Abnormal function of B lymphocytes from peripheral blood of multiple myeloma patients. Lack of correlation between the number of cells potentially able to secrete immunoglobulin M and serum immunoglobulin M levels.

Multiple myeloma patients are deficient in normal polyclonal serum immunoglobulins. To determine the reasons for this decrease, we quantitated and compared the number of surface IgM+ B lymphocytes, and the number of B cells susceptible to transformation by Epstein-Barr virus (EBV) with the concentration of IgM in serum. Serum IgM levels varied considerably in individual patients, temporally shifting from undetectable to normal amounts and then dropping again to undetectable levels. A transient rise to normal serum IgM concentrations was seen in 42% of patients assessed at two or more time points. Of 44 patients, 52% showed a lack of correlation between the number of surface IgM+ (sIgM+) B cells and serum IgM concentration. One subset of patients (25%) had detectable to normal numbers of sIgM+ B cells in blood but undetectable levels of serum IgM. Transformation of B cells from these patients indicated a block in IgM secretion that was extrinsic to the B cells that were fully able to transcribe, translate, and secrete IgM after EBV transformation. A second subset of patients (27%) had undetectable numbers of sIgM+ B cells but near normal levels of serum IgM, suggesting abundant secretion by few clones of B cells. Of 18 patients with monoclonal gammopathy of undetermined significance (MGUS), 26% showed a lack of correlation between the numbers of sIgM+ B cells and serum IgM concentration. We suggest that in patients with multiple myeloma, and in some with MGUS, there exists a mechanism(s) extrinsic to the B cell that mediates an arrest in terminal B lymphocyte maturation.

Severe deficiency of B lymphocytes in peripheral blood from multiple myeloma patients.

A major problem in the assessment of circulating B lymphocytes in multiple myeloma is the extent to which cells with passively absorbed Ig contribute to the assay. We have analyzed peripheral blood B cell numbers in 51 patients in various treatment categories by using an assay that is not subject to artifacts involving cytophilic Ig. We have defined a B lymphocyte by three different criteria (a) expression of a high surface density of Ig (b) expression of a high density of HLA.DR and (c) expression of a marker exclusive to surface Ig+ B cells. By these criteria, normal individuals have an average of 6% B cells. In multiple myeloma patients, B cell levels in purified mononuclear cell preparations are severely reduced. Untreated patients and the majority of patients on intermittent chemotherapy have 20-600-fold fewer B cells than do normal donors (average = 0.3%). This decrease was even greater in whole blood of patients as compared with normal donors (100-1,000-fold fewer B cells). The number of B cells did not correlate with disease status or paraprotein concentration. We found no evidence to support the idea that B lymphocytes in patients include a substantial monoclonal subset.

Hemopoietic origin of factor XIII A subunits in platelets, monocytes, and plasma. Evidence from bone marrow transplantation studies.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic contributions to FXIIIA in these components were studied in patients transplanted with marrows from donors with different FXIIIA phenotypes. In three patients with successful engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA in platelets and monocytes is synthesized de novo and/or from their progenitor cells. Plasma FXIIIA phenotype change after transplantation was more complex. Patient I changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115 d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but did show enrichment of the fastest band. Thus, while there is a definite contribution of donor hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to delay or prevent the phenotype change.

Addition of low-dose folinic acid to a methotrexate/cyclosporin A regimen for prevention of acute graft-versus-host disease.

A study was performed to determine whether the addition of folinic acid to a combination of methotrexate (MTX) and cyclosporin A (CsA) after allogeneic bone marrow transplantation (BMT) could improve tolerance to the regimen without inhibiting its ability to prevent graft-versus-host disease (GVHD). Sixty-nine adult BMT patients received CsA plus MTX 15 mg/m2 on day 1 and 10 mg/m2 on days +3, +6 and +11. Folinic acid 5 mg was started 24 h after each MTX dose and continued 6 hourly until 12 h before the next dose of MTX. The median age of the group was 37 years and 13 patients (19%) received bone marrow from mismatched and/or unrelated donors. No MTX doses were omitted or modified. Grade II-IV acute GVHD occurred in 18 patients (29%) and chronic GVHD in 35 of 56 (64%) patients at risk. There were no cases of grade > or = III stomatitis. Transplant-related mortality was 7% before 100 days and 20% overall (9% for low risk leukaemia) with a median follow-up of 41 months (range 24-88 months). This regimen of folinic acid rescue may contribute to a well tolerated GVHD prophylaxis protocol with reasonably low BMT-related mortality. Our results suggest that the ability of MTX to prevent acute GVHD is not abrogated by folinic acid given in this way.

