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The evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000, with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p < 0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/10(6) peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cells.

The role of the immune response in control and erradication of leukaemia remains controversial but there is increasing evidence that the principal mechanism of cure after intensive chemotherapy and allogeneic BMT is immune mediated. This is evidenced by the observations of a reduced risk of leukaemia relapse after allogeneic BMT when compared with autologous or syngeneic BMT. Furthermore, an increased incidence of leukaemic relapse has been reported after aggressive GvHD prophylaxis with cyclosporin or lymphocyte depletion. The association between GvHD and the graft-versus-leukaemia activity of allogeneic BMT is strong and has led to a number of workers concluding that GvL might be inseparable from GvHD although there is increasing evidence that this is not so. The lymphocyte subsets responsible for GvHD and GvL remain to be elucidated in man despite intensive efforts. Certainly T cells, natural killer cells and a population of in vivo activated killer cells are involved in GvL and an effective immune response probably requires a combined approach. The target antigens of GvL are also controversial. The majority of GvL studies have been conducted in the allogeneic transplant setting in which activity to leukaemia-specific peptides is easily masked by reactivity to undetected MHC mismatches and to minor histocompatibility antigens. Despite this the search for leukaemia-specific peptides has been fruitful in the case of the product of the BCR/ABL translocation in CML. The ultimate aim of a number of groups in all aspects of oncology is the development of effective and specific immunotherapy. A variety of approaches have been attempted over the last 80 years, including vaccination with irradiated leukaemic blasts and numerous trials of interleukin-2. We now know more about the mechanisms of induction of immunity than ever before and this knowledge, combined with sophisticated molecular biology and virology, promises to revolutionise the immunotherapy of leukaemia over the next ten years.

There is controversy whether adults with acute myelogenous leukemia (AML) in first remission are best treated with chemotherapy or an HLA-identical sibling bone marrow transplant. We studied 1097 adults, 16-50 years old, with AML in first remission. Results of transplants from HLA-identical siblings reported to the International Bone Marrow Transplant Registry (IBMTR; n = 901) were compared with results of chemotherapy in comparable persons treated by the German AML Cooperative Group (GAMLCG; n = 196). Preliminary analyses identified subject- and disease-related variables differing between the cohorts and associated with treatment outcome within each cohort. We adjusted for these variables and differences in time-to-treatment in subsequent comparisons of treatment-related mortality, relapse, survival and leukemia-free survival (LFS). Five-year probability of treatment-related mortality was greater for transplants than chemotherapy (43% (95% confidence interval, 37-49%) vs 7% (3-11%); P< 0.0001). Five-year relapse probability was less for transplants than chemotherapy (24% (20-28%) vs 63% (55-71%); P< 0.0001). Five-year probability of survival was similar with transplants and chemotherapy (48% (43-53%) vs 42% (33-51%); P = 0.24). Five-year LFS probability was higher for transplants than chemotherapy (46% (42-50%) vs 35% (28-41%); P= 0.01). These data indicate that bone marrow transplants from HLA-identical siblings result in comparable survival but greater LFS than chemotherapy in adults with AML in first remission.

Childhood myelofibrosis (Mf) is rare with variable outcome reported in the literature. We present clinical and investigate details of three children who presented with idiopathic Mf in early childhood. Two of these children were identical twins and have been haematologically stable over the past 7 years since their diagnosis. The third patient underwent an allogeneic bone marrow transplant (BMT) procedure as her clinical status was deteriorating. She remains engrafted at 13 months post BMT. None of the children had hepatosplenomegaly although extramedullary haemopoiesis was demonstrated on 52Fe studies in two patients. Circulating progenitors in these patients were increased in the face of reduced marrow precursors. The aetiology of childhood Mf is unclear and its natural history seems different from the adult disease. Allogeneic BMT can be an option for definitive treatment.

The presence of cytomegalovirus (CMV) in the blood has important consequences for patient management, and an external quality control study of its detection by the PCR was conducted by the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation. Forty-eight coded peripheral blood samples from bone marrow transplant recipients were processed in parallel in three European centers by using the routine in-house PCR assay. Protocols varied in choice of primers, specificity and amplificability controls, and sample processing. Results for 38 of 47 samples agreed, 35 being negative and 3 positive. Of the 12 samples reported as positive by a least one center, only 3 were found to be positive by all three centers, 1 was found to be positive by two centers, and the remaining 8 were found to be positive by one center only. The nine discrepant samples appeared to contain around 1,000-fold less viral DNA than the three concordant positive samples. CMV detection was affected both by the number of leukocytes from which DNA was extracted and by the number of cell equivalents added per PCR. External quality control schemes for CMV PCR are clearly necessary in order to compare data from different centers, and recommendation for standardizing the PCR detection of CMV in blood leukocytes are made.

We report a patient with acute myeloblastic leukaemia who presented with a pneumonia and herpes simplex viraemia associated with primary herpes simplex virus-1 infection. The importance of detecting and treating viral infections in haematology patients is discussed.

In 1993 the North London Blood Transfusion Centre received its first report of Yersinia enterocolitica transmission from a unit of red cells supplied to a local hospital. The recipient was a 23-year-old male who was neutropenic following a third cycle of chemotherapy for treatment of acute myeloblastic leukaemia (FAB type M6) and received a 34-day-old red cell unit. During transfusion the patient developed septicaemia and endotoxin-mediated shock. The transfusion was stopped immediately and broad spectrum antibiotics administered immediately on suspicion of bacteraemia from the transfused unit. This prompt action undoubtedly prevented a fatal outcome. Y. enterocolitica was isolated from the blood bag. Antibody was also detected in the bag and in a sample taken from the donor 39 days post-donation. Antibody to serotype 03 was identified, the commonest serotype reported in transfusion-transmitted Y. enterocolitica. The donor reported no gastrointestinal upset or illness prior to donation. This transfusion reaction might not have occurred had the red cells been transfused earlier in their storage period, but would not have been prevented by the exclusion of donors with a history of gastrointestinal illness as the donor was asymptomatic. Nor would it have been prevented by inspecting the blood for a change in colour, as no such change was observed. Y. enterocolitica is a significant problem in transfusion medicine and transmission is generally associated with a high mortality rate. Hospitals should be urged to investigate bacteriologically all appropriate transfusion reactions so that the true extent of the problem in the United Kingdom can be assessed.

