Before Bone Marrow Transplantation Research Articles

Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool for distinguishing permissible from non-permissible class I mismatches in T-cell-depleted allogeneic bone marrow transplantation.

Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA-identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft-versus-host-disease (GVHD). However, we have no means of defining these 'permissible' mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T-lymphocyte precursor frequency analysis (HTLp-f and CTLp-f respectively) can be applied to this end. Fifty-five recipient-donor pairs other than HLA-identical siblings, the recipient of which received a T-cell-depleted graft, were analysed by high-resolution HLA typing and/or HTLp-f/CTLp-f analysis. The predictive value of the CTLp-f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp-f assay was able to discriminate permissible from non-permissible HLA-A, -B or -Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp-f and CTLp-f together had a high negative predictive value, HTLp-f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp-f or CTLp-f can identify permissible DRB/DQB mismatches, high-resolution matching for these antigens remains the best option. The combination of high-resolution DRB/DQB typing and the CTLp-f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T- cells in the graft accordingly to improve clinical outcome.

Cyclophosphamide prevents systemic keratinocyte growth factor-induced up-regulation of surfactant protein A after allogeneic transplant in mice.

We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.

Biological dosimetry after total body irradiation (TBI) for hematologic malignancy patients

Purpose: Biological dosimetry based on scoring chromosomal aberrations in peripheral lymphocytes was compared to physical dosimetry done for total body irradiation (TBI) before bone marrow transplantation (BMT) in patients with hematologic malignancies. Patients and Methods: Fifteen patients undergoing TBI were included in the study. A total dose of 12 Gy in 2.5 days was fractionated into 2 or 3 daily doses of 1.8 Gy delivered by a 18 MV linear accelerator (dose rate: 15.8 cGy · min −1). Blood samples were obtained from patients before irradiation and after the first fraction of 1.8 Gy. A standard dose-effect curve was established by in vitro irradiation of healthy volunteer lymphocytes. Chromosomal aberrations were scored by the conventional cytogenetics (CCG) method for unstable anomalies and by fluorescent in situ hybridization (FISH) for stable anomalies. Results: Healthy donor lymphocytes before irradiation yielded 0.1% dicentrics and 0.3% translocations of chromosome 4 (Chr. 4), that is 2.5% for the whole genome. Patients before irradiation had 2% of dicentrics and 1.1% of chromosome 4 translocations. The biologically estimated dose of the 15 patients after exposure to 1.8 Gy was 1.93 Gy (95% CI: 1.85–2.05) according to CCG, and 2.06 Gy (95% CI: 1.75–2.15) by FISH. Conclusion: The dose estimated by biological dosimetry, in this case of homogeneously distributed radiation of TBI, agrees well with the absorbed radiation dose calculated by physical dosimetry.

Total body irradiation (TBI) before bone marrow transplantation (BMT): technique and acute toxicity

Total body irradiation (TBI) before bone marrow transplantation (BMT): technique and acute toxicity

Total body irradiation: techniques, dosimetry, and complications

Total-body irradiation (TBI) has an established role in many preparative regimens used before bone marrow transplantation (BMT) in the treatment of hematological malignancies in children and adults. Better choice in TBI techniques and dosimetry have permitted better homogeneity of dose, and therefore a significant sparing of critical tissues. Advances in treatments over the past 20 years have greatly improved survival; therefore, the evaluation of early and late complications, with a sufficient follow-up, according to different conditioning regimens is important. In this article, we review and compare different TBI techniques and dosimetry, and their influence on the distribution and homogeneity of dose, and the possible relationship to the risk of complications. We also describe the acute and late effects of TBI in children and adults appearing in the first month post-BMT as veno-occlusive disease, interstitial pneumonitis, or after 3 months, i.e., endocrinal late effects and growth in children, cataracts, neurological and bone or other complications, secondary tumors and alteration in the quality of life. The responsibility of TBI in the increased rate of certain complications is difficult to assess from chemotherapy or allograft side effects (chronic graft vs. host disease) or from other associated medical treatments, such as long term steroid therapy.