Treatment of advanced Hodgkin's disease with high dose melphalan and autologous bone marrow transplantation.

Twenty patients with Hodgkin's disease which had relapsed at least once after chemotherapy, were treated with melphalan 140-220 mg/m2 i.v. followed by reinfusion of non-cryopreserved autologous bone marrow. Four patients (20%) remain alive and disease-free 28, 45, 52, and 96 months after treatment respectively. There were no treatment-related deaths. This appears to be the only reported series of patients treated with a single agent in this situation. The results are comparable to those achieved by multi-agent regimens with autologous or allogeneic bone marrow transplantation.

Superior autologous blood stem cell mobilization from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-CSF than from less intensive chemotherapy regimens.

The study purpose was to determine if G-CSF plus dose-intensive cyclophosphamide 5.25 g/m2, etoposide 1.05 g/m2 and cisplatin 105 mg/m2 (DICEP) results in superior autologous blood stem cell mobilization (BSCM) than less intensive chemotherapy. From January 1993 until May 1997, 152 consecutive patients with non-Hodgkin's lymphoma (n = 55), breast cancer (n = 47), Hodgkin's disease (n = 14), multiple myeloma (n = 9), AML (n = 9), or other cancers (n = 18) initially underwent BSCM by one of three methods: Group 1: G-CSF alone x 4 days (n = 30). Group 2: disease-oriented chemotherapy, dosed to avoid blood transfusions, followed by G-CSF starting day 7 or 8, and apheresis day 13 or 14 (n = 82). Group 3: DICEP days 1-3, G-CSF starting day 14, and apheresis planned day 19, 20 or 21 (n = 40). A multivariate analysis was performed to determine which factors independently predicted BSCM. The median peripheral blood CD34+ (PB CD34+) cell count the morning of apheresis linearly correlated with the number of CD34+ cells removed per litre of apheresis that day. The median PB CD34+ cell count and median CD34+ cells x 10(6) removed per litre of apheresis were highest for Group 3, intermediate for Group 2, and lowest for Group 1. By multivariate analysis, mobilization group (3 > 2 > 1), disease other than AML, no prior melphalan or mitomycin-C, and less than two prior chemotherapy regimens predicted better BSCM. Out of 15 Group 3 patients who had infiltrated marrows, 11 had no detectable cancer in marrow and apheresis products after DICEP. These data suggest that DICEP results in superior BSCM than less intensive chemotherapy regimens.

Allogeneic blood stem cell and bone marrow transplantation for acute myelogenous leukemia and myelodysplasia: influence of stem cell source on outcome.

We have compared the outcomes of 87 patients with acute myelogenous leukemia (AML) and myelodysplasia (MDS) receiving matched sibling transplants with stem cells from peripheral blood (blood cell transplant, BCT) or bone marrow (BMT). In good risk patients (AML in CR1) granulocytes recovered to 0.5 x 10(9)/l a median of 14 days after BCT compared with 19 days after BMT (P < 0.0001). For patients with poor risk disease (AML beyond CR1 and MDS) corresponding figures were 16 vs 26 days (P < 0.0001). Platelet recovery to 20 x 10(9)/l was also faster after BCT (good risk 12 vs 20 days, P < 0.0001; poor risk 17 vs 22 days, P = 0.04). Red cell transfusions were unaffected by cell source, but BCT recipients required less platelet transfusions (good risk 1 vs 5, P = 0.002; poor risk 5 vs 11, P = 0.004). Blood cell transplants resulted in more chronic GVHD (86% vs 48%, P = 0.005) and a significantly higher proportion of recipients with KPS of 80% or less (48% vs 5%, P = 0.004). Disease-free survival at 4 years was 23% for both groups of poor risk patients but outcome in good risk patients was better after BCT (93% vs 62%, P = 0.047) related mainly to less relapse. While disease-free survival may be better after BCT than BMT for AML in CR1, quality of life may be relatively impaired.

Giant lymph node hyperplasia (Castleman's disease) of hyaline vascular type. Clinical heterogeneity with immunohistologic uniformity.

Of three patients with giant lymph node hyperplasia (GLNH) of hyaline vascular type, one had multicentric involvement and systemic symptoms, and had the nephrotic syndrome develop. The two others had localized adenopathy and were asymptomatic. Immunohistochemistry carried out on frozen and paraffin-embedded tissue was uniform in all cases. For the most part this revealed a polyclonal B-cell population and a reactive T-cell population in keeping with reactive follicular hyperplasia. In contrast to the latter, however, was a total absence of Leu-7 (natural killer cell) activity. The significance of this is unclear but may represent an abnormal immunologic response in the face of overwhelming antigenic stimulation. GLNH is a clinically and pathologically heterogeneous disorder, the management of which is dependent on both the clinical presentation of the patient and the histologic appearances of the involved lymph nodes.