A patient with relapsed refractory acute myeloid leukaemia developed typical fungal lung lesions despite intravenous amphotericin B prophylaxis. Chaetomium globosum was cultured from the resected right lower lobe. Histology showed branching hyphae negative for common Aspergillus species by immunohistochemical staining. Previous reports of invasive disease caused by Chaetomium and some applications of immunohistochemical staining for Aspergillus are discussed.

To evaluate whether the results of bone marrow transplantation have improved in Europe with time, we analyzed the outcome for 2195 patients with acute leukemia. 1405 had acute myeloid leukemia (AML) and 790 had acute lymphoblastic leukemia (ALL), and were allografted in first complete remission between September 1979 and December 1991 with marrow from an HLA-identical sibling donor. We found a continuing improvement more evident since 1987 for AML and since 1986 for ALL. A substantial reduction in the 3 years transplant related mortality (TRM): 26 vs 39% for AML (P = 10(-4)), and 25 vs 39% for ALL (P = 10(-4)), has resulted in an increase of the 5-year actuarial leukemia-free survival (LFS). 57 vs 45% for AML (P < 10(-4)) and 54 vs 45% (P = 10(-4)) for ALL. Four important changes have occurred. (1) Graft-versus-host disease (GVHD) prevention has involved an increased use of cyclosporin A (CsA) alone and subsequently its use in combination with methotrexate: this was associated with lower TRM both in AML and ALL; (2) Use of total body irradiation as pretransplant regimen has decreased; (3) a shorter interval from remission to BMT is more common; (4) an older population of patients has undergone BMT. Multivariate analyses were performed separately in AML and ALL. In AML four variables significantly influenced TRM favorably: year of BMT (P = 10(-4)), younger age at BMT (P = 10(-4)), prevention of GVHD including CsA (P = 0.008), sex match other than female donor to male recipient (P = 0.002). The relapse incidence (RI) was lower in patients with FAB M1-2-3 vs M4-5 (P = 0.0004). The LFS improved by year of BMT (P = 0.0004), younger age at BMT (P = 10(-4)), prevention of GVHD including CsA (P = 0.01), FAB M1-2-3 (P = 0.03). In ALL, three variables were associated with a lower TRM: year of BMT (P = 10(-4)), younger age at BMT (P = 10(-4)), sex combination other than female to male (P = 0.008). The LFS was better after 1986 (P = 0.0004) and in younger patients (P = 10(-4)). However a better outcome after 1986/87 was observed in patients receiving the same GVHD prophylaxis: therefore, other unidentified factors resulting in better patient care have also contributed to this. The improved results of allogeneic BMT are entirely related to a reduction in TRM without loss of the antileukemic effect since relapse incidence has not changed over the years.

The best therapy for persons with acute myelogenous leukemia (AML) in 2nd remission is unknown. Bone marrow transplants from an HLA-identical sibling are reported to be better than chemotherapy but this is controversial. The objective of the study was to compare 3-year leukemia-free survival (LFS) in comparable subjects receiving chemotherapy or a transplant. 485 persons with AML in 2nd remission were studied. The chemotherapy cohort included 244 persons treated on trials of the British Medical Research Council, Eastern Cooperative Oncology Group and MD Anderson Hospital. The transplant cohort included 257 persons transplanted worldwide and reported to the international Bone Marrow Transplant Registry (16 were also chemotherapy subjects.) Subjects were selected for comparable age and year of treatment. Preliminary analyses identified two factors correlated with LFS: age < or = or > 30 years and 1st remission duration < or = or > 1 year; subsequent analyses were partitioned accordingly. Three-year probabilities of treatment-related mortality with chemotherapy and transplants were 7% (95% confidence interval, 3-15%) vs 56% (49-63%). Three-year leukemia relapse probabilities were 81% (74-86%) vs 41% (33-49%). Three-year probabilities of LFS were 17% (12-23%) vs 26 (20-32%). Cohort analysis showed significantly higher LFS with transplants vs chemotherapy in persons < or = 30 years and 1st remissions > 1 year (41% (29-53%) vs 17% (7-32%); P = 0.017) and those in > 30 years with 1st remissions < or = 1 year (18% (9-29%) vs 7% (2-16%); P = 0.046). Others had comparable LFS with both treatments. These data indicate better LFS with HLA-identical sibling transplants than chemotherapy in some persons with AML in 2nd remission.