Norethisterone Treatment, a Major Risk-Factor for Veno-Occlusive Disease in the Liver After Allogeneic Bone Marrow Transplantation

AbstractIn this single-center study, we retrospectively analyzed incidence and risk factors for hepatic veno-occlusive disease (VOD) in 249 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation between January 1990 and June 1995. Twenty-four of the 249 transplanted patients developed VOD. The probabilities of developing VOD were 17% among women and 7% in men (P = .01). In women treated with norethisterone, the incidence was 27% compared with 3% in women without this treatment (P = .007). One-year survival rates were 17% and 73% in patients with (n = 24) or without VOD (n = 225), respectively. The use of heparin prophylaxis (100 IE/kg/24 hours for 1 month) did not alter the incidence or 1-year mortality of VOD. In multivariate analysis, the following risk factors were significant: norethisterone treatment (P < .001), bilirubin >26 μmol/L before bone marrow transplantation (BMT) (P = .002), one HLA-antigen mismatch (P = .003), previous abdominal irradiation (P = .02), and conditioning with busulphan (P = .02). Our conclusion is that norethisterone treatment should not be used in patients undergoing BMT and heparin prophylaxis did not affect the incidence or mortality of VOD.

Norethisterone Treatment, a Major Risk-Factor for Veno-Occlusive Disease in the Liver After Allogeneic Bone Marrow Transplantation

In this single-center study, we retrospectively analyzed incidence and risk factors for hepatic veno-occlusive disease (VOD) in 249 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation between January 1990 and June 1995. Twenty-four of the 249 transplanted patients developed VOD. The probabilities of developing VOD were 17% among women and 7% in men (P = .01). In women treated with norethisterone, the incidence was 27% compared with 3% in women without this treatment (P = .007). One-year survival rates were 17% and 73% in patients with (n = 24) or without VOD (n = 225), respectively. The use of heparin prophylaxis (100 IE/kg/24 hours for 1 month) did not alter the incidence or 1-year mortality of VOD. In multivariate analysis, the following risk factors were significant: norethisterone treatment (P &lt; .001), bilirubin &gt;26 μmol/L before bone marrow transplantation (BMT) (P = .002), one HLA-antigen mismatch (P = .003), previous abdominal irradiation (P = .02), and conditioning with busulphan (P = .02). Our conclusion is that norethisterone treatment should not be used in patients undergoing BMT and heparin prophylaxis did not affect the incidence or mortality of VOD.

Absorbed doses in the head and oral cavity during total body irradiation.

Conditioning before bone marrow transplantation (BMT) with total body irradiation (TBI) and cyclophosphamide carries the risk of severe long-term complications. The aim of the present study was to determine absorbed doses in the craniofacial area during TBI, using thermoluminescent dosimeters for both in vivo techniques and phantom measurements. The results from the phantom study showed that the mean deviation from the reference dose varied between -5.1 and +11.1%. The superficial part of left parotid gland received 11.1 +/- 1.5% more radiation compared with the reference. In patients, deviations from the reference dose varied in individual patients between -22.4 and +20.1% in various intra-oral sites. Since a small increase in dose can result in a significantly increased risk of pathology, the observed dose inhomogeneities for structures receiving tangentially directed radiation therapy, such as the parotid glands, may contribute to salivary dysfunction after TBI.

Dose homogeneity in the group of 15 patients undergoing fractionated total body irradiation

Methods Fractionated total body irradiation (FTBI) before bone marrow transplantation (BMT) was performed in 15 patients with different proliferative diseases. Common regimen of 12 Gy in 8 fractions (four days) was applied with the reduction of dose to lungs to 9.4 Gy. Results Midline dose discrepancies ranged from −15.9% to +9.9% and exceeded ±10% of prescribed dose in one patient. Outside midline dose discrepancies ranged from −16.5% to +19.8% and exceeded ±10% of prescribed dose in six patients. Dose determination error was estimated for between 2.8% and 5% depending on the body part. Conclusions Estimated error of the dose determination increased dose deviations from prescribed values from between −1.6% and +4.8% to between −5.2% and +9.3% in midline and from −8.4% and +19.8% to between −12.0% and +24.8% across transverses.