Red blood cell alloimmunization complicating plasma transfusion.

Well-known adverse effects of plasma transfusion include viral transmission, allergic complications, and rare anaphylactic reactions. In making clinical decisions to transfuse plasma, a seldom-considered complication is that of red blood cell (RBC) alloimmunization. The authors report a patient in whom strong IgM and IgG anti-E and weak IgG anti-JKa RBC antibodies developed 15 days after infusion of two units of fresh-frozen plasma for volume expansion. These antibodies are potentially hemolytic. This case underscores the importance of considering risks of plasma infusion. Plasma should not be used casually, especially for indications for which alternate therapies, such as crystalloid and colloid solutions, are available.

Staging laparotomy in Hodgkin's disease: a community hospital experience.

Between 1977 and 1984, 50 patients with Hodgkin's disease underwent a staging laparotomy performed by nine surgeons in a community hospital. Adequate procedures were performed in 80% of cases compared to staging laparotomies done between 1969 and 1976 when only 40% were properly performed. Abdominal lymphangiogram had a false-negative rate of 0 but a false positive rate of 70%. Clinical stage III disease was significantly downstaged at laparotomy (65% of cases). Postoperative complication rate was 4% and there were no operative deaths. A subset of patients not requiring laparotomy have been identified. Because the quality of staging laparotomy and lymphangiography was variable, we encourage all centres treating patients with Hodgkin's disease to review their own experience with these techniques before making individual patient treatment decisions.

Pre-B cells in peripheral blood of multiple myeloma patients.

Although multiple myeloma is a disease of plasma cells, abnormalities have been detected in both B and T lymphocytes in peripheral blood. Although multiple myeloma patients are deficient in surface Ig (sIg)-positive B lymphocytes, analysis of lymphocytes present in blood indicates an abnormally large pool of circulating pre-B cells. These pre-B cells express BA-1, do not bear sIg, and contain cytoplasmic mu chains. High numbers of pre-B cells occur in 88% of individuals with frank myeloma and in 44% of individuals with monoclonal gammopathy of undetermined significance. Pre-B cells bearing BA-1 differ between patients in their expression of HLA-DR and receptors for peanut agglutinin (PNA). Those pre-B cells in myeloma patients are either BA-1+ PNA- HLA-DR+ (54% of patients) or BA-1+ PNA+ HLA-DR- (30% of patients), or have a mixture of phenotypes (14% of patients). Pre-B cells of the PNA- phenotype are almost always HLA-DR+, and PNA+ pre-B cells are HLA-DR-. Within the same patient, the pre-B cell population varies by both quantitative and qualitative definitions. The number of pre-B cells may increase 460-fold and temporal shifts of surface phenotype from BA-1+ PNA- to BA-1+ PNA+ or vice versa have been detected. These observations indicate an abnormality in the B lymphocyte differentiation pathway leading to pre-B cells in the periphery that vary in number and cell surface phenotype, and that are unable to express sIg.

Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia.

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.

Double autologous bone marrow transplantation for acute myelogenous leukemia in a patient treated for Hodgkin's disease.

A 30-year-old man developed acute myelogenous leukemia nearly 3 years after treatment of Hodgkin's disease with radiation and three chemotherapy combinations. Remission was induced with one cycle of high-dose Ara-C therapy. Three cycles of consolidation chemotherapy were given. The patient then had two autologous bone marrow transplants, the first after conditioning with 5 Gy total body irradiation, the second after Melphalan 140 mg/m2. The procedures were well tolerated, although hematological reconstitution was very slow after the second autotransplant. The patient has been disease-free for over 4 years. Such patients may be more vulnerable to transplant-related complications because of their previous exposure to chemotherapy and radiation, which may damage several organs including the bone marrow. This report demonstrates that patients with secondary acute myelogenous leukemia may tolerate a double autotransplant procedure and achieve durable remissions.

Humoral immune deficiency in multiple myeloma patients due to compromised B-cell function.