The objective was to analyze risk factors for veno-occlusive disease of the liver (VOD) after allogeneic bone marrow transplantation. A cohort of 1717 recipients of HLA-identical sibling transplants for leukemia between 1988 and 1990, in 200 transplant teams worldwide, was studied. Patients were scored as having VOD if liver tissue showed typical histologic features or if they had all three of the following: (1) jaundice; (2) hepatomegaly and right upper quadrant abdominal pain; and (3) ascites and/or unexplained weight gain. Patients surviving more than 7 days post-transplant without histologic or any of these clinical features of VOD were classified as not having VOD. Patient-, disease- and transplant-related characteristics of 95 patients with VOD were compared to those of 1514 without VOD. Variables correlated with an increased risk of VOD were: pretransplant conditioning with busulfan and cyclophosphamide compared to total body radiation (relative risk (RR) 2.8; P < 0.0001), pretransplant fungal infection (RR 4.1; P = 0.011), pretransplant Karnofsky performance score < 90% (RR 1.9; P = 0.012), prior liver disease (RR 1.9; P = 0.05) and age > 20 years (RR 1.8; P = 0.05). In patients receiving radiation for conditioning, intravenous immune globulin decreased VOD risk (RR 0.26; P = 0.003). This analysis identifies risk factors for VOD. The data suggest several strategies for modifying transplant regimens to reduce VOD risk and which patients might be suitable subjects for trials of strategies of VOD prevention.

Graft-vs.-leukemia (GVL) is postulated to be the principal mechanism responsible for continued remission after allogeneic bone marrow transplantation (BMT). The specific cytotoxic effectors mediating this effect are as yet undefined, but the major histocompatibility complex (MHC)-nonrestricted lysis of tumor cell lines by natural killer (NK) and lymphokine-activated killer (LAK) cells from recipients of allogeneic BMTs has been proposed as an in vitro correlate of GVL. In vitro culture or treatment in vivo with interleukin-2 (IL-2) is associated with enhanced NK cytotoxicity and lysis of NK-resistant targets (LAK cytotoxicity). NK, LAK, and cytotoxic T lymphocytes (CTL) have cytotoxic properties against autologous and allogeneic leukemic targets. These immune effector cells require receptor-ligand interaction for target recognition and adhesion via specific molecules such as integrins, a group of heterodimeric transmembrane glycoproteins. The integrins include the very late activation (VLA) subfamily, which all share the same beta 1 subunit but have distinct chains. VLA-6 (CDw49f) has been identified on NK cells and binds to laminin, a basement membrane protein found on malignant tumor cells but not normal cells. Monoclonal antibodies (mAbs) to laminin have been found to inhibit in vitro cytotoxicity of the tumor cell line K562, suggesting an important role for VLA-6 in this interaction. The specific aim of this study was to investigate the role of VLA-6 in the interactions of the tumor cell lines K562 and Daudi with peripheral blood lymphocytes (PBL) acting as effectors in cell-mediated cytotoxicity from normal volunteers, patients recovering from chemotherapy, and patients recovering from autologous or allogeneic BMT. In over 96% of assays, incubation of effector cells with anti-CDw49f mAbs led to detectable inhibition of NK and LAK cell-mediated cytotoxicity. More notably, the degree of anti-VLA6-induced suppression of LAK activity was significantly greater in the normal donors than in any of the patient groups, despite a significantly lower incidence of expression of VLA-6 on NK cells from controls than from patients. This implies a reduced role for this adhesion molecule in LAK activity following some form of in vivo stimulation. This hypothesis is supported by the observation that addition of exogenous IL-2 to the cultures ameliorated the effect of VLA-6 blockade, although the incidence and level of VLA-6 expression was unchanged by IL-2. In contrast, VLA-6 blocking led to a greater reduction in NK activity of BMT recipients than of normal donors, demonstrating that the VLA-6 adhesion pathway is important in this group of patients. These results indicate that the VLA-6-laminin interaction is important in normal NK-target interaction but may play a less significant role in the innate cytotoxic response post-BMT, perhaps reflecting subtle differences in the subsets of NK cells present in BMT recipients compared with normal donors.

Chronic parvovirus B19 infection in the immunocompromised host may cause severe anaemia secondary to failure of erythropoiesis. This has been previously documented in patients with the Acquired Immune Deficiency Syndrome (AIDS), congenital immunodeficiencies and in children with acute lymphoblastic leukaemia during maintenance chemotherapy. We describe persistent parvovirus infection in a 14-year-old boy after HLA-matched sibling allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in second remission.

A double-blind double-dummy, comparative study was carried out in 30 patients receiving highly emetogenic fast dose rate, single fraction total body irradiation prior to bone marrow transplantation. Patients were randomised into one of two groups, receiving either granisetron, a specific 5-HT3 antagonist or a combination of metoclopramide, dexamethasone phosphate and lorazepam to assess the comparative efficacy of the two regimens in the control of irradiation-induced nausea and vomiting. After 24 h eight patient (53%) treated with granisetron showed a complete response compared with two patients (13%) in the comparator group. The control of vomiting by granisetron, over both 24 h and 7-day periods (P = 0.001 and P = 0.004), was significantly better than that seen with the comparator and required significantly fewer rescue doses. The safety profiles in the two groups appeared similar, with the exception that granisetron produced less drowsiness than the comparator.

A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to investigate and compare transcription levels of the human multidrug resistance gene (MDR1) and the recently described multidrug resistance-associated protein (MRP) in 105 samples from patients with acute leukaemia at presentation and relapse. MRP gene expression was significantly greater in samples from patients with acute lymphoblastic leukaemia (ALL) compared with samples from normal peripheral mononuclear cells (PBMC) and patients with de novo acute myeloid leukaemia (AML). MRP gene expression was found to be higher in patients with relapsed de novo AML compared to those at presentation but prior therapy did not affect MRP gene expression in ALL. MDR1 gene expression was significantly lower in ALL patients compared to normal PBMC and AML samples. Samples from patients with secondary AML had higher levels of MDR1 expression than those of de novo AML. No changes of MDR1 expression were observed in AML or ALL at relapse. No correlation was observed between MDR1 and MRP gene expression in this group of patients. Our results suggest that MRP expression may be of prognostic importance in AML but the significance of the increased levels we have detected remain unclear.