Total body irradiation prior to bone marrow transplantation: Efficacy and safety of granisetron in the prophylaxis and control of radiation-induced emesis

Purpose : Radiation-induced emesis is one of the most disturbing side effects of total body irradiation (TBI). To evaluate the efficacy and to determine the best schedule of granisetron (a selective 5-hydroxytryptamine 3serotonin receptor antagonist) administration in the prevention of radiation-induced nausea and vomiting, we conducted a trial involving patients receiving single-dose TBI before bone marrow transplantation (BMT). Methods and Materials: Thirty-six patients with non-Hodgkin's lymphoma ( n = 12, multiple myeloma ( n = 8), acute lymphoblastic leukemia ( n = 7), acute nonlymphoblastic leukemia ( n = 6, and chronic myeloid leukemia ( n = 3), referred to our department between March 1992 and February 1994 were enrolled in this study to assess the efficacy of granisetron during single-dose TBI before autologous BMT ( n = 26), allogeneic BMT ( n = 8), or syngeneic BMT ( n = 2). The male-to-female ratio was 22:14 (1.57), and the mean age was 41 ± 11 years (range 16–58). Before TBI, conditioning chemotherapy consisted of cyclophosphamide (CY) alone (60 mg/kg per day on 2 successive days) in 24 patients, CY combined with other drugs in 6, and combinations without CY in 6. All patients received single-dose TBI (10 Gy administered to the midplane at L4, and 8 Gy to the lungs). The mean instantaneous and average does rates were 0.039 ± 0.012 Gy/min (range 0.031–0.058), and 0.025-0.006 Gy/min (range 2.08–3.96), respectively. Granisetron was administered 30–45 min before TBI according to two different modalities: a total dose of 3 mg as a 5-min intravenous (i.v.) infusion (Treatment A, n = 15; 42%) or the same treatment plus 3 mg of granisetron as a 24-h continuous i.v. infusion (total dose: 6 mg, Treatment, n = 21; 58%). Depending on the BMT teams, hyperdiuresis was continued ( n = 19. 53%) or suspended ( n = 17, 47%) during TBI. Nausea and vomiting were assessed during the TBI session and the following 12 h, and were scored as follows: S1 = no nausea or vomiting; S2 = moderate nausea; S3 = severe nausea and/or single episode of vomiting; and S4 = multiple episodes of vomiting. Results : During TBI, 18 (50%) patients were scored as complete responders (S1), 1 (3%) as a major responder (S2), 9 (25%) as minor responders (S3), and 8 (22%) as nonresponders (S4). During the following 12 h, 28 (78%) patients were free of severe nausea and vomiting (S1 or S2), whereas 8 (22%) vomited (S3 or S4). In univariate analyses, the 12-h probability of emesis was significantly higher in patients undergoing hyperdiuresis (63% vs. 30%; p = 0.05), and in patients older than 45 years (65% for age >45 vs. 33% for age ≤45; p = 0.05). The probability of S3 and S4 emesis was 50% with Treatment A and 47% with Treatment B ( p = 0.86). Sex, body weight, and type of conditioning chemotherapy did not influence the 12-h probability of emesis. Multivariate analysis revealed that hyperdiuresis ( p = 0.02) and Treatment A ( p = 0.04) were independently associated with radiation-induced emesis, whereas sex ( p = 0.85), body weight ( p = 0.13), age ( p = 0.12), and type of conditioning chemotherapy ( p = 0.92) were not. No early toxicity related to granisetron was observed. Conclusion : Granisetron is a well-tolerated and effective antiemetic agent that can be used as monotherapy during single-dose TBI. Good control of nausea and vomiting is obtained with this antiemetic drug, and its effect is increased when hyperdiuresis is suspended during TBI.

Oral ribavirin for prevention of severe liver disease caused by hepatitis C virus during allogeneic bone marrow transplantation.

Four allogeneic bone marrow transplant patients who were infected with hepatitis C virus (HCV) before bone marrow transplantation (BMT) were treated with oral ribavirin during and after transplantation. None of the patients had severe liver complications. One patient died early. Polymerase chain reaction analysis revealed that two patients who were HCV RNA-positive before BMT became HCV RNA-negative during therapy and remained HCV RNA-negative during 6 and 12 months of follow-up, respectively. No severe side effects of ribavirin were documented. Ribavirin therapy can be given safely to patients undergoing BMT and can result in clearance of HCV RNA.