Patients with multiple myeloma are generally immunodeficient, with pronounced depression in primary antibody responses. We have attempted to delineate the reasons for the humoral immunodeficiency by analyzing the specificity repertoire of the surface immunoglobulin (Ig)-positive B cells in patients with multiple myeloma or monoclonal gammopathy of undetermined significance (MGUS), in comparison with normal donors. B lymphocytes from 26 patients with multiple myeloma, 12 patients with MGUS, and 8 normal donors were transformed with Epstein-Barr virus (EBV) and cultured at limiting dilution for clonal analysis. The Ig secreted by each clone was analyzed for class and anti-tetanus toxoid (TT) specificity to determine the frequencies of IgM, IgG, anti-TT IgM, and anti-TT IgG antibody-secreting clones. Our objective was to establish whether the inability to mount humoral responses to common environmental pathogens was due to a lack of specific B cells or to inhibition of B-cell function. Our results indicate that the quantitative B-cell deficiency in patients was due to a nonrandom loss of selected sets of B cells. Although most patients had a reduced aggregate number of B cells, the number of TT-specific B cells was normal. There was, on average, a threefold increase in the proportion of the B-cell specificity repertoire devoted to recognition of TT. Forty-four percent of the patients with MGUS were also affected. In addition, the TT-specific B cells in multiple myeloma patients were severely compromised in their ability to secrete antibody or to differentiate to antibody-secreting cells in vivo. This arrest in differentiation appears to be extrinsic to the B cells, as they were fully able to secrete anti-TT antibody after transformation and culture in vitro. We postulate the existence of an autoimmune inhibitory network mediating the arrest in B-cell differentiation and the humoral immune deficiency.

Expression of multiple beta 1 integrins on circulating monoclonal B cells in patients with multiple myeloma.

We have previously reported the presence of monoclonal, tumor-related B lineage cells in the blood of myeloma patients. The cells are continuously differentiating, and the majority are at a very late stage of B cell differentiation into plasma cells, consistent with the hypothesis that they comprise a precursor cell subset responsible for disseminating and possibly for relapse of the disease. The pattern of beta 1 integrin expression on monoclonal B lineage cells from blood and bone marrow of myeloma patients was evaluated using multiparameter flow cytometry in comparison to normal blood or tissue B cells and malignant B cells from B-CLL, B lymphoma, or plasma cell leukemia. The alpha 4 and beta 1 chains were found on the majority of normal B cells, usually with a higher expression of alpha 4 compared to beta 1. alpha 5 was detectable at low density on B cells from lymph node, bone marrow, and lamina propria. the alpha 2 and alpha 6 chains are absent on B cells localized in normal lymphoid tissues as well as on normal blood B cells and in vitro activated B cells. In myeloma, the blood B cells express alpha 2, alpha 5, and alpha 6, suggesting important functional differences between these tumor-related B cells and their normal counterparts. The plasma cells located in myeloma bone marrow express no alpha 2, and almost no alpha 6, although they have variable expression of alpha 4, alpha 5, and beta 1. Thus the end-stage plasma cells appear to lack receptors that would support a propensity for invasion of basement membranes and exit to extravascular spaces. In contrast, the circulating plasmablasts in a patient with plasma cell leukemia make up a large subset of early plasma cells expressing all integrin receptors analyzed, including alpha 2 and alpha 6. Malignant cells from B-CLL and B lymphoma express only the alpha 4 and beta 1 integrins, and some B-CLL have very low levels of alpha 3, but no alpha 2, alpha 5, or alpha 6, suggesting that they may be limited to the vascular spaces and do not extravasate, at least for the stages of disease analyzed here.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective expression of CD45 isoforms defines CALLA+ monoclonal B-lineage cells in peripheral blood from myeloma patients as late stage B cells.

The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B-lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.

Allogeneic bone-marrow transplantation without protective isolation in adults with malignant disease.

Bone-marrow transplant (BMT) patients are severely immunocompromised immediately after the procedure and they are commonly nursed in strict protective isolation to reduce the risk of both infection and graft-versus-host disease (GvHD). We have studied a consecutive series of patients to see whether protective isolation is of benefit as prophylaxis against infectious complications of BMT. 50 consecutive patients who had malignant disease and received their first BMT from siblings or unrelated donors were nursed in standard single rooms with visitors instructed to wash their hands. A subset of 20 patients living locally spent a median of 25 days in hospital after BMT; they also spent some time at home on a median of 8 days before engraftment and 3 patients went home on more than 90% of their hospital days. 16 patients (32%) had positive bacterial cultures and/or focal infection. Gram-positive bacteraemia was found in 12 subjects (24%) but there were no gram-negative or deep fungal infections. Grade II or III acute GvHD developed in 17 patients (34%). There were no deaths from infection or acute GvHD. Transplant-related mortality was 6% in the first 100 days and 18% overall with a median follow-up of 22 months. Our mortality data compare favourably with those from institutions with strict isolation procedures. We conclude that BMT may be safely completed in some institutions without either protective isolation or the need to confine patients continuously in hospital.