Twenty-five patients with loss of part or all of 17p were selected from 701 patients with hematologic malignancies karyotyped either at diagnosis and/or at relapse and/or in refractory disease. Loss of 17p resulted from partial arm deletion (eight cases), unbalanced translocation (12 cases), i(17)(q10)(five cases) or monosomy 17 (four cases). In three cases, both 17ps were missing; in one case, two sublines independently acquired loss of 17p. In eight cases, loss of 17p was confirmed as a secondary change. The karyotypes generally were complex, with an average of 4.0 structurally abnormal chromosomes in the simplest lines showing 17p loss. The incidence at diagnosis was acute lymphoblastic leukemia (ALL) 2.2%, acute myeloid leukemia (AML) 2.4%, and myelodysplastic syndrome (MDS) 3.4%. The incidence in relapse and refractory disease was ALL 8.9%, AML 3.3%, and MDS 6.3%. The increased incidence of 17p loss in relapse and refractory disease was statistically significant (p < 0.05). Loss of 17p appears to be a feature of late-stage or resistant disease in hematologic malignancy.

Three cases of acute lymphoblastic leukemia (ALL) with the rare t(17;19)(q22;p13) translocation were investigated for E2A/HLF fusion genes using reverse transcription coupled with polymerase chain reaction (RT-PCR). The patients had C-ALL, F/17 years (case 1) or pre-B ALL, M/11 years (case 2) and M/13 years (case 3). Case 1 had an event-free survival (EFS) of 42 months. Case 2 was ultimately refractory to treatment. Case 3 presented following EFS of 16 months in morphological remission (1% blasts), but with immunological and cytogenetic evidence of active disease, then relapsed, remitted and relapsed. Type II E2A/HLF fusion cDNA was found at diagnosis (cases 1, 2), at presentation (case 3) and in all samples tested, whether with active disease or in complete remission (CR). Case 3 showed, in addition, type I fusion E2A/HLF cDNA at presentation, through induction therapy when there was evidence of active disease, but not in CR. Cases 1 and 3 had bone marrow transplantation while in CR but with residual disease detectable by RT-PCR. All patients have died of ALL. Two cases (2 and 3) had hypercalcemia with bone lesions. No case had any evidence of disseminated intravascular coagulation. This is the first demonstration of the value of RT-PCR for the detection of minimal residual disease in t(17;19) ALL.

We investigated the occurrence of late events (beyond 2 years) in patients with acute leukaemia who received an allogeneic (BMT) (n = 1059), or an autologous bone marrow transplantation (ABMT) (n = 656) in Europe during the period from January 1979 to December 1990. Patients with no recurrence of leukaemia at 2 years had overall 82% chance of being alive in complete remission at 9 years following transplantation regardless of the nature of the leukaemia, the status at transplant, and the type of transplant. The incidence of late relapses continuously decreased with time. The latest relapses in acute myelogenous leukaemia (AML) were observed following BMT at 6.6 years in a patient transplanted in first remission (CR1) and at 3.7 years in a patient transplanted in second remission (CR2), and following ABMT at 6 years and 5.1 years respectively. The latest relapses in acute lymphoblastic leukaemia (ALL) were observed following BMT at 4 years in a patient transplanted in first remission (CR1) and at 6.8 years in a patient transplanted in second remission (CR2), and following ABMT at 5.3 years and 4.5 years respectively. Several factors predictive for late relapse or death were identified. Patients allografted experienced a lower frequency of late relapse than patients autografted. Of the numerous other prognostic factors studied, female sex in AML, the use of total body irradiation (TBI) in ALL and status in CR1, rather than CR2-3, for both ALL and AML allografted were correlated with a lower relapse incidence. The use of TBI in ALL was also associated with a better LFS and survival. The absence of acute graft-versus-host disease (GVHD) in allografted AML correlated with better LFS and better survival, but had no influence on the relapse incidence. This study indicates that patients alive and well at 2 years post transplant have a very high probability of being cured, but the possibility of late relapse still remains.

Haemopoietic growth factors are accepted as accelerating haemopoietic recovery after bone-marrow grafting, yet no large randomised trials have been published that convincingly show benefit. Lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) was given to 315 patients after bone-marrow transplantation in a prospective randomised placebo-controlled multicentre trial. 1 day after bone-marrow infusion, 163 patients received lenograstim 5 micrograms/kg per day by 30-min infusion, and 152 patients received placebo daily for 28 days or until neutrophil recovery. 137 patients had lymphoma, 35 myeloma, 85 acute lymphoblastic leukaemia, and 58 a solid tumour. Patients were stratified by age and by type of bone-marrow transplantation (BMT). Neutrophil recovery to above 10(9)/L for 3 consecutive days was seen earlier in lenograstim-treated patients (16 vs 27 days, p < 0.001). Time to neutrophil recovery above 0.5 x 10(9)/L was reduced (14 vs 20 days, p < 0.001). The difference was significant both in autograft (20 vs 14 days, p < 0.001) and allograft (20 vs 14 days, p < 0.01) patients, in children (20 vs 13 days, p < 0.001), and adults. Lenograstim-treated patients had fewer days of infection, and of antibiotic administration, and also spent less time in hospital. However, clinical and microbiological sepsis was similar in both groups. There was no significant toxicity ascribed to lenograstim. Survival was the same at days 100 and 365. In patients undergoing autologous or allogeneic BMT for neoplastic disease, lenograstim significantly reduced duration of neutropenia and led to earlier hospital discharge.