Loss of protective immunity to polio, diphtheria andHaemophilus influenzaetype b after allogeneic bone marrow transplantation

Immunity to poliovirus, diphtheria and Haemophilus influenzae type b (Hib) was studied in 16 adult recipients of a bone marrow transplant from an HLA-identical sibling donor in order to evaluate the need for revaccinations. T-cell depletion was not done in any case. The donors and patients were studied before bone marrow transplantation (BMT) and the patients 1, 3, 6, and 12 months later. Prior to the BMT 10 of 11 patients were immune (titre > or = 4) to all vaccine poliovirus types by a standard microneutralization assay. At 12 months after BMT only two of seven patients were immune to all vaccine types, and none had immunity against an antigenically altered poliovirus type 3 strain Finland. The geometric means of antibody titres against poliovirus types 1, 2, and 3 strain Saukett and strain Finland declined gradually after 1 month postgrafting, being 4.4, 5.4, 3.3, and 1.3 respectively at 12 months after BMT. At 1 year 6 of 11 patients had immunity against diphtheria by a toxin neutralization method, but the antitoxin geometric mean level had decreased to a barely protective level, 0.01 IU/ml. The geometric mean Hib antibody concentration decreased during the first 6 months after BMT and thereafter increased slightly. A significant proportion of BMT recipients lose their protection against polio, diphtheria and Hib, and revaccinations are necessary.

Combined transplantation of allogeneic bone marrow and CD34+ blood cells

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA-identical and MLC-sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti-CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to &gt; 100, &gt; 500, and &gt; 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Combined Transplantation of Allogeneic Bone Marrow and CD34+ Blood Cells

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA-identical and MLC- sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti-CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively. Group I patients were given platelet transfusions within 18 days and red blood cells within 10 days, whereas for group II patients, these time points were 26 and 17 days, respectively. These same transfusions could be ceased within 38 and 24 days, respectively, in control patients. The addition of about 2% more peripheral blood CD3+ cells (group I patients) did not result in higher grades of acute GVHD (median grade II) as compared with the controls (median grade II). Four of five group II patients showed no signs of acute GVHD. These data suggest that the addition of immunoselected allogeneic CD34+ progenitor cells to BM cells may accelerate hematopoietic recovery.

High dose chemotherapy and radiotherapy (HDCR) before bone marrow transplantation (BMT) results in selective loss of integrity of small intestinal (SI) but not gastroduodenal (GD) mucosa: Beneficial effects of enteral nutrition (EN)

High dose chemotherapy and radiotherapy (HDCR) before bone marrow transplantation (BMT) results in selective loss of integrity of small intestinal (SI) but not gastroduodenal (GD) mucosa: Beneficial effects of enteral nutrition (EN)

Pharmacokinetics of undiluted or diluted high-dose etoposide with or without busulfan administered to patients with hematologic malignancies.

We used two different methods to administer high-dose etoposide (VP-16) during a myeloablative conditioning chemotherapy regimen before bone marrow transplantation (BMT). VP-16 was administered either diluted (0.5 mg/mL) or undiluted (20 mg/mL), with or without busulfan. Blood samples were drawn from 17 patients during the infusion (6 hours) and thereafter daily until 2 days after BMT. VP-16 concentrations were measured in all plasma samples by high-performance liquid chromatography (HPLC) and electrochemical detection, and the results were analyzed with a pharmacokinetic data analysis system. All calculated pharmacokinetic parameters in the two patient groups (VP-16 administered diluted or undiluted) were similar. There were no statistically significant differences in half-lives, mean residence time (MRT), volume of distribution, total clearance, or area under the curve (AUC). VP-16 was found in blood samples from eight of 17 patients at the time of BMT. Significant differences in systemic drug exposure and systemic clearance were found when patients were grouped according to treatment with busulfan or with total-body irradiation (TBI). The two administration modes for VP-16, either diluted or undiluted, are bioequivalent in pharmacokinetic terms. The terminal half-lives were longer than expected and resulted in significant VP-16 plasma levels at the time of BMT. The biologic significance remains unclear. Busulfan and/or concomitant medication with phenytoin influence plasma clearance (clp) and systemic drug exposure significantly.

