsy-confirmed acute gut GvHD after allogeneic bone marrow transplantation received octreotide 50 to 250 micrograms three times a day subcutaneously. RESULTS: Three of the six treated patients had a prompt and dramatic reduction in stool volume within 1 to 3 days of initiation of octreotide therapy. CONCLUSIONS: Somatostatin and its analogues have been used successfully in diarrheal states by antagonism of neuropeptide overproduction, although other potential therapeutic mechanisms include inhibition of fluid secretion, enhanced salt absorption, and inhibition of gut motility. Somatostatin and its analogues may be promising adjunctive agents in the treatment of gastrointestinal GvHD, although assessment in a controlled trial will be required to confirm their therapeutic efficacy.

The bowel bypass syndrome: a response to bacterial peptidoglycans.

A characteristic intermittent neutrophilic dermatosis, associated with polyarthritis, tenosynovitis, malaise, fever, and cryoglobulinemia, occurs in 20% of patients who undergo ileojejunal bypass surgery for the treatment of morbid obesity. The clinical syndrome may mimic gonococcal sepsis. The histologic changes in the skin are those of Sweet's syndrome. The syndrome remits spontaneously in most cases, but it may recur intermittently over a period of years. Treatment with low-dose steroids, tetracycline, or metronidazole suppresses symptoms in most cases, and restoration of normal bowel anatomy is curative. Skin testing with Streptococcus pyogenes antigen causes an excerbation of symptoms, or may provoke the entire syndrome de novo. Bacterial peptidoglycans, especially those of group A streptococci, produce similar arthritis and skin lesions in animal models. Peptidoglycans from numerous intestinal bacteria share common structural and antigenic features with S. pyrogenes peptidoglycan and are suggested as causative of the toxic and immunologic features of this syndrome.