Analysis of the results of different methods of post remission therapy in acute myeloid leukemia (AML) has been used to study their impact on outcome, with a particular emphasis on antileukemic effects by contrasting the risk of relapse. We have compared consolidation chemotherapy (CT), autologous bone marrow transplantation (ABMT) with and without subsequent interleukin-2 (IL-2) and allogeneic bone marrow transplantation (BMT). Additionally we have studied the immunological consequences of each of these maneuvers with particular regard to the cellular components having proven or suspected antileukemic properties and the secondary cytokines released by these cells in vitro and in vivo.

Donor-specific bone marrow infusion after organ grafting can induce tolerance in animals. In this randomised controlled study we show it has no benefit in patients undergoing liver transplantation. Of 25 patients, 9 received bone marrow 5 days after a 10 day course of antithymocyte globulin. Immunosuppression was maintained with cyclosporin only. An average of 3.0 rejection episodes per patient was seen in the bone marrow group compared to 3.1 in the controls. Chimerism was not found in peripheral blood or bone marrow of recipients using erythrocyte antigen markers, PCR for donor class II DNA or Y-probe in-situ hybridisation in one female recipient of male liver and bone marrow.

The results of 5018 surveillance cultures for the detection of cytomegalovirus (CMV) from 177 patients undergoing allogeneic T cell-depleted bone marrow transplantation (BMT) were analysed to determine their value in predicting future development of CMV pneumonitis. Twenty-two patients were diagnosed as having CMV pneumonitis. The median times of a positive CMV culture result from urine, saliva or blood before disease in these patients were 32, 8 and 23 days, respectively. The positive predictive value of CMV viraemia for the development of CMV pneumonitis was 0.64. Multivariate analysis showed that recipient pretransplant CMV seropositivity and increasing recipient age were independent risk factors for CMV pneumonitis. By contrast, donor CMV seropositivity was protective against disease, supporting the hypothesis that immunity against CMV can be adopted from donor T cell-depleted marrow. It is concluded that detection of CMV viraemia in a surveillance protocol can predict subsequent CMV pneumonitis but that it is of low sensitivity. This might be improved by more frequent sampling or by the use of a more sensitive assay, such as the polymerase chain reaction, thus identifying individuals who may benefit from 'pre-emptive' therapy.

Two males with Down syndrome and acute lymphoblastic leukemia with the acquired translocation, t(8;14)(q11;q32), are described. In each case the constitutional karyotype was 47,XY,+21. The patients were, respectively, aged 3 years 11 months and 32 years, with presenting white blood counts 34 and 1.9 x 10(9)/L with blasts of FAB L1 and L2. In each case immunophenotype of the blasts was C-ALL. The child is alive and well and in first remission 6 years from diagnosis. In contrast, the adult patient died in first remission 8.5 months from diagnosis with severe pancytopenia. These are to our knowledge the second and third cases of ALL with t(8;14)(q11-12;q32) associated with a constitutional genetic disorder.

Forty one patients with acute myeloid leukemia (AML), including 27 at presentation and 14 relapsed or resistant cases, were assessed for laboratory evidence of the MDR phenotype. Leukaemic cells from all 41 cases were studied by immunocytochemistry using the JSB-1 monoclonal antibody and simultaneously by reverse transcription polymerase chain reaction (RT-PCR) to evaluate expression of the mdr 1 gene. Cells from 32/41 cases were also assessed for daunorubicin (DNR) accumulation and retention by flow cytometry (FC). Nineteen of the 41 (46%) patients were positive for MDR by JSB-1 immunocytochemistry (11/27 [41%] at presentation and 8/14 [57%] relapsed or resistant cases). Nine of the 19 (47%) P-gp positive, de novo patients achieved complete remission. 22 patients were negative by JSB-1 immunocytochemistry (16/27 [59%] at presentation and 6/14 [43%] of the relapsed or resistant cases) and 11/22 (50%) P-gp negative patients achieved a complete remission. Of the 32 patients assessed by FC, 7 (22%) were positive for the MDR phenotype with increased DNR accumulation and retention in the presence of the MDR reversing agent verapamil (VPM). 6/7 of the FC positive cases were also JSB-1 positive, and 6 had additional poor risk features. Of the twenty five FC negative patients, 6 had received previous chemotherapy and 15 (60%) achieved complete remission. Mdr 1 mRNA levels were increased in all seven FC positive cases whereas only 7/19 JSB-1 positive cases had raised mdr 1 mRNA levels. These results suggest that the assessment of MDR status by immunocytochemistry using JSB-1 is not predictive of response to chemotherapy. Flow cytometric analysis of blast cells appears to correlate well with mdr 1 mRNA levels and may be a better predictor of treatment outcome.

Eighty-eight patients who received single fraction total body irradiation (sfTBI) as part of their conditioning for allogeneic BMT have been evaluated for the risk of cataract formation. Thirty-eight (43%) have developed cataracts; 11 required surgery. With 9.5-13.6 years follow-up (median 10.7 years), all 12 recipients of unmanipulated marrow allografts have developed cataracts; the actuarial risk of needing surgery was 32 (+/- 18%, 95% confidence intervals (CI)). Ten of these 12 required high-dose steroids (prednisolone > 1 mg/kg/day) for the treatment of GVHD. Seventy-six patients received T cell-depleted allografts; 14 of 76 required post-transplant immunosuppression with high-dose steroids. With 1-9.4 years follow-up (median 5 years), the actuarial risk of cataract formation in T cell-depleted allograft recipients is 72% (+/- 52% CI), the actuarial risk for needing surgery is 20% (+/- 9% CI). Recipients of sfTBI and non-T cell-depleted allografts had a significantly greater risk of developing cataracts (p = 0.003, long rank test) and of needing surgery (p < 0.05, log rank test) than patients receiving T cell-depleted BM. Cataracts occurred more frequently in patients requiring post-transplant immunosuppression with steroids (relative risk 2.12, p < 0.01 log rank test).