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.

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.

Improved survival at 2 and 5 years in the LMCE1 unselected group of 72 children with stage IV neuroblastoma older than 1 year of age at diagnosis: is cure possible in a small subgroup?

The objectives of this study were to determine (1) the role of selection before bone marrow transplantation (BMT), (2) the role of vincristine, melphalan, and total body irradiation (TBI) as consolidation of induction therapy for stage IV over 12 months at diagnosis, and (3) the role of immunomagnetic purging in metastatic neuroblastoma. Among 72 consecutive unselected patients, 10 were not grafted (four died at induction: two in complete remission [CR], two in partial remission [PR]); three had bone marrow progression before harvest; one had uncontrolled progression; and two had parental refusal). Sixty-two patients were grafted (23 in CR/very good PR [VGPR] and 39 in PR). Among the 62, 33 were consolidated with at least 90% excision of their initial tumor excised (53.2%), 15 with catecholamine secretions (24.2%), 22 with minor bone marrow involvement (35.5%), and 31 with positive bone scan (50%). Median observation time is 59 months. Progression-free survival (PFS) for the 10 excluded patients was 20% at 2 years and 0% at 4 years. PFS for the grafted population (n = 62) is 40% at 2 years, 20% at 4 years, and 13% at 7 years. No difference was observed between patients grafted in CR/VGPR or in PR. However, a group of 19 children was grafted resulting in complete normalization of metastasis (regardless of primary-site tumor status). In this group, PFS at 59 months was 38% with no relapses up to 7 years post-BMT. A group of 31 patients with no bone involvement at BMT was also identified. PFS at 5 years is 30% compared with 12% for bone-positive patients at BMT. Moreover, the 11 children presenting at diagnosis with no bone involvement (Evans stage IVS or stage C Memphis) and consolidated with BMT had PFS at 5 years of 50% with no late relapses. A subgroup of stage IV neuroblastoma patients older than 1 year of age at diagnosis may be curable with this therapeutic approach, and the use of multivariate analyses to search for prognostic factors is warranted in currently existing international registries.

Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients

Three radiation protocols currently used in treatment of leukemia patients before bone marrow transplantation (BMT) were investigated in a murine model (C57BL/6----C3H/HeJ) for BM allograft rejection. These include (a) a single dose of total body irradiation (8.5 Gy TBI delivered at a dose rate of 0.2 Gy/min), (b) fractionated TBI (12 Gy administered in six fractions, 2 Gy twice a day in 3 days, delivered at a dose rate of 0.1 Gy/min, and (c) hyperfractionated TBI (14.4 Gy administered in 12 fractions, 1.2 Gy three times a day in 3 days, delivered at a dose rate of 0.1 Gy/min). Donor-type chimerism 6 to 8 weeks after BMT and hematologic reconstitution on day 12 after BMT found in these groups were compared with results obtained in mice conditioned with 8 Gy TBI delivered at a dose rate of 0.67 Gy/min, routinely used in this murine model. The results in both parameters showed a marked advantage for the single dose 8.5 Gy TBI over all the other treatments. This advantage was found to be equivalent to three- to fourfold increment in the BM inoculum when compared with hyperfractionated radiation, which afforded the least favorable conditions for development of donor-type chimerism. The fractionated radiation protocol was equivalent in its efficacy to results obtained in mice irradiated by single-dose 8 Gy TBI, both of which afforded a smaller but not significant advantage over the hyperfractionated protocol. This model was also used to test the effect of radiation dose rate on the development of donor-type chimerism. A significant enhancement was found after an increase in dose rate from 0.1 to 0.7 Gy/min. Further enhancement could be achieved when the dose rate was increased to 1.3 Gy/min, but survival at this high dose rate was reduced. These results demonstrated indirectly that dose rate affects the expression of host-type pluripotent stem cells, the progeny of which appear 3 to 6 weeks after treatment with 8 Gy TBI delivered at a dose rate of 0.1 Gy/min, but which are eradicated if radiation is delivered at a dose rate of 1.3 Gy/min.