Over 13 years, we have seen 16 cases of proven invasive aspergillosis in 446 bone marrow transplant recipients, an incidence of 3.6%. The incidence of infection is low in patients with uncomplicated allogeneic or autologous bone-marrow transplants (< 2% and 0, respectively). Of the 16 episodes following transplantation, 10 occurred in patients with late transplant complications who were no longer in protective isolation. In patients who had focal pulmonary lesions (as diagnosed by computed tomographic scanning), culture of bronchoalveolar lavage (BAL) fluid was not an effective diagnostic procedure. In diffuse pulmonary disease due to Aspergillus, culture of BAL fluid had a sensitivity of 100%. Aspergillus species were isolated from an additional six patients who had no evidence of invasive aspergillosis. Graft rejection was a significant predisposing factor for the development of invasive aspergillosis (P < .001, log-rank test), and in our hospital, these patients now receive intravenous amphotericin B as prophylaxis. None of the six patients whose chest roentgenograms showed abnormalities before transplantation and who underwent surgical resection as part of the treatment for invasive aspergillosis developed recurrent infection.

Donor cell leukemia after BMT has been documented in a small number of cases mainly by cytogenetic studies. We describe a case of leukemia relapse in a 16-year-old girl 1 year after BMT from her histocompatible brother. Relapse in donor cells was initially suspected on the basis of cytogenetic analysis and confirmed by DNA in situ hybridization in blast cells using a Y chromosome-specific probe.

Myeloablative chemo (+/- radio) therapy and rescue with ABMT has been used as final consolidation therapy in 18 patients with AML in first remission. In seven (6 autologous, 1 syngeneic) marrow reinfusion was followed by iv IL-2. One patient, who commenced IL-2 4 days after BMT, died from pulmonary oedema due to the capillary leak syndrome. Thereafter, treatment with IL-2 was delayed until the platelet count reached 30 x 10(9)/l. All patients developed reversible hypotension (treated with infusion of colloid), but treatment was otherwise well tolerated. With 21-58 months (median 32 months) from the time of ABMT there has been one relapse (actuarial risk 17%, 95% confidence intervals (CI) 3-31%). The disease-free survival is 71% (95% CI 38-100%). Eleven patients with comparable remission induction and consolidation therapy, and an identical interval between diagnosis and ABMT (5-11 months, median 6 months) received an autograft without immunotherapy. With 24-45 months (median 29 months) follow-up the actuarial disease-free survival is 36% (95% CI 8-64%), the actuarial relapse risk is 54% (95% CI 18-90%). We conclude that immunotherapy given after ABMT to patients with AML in first remission when the platelet count exceeds 30 x 10(9)/l is safe and may induce an immunological environment which results in the elimination of residual leukaemia.

Studies of peripheral blood lymphocytes (PBL) and plasma from patients with malignant lymphoma [Hodgkin's disease (HD) and non Hodgkin's lymphoma (NHL)] show that plasma soluble interleukin 2 receptor (sIL2R) levels are closely linked with disease status [normal volunteers (n = 15) 402 +/- 158 u/ml; patients with Hodgkin's disease in remission (n = 4) 525 +/- 195 u/ml or with active disease (n = 11) 3026 +/- 1602 u/ml (p < 0.001); patients with non Hodgkin's lymphoma in remission (n = 6) 462 +/- 202 u/ml, active disease (n = 15) 2713 +/- 1755 u/ml, (p < 0.001)] but no correlation between sIL2R and the inhibition of interleukin 2 (IL2) generated cytotoxicity for the cell line K562. In only 1 of 15 patient plasma samples studied was there a dose dependent inhibition of IL2 generated cell killing. In a further patient, IL2 generated K562 killing was inhibited at all doses (500-3000 brmp units/ml); treatment of this plasma with anti-Interleukin 4 (alpha IL4) had no effect on the potent inhibitory activity of the plasma. Plasma sIL2R levels were markedly elevated in patients receiving IL2 in vivo (pre treatment 520 +/- 170 IU/ml, during treatment 5578 +/- 2564 IU/ml, p = 0.05). The aetiology of immunosuppression in patients with lymphoma appears to be multi-factorial; although sIL2R correlates with disease activity it does not appear to directly mediate immunosuppression in most patients with malignant lymphoma.

Rearrangement of the immunoglobulin heavy chain (IgH) gene can be utilized as a marker of clonality in a number of B-lineage lymphoproliferative disorders including acute lymphoblastic leukaemia (ALL). We have used a PCR technique involving a panel of amplimers for the 6 different Variable (VH) region families and for a consensus sequence of the Joining (JH) segment to detect clonal IgH rearrangements in the peripheral blood (PB) and/or bone marrow (BM) of 28 patients (17 children and 11 adults) with B-lineage ALL at presentation (20 patients) or with overt relapse (8 patients). The age range of the patients was 2-65 years (mean 15.7 years). Follow up remission BM samples were analysed in 22 patients during and after therapy (2-7 samples per patient), 1-50 months after presentation or relapse. In 1 relapsed case, previously stored complete remission (CR) samples were analysed retrospectively. Clonal IgH chain rearrangements were detected by PCR in 90% of patients studied initially. The 2 VH region families most commonly used were the large VH3 family (65%) and the smaller more JH-proximal VH4 family (22%). More than one VH clone was detectable in 25% of the cases. A gene "fingerprinting" modification of a previously described method was applied to the detection of minimal residual disease (MRD) in follow up BM samples with a sensitivity of 10(-3) to 10(-4). In 8 of 14 patients remaining in complete remission (CR) during the time of study, all PCR analyses on BM samples in the first 6 months were negative, in some cases as early as 2 weeks post-induction therapy, and a further patient reverted from being PCR positive in the first month after the commencement of therapy to sustained PCR negativity. One adult remains in CR at 50 months after presentation and has been PCR negative at 2 time points after cessation of maintenance therapy (30 and 50 months). Eight patients relapsed in the study period comprising 6 BM and 2 isolated CNS relapses. In 4 cases of BM relapse occurring within 7 months of the start of therapy, all BM remission samples tested in this period were PCR positive. In 2 other patients BM samples tested 7 and 2 months respectively prior to relapse were PCR negative. In the 2 patients with isolated CNS relapse, PCR of BM samples from 2 and 10 months before the relapse were negative.(ABSTRACT TRUNCATED AT 400 WORDS)

The identification of risk factors for viral reactivation following transplantation is essential in order that antiviral prophylactic regimes may be allocated rationally. In the pretransplant period qualitative serological assessment of recipient and donor is informative with regard to the risk of post-transplant reactivation and disease. In addition, the quantitative level of herpes simplex virus (HSV) IgG in the pretransplant recipient is predictive of post-transplant HSV excretion, although this relationship does not exist for other viruses of the herpes group. We discuss the value of surveillance cultures for cytomegalovirus in the post-transplant period in predicting the development of CMV disease, and how this may allow effective intervention to prevent what is a major cause of morbidity and mortality in the transplant population.

Mycobacterium chelonae, a rapidly growing species, is a significant cause of fever in neutropenic patients. We describe three febrile neutropenic patients at the Royal Free Hospital from whom this organism was isolated on several occasions. The condition of the first patient improved as the neutrophil count recovered. The second patient developed pulmonary disease and required surgical resection of a pulmonary lesion. The third patient, who had rapidly progressive, diffuse pulmonary disease, responded to an antibiotic regimen including erythromycin and ciprofloxacin. Both our findings and reports in the literature suggest that neutropenia may be a major risk factor for disseminated infection due to M. chelonae and that treatment is effective only after the recovery of the neutrophil count.

Background: Lung function often deteriorates after bone marrow transplantation for haematological malignancies. Whether pulmonary function measurements are useful for monitoring patients' progress after transplantation and for alerting clinicians to the development of pneumonitis is uncertain., Methods: Serial pulmonary function measurements were made in 39 patients with a haematological malignancy, and the values from 18 recipients of T cell depleted allogeneic (n = 17) or autologous (n = 1) bone marrow transplants who developed interstitial pneumonitis were compared retrospectively with values from 21 recipients of allogeneic (n = 17) or autologous (n = 4) transplants who did not develop pneumonitis. Lung function was measured at the onset of a further 18 episodes of pneumonitis., Results: Measurements made before transplantation showed no difference in forced expiratory volume in one second (FEV1), transfer factor for carbon monoxide (TLCO), or total lung capacity between the two groups, but the forced vital capacity (FVC) was slightly higher in those who developed pneumonitis (mean (SD)% predicted 104 (12)) than in those who did not (93 (17%)). Six weeks and three months after transplantation all pulmonary function measurements had fallen slightly in both groups but TLCO had fallen considerably more in those who later developed pneumonitis, being 71% (SD 11%) and 77% (7%) of pretransplant values in patients who later developed pneumonitis compared with 109% (38%) and 96% (26%) in those who did not. All lung function measurements were significantly lower at the onset of pneumonitis than three months after transplantation, even in patients with no abnormal signs and a normal chest radiograph., Conclusions: Serial measurements of gas transfer before and after bone marrow transplantation may be useful for predicting which patients will be at risk of developing pneumonitis and may help to diagnose pneumonitis in breathless patients with no abnormal signs.

We analysed data from 114 recipients of HLA-identical sibling transplants who relapsed and received a second transplant between 1978 and 1989. Twenty-nine patients had acute lymphoblastic leukemia, 46 acute myeloid leukemia and 39 chronic myelogenous leukemia. Median (range) interval between first and second transplants was 15 (1-80) months. Following the second transplant, graft failure occurred in 2%, acute graft-versus-host disease (GVHD) in 27% and chronic GVHD in 21% of patients at risk. Risks of interstitial pneumonia and hepatic veno-occlusive disease were higher after the second than the first transplant. Two-year probabilities (95% confidence interval) of treatment-related mortality, relapse and leukemia-free survival were 41% (30-53%), 65% (53-75%) and 21% (14-30%), respectively. Leukemia-free survival was 7% (2-19%) among patients relapsing less than 6 months after their first transplant, with high rates of both relapse, 77% (49-92%), and treatment-related mortality 69% (46-85%). In contrast, leukemia-free survival was 28% (19-41%) in those relapsing more than 6 months after the first transplant; in this group the probability of relapse was 59% (45-72%) and treatment-related mortality 30% (20-43%). Factors correlated with better outcome included a diagnosis of chronic myelogenous leukemia, relapse more than 6 months after the first transplant, acute leukemia in remission prior to the second transplant and good performance status.

Stomatococcus mucilaginosus was isolated from eight neutropenic patients during nine febrile episodes over a 13-month period. Five of these isolates were from definite infections, including one case of fatal meningitis. This slime-producing, catalase-variable, gram-positive coccus is a component of the normal oral flora of humans. Its biochemical profile may result in misidentification; however, unlike most micrococci, it characteristically fails to grow on media containing 5% NaCl. All but one of our isolates were sensitive to benzylpenicillin, and all were sensitive to vancomycin. S. mucilaginosus may prove to be an important pathogen in severely immunocompromised patients.

Although IL-2 infusion enhances cell-mediated cytotoxicity in patients with neoplastic disease, administration is paradoxically associated with a modest fall in total serum IgG and an increased risk of infection. We now show that the adverse effects of IL-2 infusion on the humoral immune system are substantial. Although IL-2 induces the B cell growth and differentiating factors IL-4 and IL-6, infusion abrogates primary antibody responses entirely and reduces secondary antibody responses 50-fold following antigen challenge. There is no evidence of the generation of cells with suppressive activity on B cells but IL-2 increases the ratio of circulating virgin:memory cells. These results may help to explain the increased rate of bacterial infection in patients receiving IL-2. As IL-2 plays a central role in the generation of an immune response, the finding that it is also sufficiently immunosuppressive to inhibit primary- and secondary-type antibody responses suggests that exploration of the underlying mechanisms may provide insights into immune system homeostasis and may offer new approaches to therapeutic immunosuppression.

AmBisome is a commercially available preparation of amphotericin B incorporated in small unilamellar liposomes. The analysis of safety data provided in a multicentre compassionate phase II/III study evaluating 133 episodes of therapy with AmBisome in patients with severe underlying disease is very encouraging. Rapid (30-60 min) administration of a high daily dose of AmBisome is feasible without the well known side effects observed with the conventional formulation of amphotericin B (Fungizone). Although eleven of 71 patients with initially normal serum creatinine concentrations showed increased values after AmBisome therapy, 17 patients among 50 with initially high creatinine concentrations recovered normal renal function during AmBisome treatment. Hypokalaemia was observed in 24 episodes of treatment (18%). Increases in serum glutamyl oxaloacetic transaminase and alkaline phosphatase were also quite common in this selected population with severe underlying disease and the contribution of AmBisome to these changes is difficult to establish. AmBisome appears a safe alternative to conventional amphotericin B, and its efficacy should be further investigated.

The results in 34 adult patients with acute myeloid leukemia (AML) who have undergone autologous bone marrow transplantation (ABMT) using busulfan and cyclophosphamide (Bu/Cy) in 12 United Kingdom (UK) centers have been analyzed. There were 19 females and 15 males; median age was 40 years (range, 21 to 62 years). Nine patients were in first relapse; 25 were in second remission. The median time of first remission for the whole group was 11.5 months (range, 1 to 56 months). All the patients in first relapse and six patients in second remission received first remission marrow. The leukemia-free survival (LFS) for the patients in first relapse was 33%, with a median follow-up of 20 months. The LFS for the patients in second remission was 48% with a median follow-up of 26 months. The length of second remission exceeds the length of first remission in 14 patients. Considerable toxicity with hemorrhagic cystitis (four patients; none fatal), venoocclusive disease (four patients; one fatal), pneumonitis (four patients; one fatal), intracranial hemorrhage (two patients; two fatal) has occurred. There have been four procedure-related deaths (12%). Hematologic recovery was satisfactory for neutrophils (median time to 0.5 x 10(9)/L, 22 days [range, 11 to 101 days]), but very slow for platelets (median time to 50 x 10(9)/L, 62 days [range, 15 to 1,080 days]). This study suggests that the use of Bu/Cy with ABMT for patients beyond first remission in AML compares favorably with chemotherapy, and although the procedure-related mortality is acceptable, it is associated with protracted platelet recovery.

Chromosome analysis of bone marrow cells from a patient with acute nonlymphocytic leukemia M2 revealed the translocation t(11;22)(q24;q12) usually associated with Ewing's sarcoma. Molecular investigations ruled out the possibility that this was a variant Philadelphia translocation with breakpoints in the major breakpoint cluster region. Although cytogenetic analysis was not available at diagnosis, this abnormality was found both pre- and postallogeneic bone marrow transplant.

Patients in first remission of acute lymphoblastic leukaemia (ALL) considered to be at high risk of relapse were offered autologous bone marrow transplantation (ABMT) using purged marrow as a therapeutic alternative to cranial irradiation and maintenance chemotherapy. Twenty-seven bone marrows taken in remission, were purged using monoclonal antibodies (anti CD7 for T lineage and anti CD10 and/or anti CD19 for B lineage leukaemias) plus rabbit complement. Retrospective analysis of 19 purged marrows by immunophenotyping or immunoglobulin gene rearrangement studies demonstrated no evidence of disease. Engraftment was seen in 26 of the patients. No correlation was found between the numbers of infused nucleated cells or colony forming units-granulocyte-macrophage (CFU-GM) and subsequent engraftment kinetics. The actuarial disease-free survival (DFS) is 32% at 7 years (median follow-up 3.4 years). There were two transplant related deaths (actuarial risk 8%); the main cause of treatment failure has been disease recurrence with an overall actuarial risk of 67%; 76% for T-ALL (five of nine), 62% for common ALL (five of 10), two of five pre B and none of three patients with B-ALL. In these 27 high risk patients in vitro purging of remission marrow as part of ABMT appears not to improve patient outcome, although confirmation of this would require a randomized trial.

The antiemetic efficacy of granisetron was tested in an open trial in patients undergoing highly emetogenic treatment by single fraction total body irradiation. Thirty-two consecutive patients were entered. Results were both patient- and observer-rated. Following a single intravenous dose of granisetron 18 patients (56.3%) experienced total protection and a further 13 (40.6%) had major antiemetic protection with four of these patients experiencing nausea only. One patient experienced an anaphylactic reaction on infusion of monoclonal antibody-treated donor marrow 5 h after administration of the trial drug and vomited on multiple occasions. The reaction was associated with hypotension. A further patient experienced transient hypotension secondary to septicaemia 8 h after receiving granisetron. Three patients required a second dose. Headache was the most frequent side-effect occurring in three patients, but in to of these patients the test drug was not thought to be implicated. In conclusion granisetron is a highly effective agent in controlling radiation induced emesis with a favourable toxicity profile.