Summary:We investigated the use of 'prophylactic' donor lymphocyte infusions (DLI) containing 1 ؋ 10 7 CD3 ؉ cells, given at 30, 60 and 90 days post-allogeneic blood and marrow transplantation (BMT), following conditioning with fludarabine 30 mg/m 2 /4 days and melphalan 70 mg/m 2 /2 days. GVHD prophylaxis consisted of cyclosporin A (CsA) 2 mg/kg daily with early tapering by day 60. Our goals were the rapid achievement of chimerism and disease control, providing an immunological platform for DLIs to treat refractory patients with hematological malignancies. Twelve heavily pre-treated patients with life expectancy less than 6 months were studied; none were in remission. Diagnoses were AML (n ‫؍‬ 4), MDS (n ‫؍‬ 1), ALL (n ‫؍‬ 3), CML (n ‫؍‬ 3) and multiple myeloma (n ‫؍‬ 1). Response rate was 75%. Three patients are alive at a median of 450 days (range, 450-540). Two patients are in remission of CML in blast crisis and AML for more than 14 months. Median survival is 116 days (range, 25-648). Six patients received 12 DLIs; three patients developed acute GVHD after the first infusion and were excluded from further DLIs, but no GVHD occurred among patients receiving subsequent DLIs. One patient with CML in blast crisis went into CR after the first DLI. The overall incidence of acute GVHD was 70%. Primary causes of death were infections (n ‫؍‬ 3), acute GVHD (n ‫؍‬ 3), chronic GVHD (n ‫؍‬ 1) and disease relapse (n ‫؍‬ 2). We observed high response and chimerism rates at the expense of an excessive incidence of GVHD. DLI given at day ؉30 post BMT caused GVHD in 50% of the patients, and its role in this setting remains unclear. The therapeutic benefit of allogeneic BMT is in part related to an immunological graft-versus-leukemia (GVL) effect that frequently evolves in the context of graft-versus-host disease (GVHD). The ability of donor lymphocytes to induce remission in patients relapsing after allogeneic transplantation illustrates the potency of this effect.1 Establishing donor-recipient tolerance with less toxic regimens may provide the basis for further immunological manipulations in order to maximize the GVL effect. However, rapidly evolving diseases may not be amenable to this strategy, considering that the immune-mediated elimination of malignant cells may take weeks or months to occur. This fact suggests the need for strategies to reinforce the immune-mediated phenomena in the post-transplant period.Groups in Jerusalem and Houston pioneered the use of sub-lethal doses of fludarabine-based conditioning regimens. These regimens have been shown to be less toxic and to provide enough immunosuppression to prevent graft rejection and establish stable mixed or complete chimerism.2,3 The combination of melphalan and fludarabine has enabled allogeneic stem cell engraftment in the majority of patients treated, at least in the setting of HLA-identical transplantation. Patients with refractory relapses of advanced leukemias appear to benefit the least.

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Summary:In order to assess the effect of delaying G-CSF administration after autologous peripheral blood progenitor cell (PBPC) transplantation on the duration of neutropenia, 87 patients were randomized to receive G-CSF 5 g/kg/day starting on day +1 (n = 45) or +5 (n = 42) following PBPC transplantation, until recovery of the neutrophils. The duration of neutropenia (Ͻ0.5 ؋ 10 9 /l) was shorter in the day +1 group (7 vs 8 days; P = 0.02), especially in patients receiving melphalan 200 mg/m 2 and CD34 + cell doses Ͼ3.0 ؋ 10 6 /kg. These patients had a later onset of neutropenia after transplant. There were no differences in time to neutrophil and platelet engraftment, or in the incidence of fever and documentation of infection. Although the duration of antibiotic therapy (7 vs 10.5 days; P = 0.01) and time to hospital discharge (13 vs 15 days; P = 0.02) were shorter in the day +1 group, these differences could not be predicted by the day of G-CSF initiation in multivariate analysis. Starting G-CSF on day +1 does not result in faster neutrophil engraftment but in later onset and consequently, slightly shorter duration of neutropenia in patients who receive melphalan 200 mg/m 2 and CD34 + cell doses Ͼ3.0 ؋ 10 6 /kg. Bone Marrow Transplantation (2002) 29, 745-751.

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Summary:We report a case of capillary leak syndrome in a 37-year-old female PBPC donor who received G-CSF 900 g/day for 4 days and underwent leukapheresis. This lady had remained well and stable despite marked leukocytosis during G-CSF treatment, but developed hypotension during leukapheresis, quickly followed by hypoxemia, ascites, pericardial and pleural effusion, shock, edema, neurologic changes and hepatocellular injury. Upon G-CSF withdrawal, dopamine and crystalloid infusion, methylprednisolone treatment and suspension of apheresis, the clinical situation fully reversed. We hypothesize that leukapheresis, in the presence of marked leukocytosis and high doses of G-CSF, may have triggered neutrophil activation and the release of inflammatory mediators, resulting in tissue damage and systemic manifestations of increased capillary permeability. Bone Marrow Transplantation (2001) 28, 311-312. Keywords: G-CSF; capillary leak syndrome; leukapheresis; peripheral blood progenitor cell transplantation A 37-year-old white female agreed to donate allogeneic G-CSF-mobilized PBPC for her brother. She was asymptomatic, in good health, and used no medications. Her past medical history was unremarkable. Physical examination was normal except for moderate obesity and bradycardia. She had a normal chest film and echocardiogram. ECG showed sinus bradycardia. Serum biochemistry and coagulation tests were normal, Hb was 12.9 g/dl, hematocrit 38%, WBC 6.4 × 10 9 /l (ANC 3.1 × 10 9 /l) and platelets 322 × 10 9 /l. She received G-CSF 900 g s.c. daily in two divided doses for 4 days (10 g/kg/day). On the fifth day, the WBC count was 90.5 × 10 9 /l. Her only symptom thus far had been moderate headache which subsided with acetaminophen. A last dose of G-CSF was given. Three hours after the start of apheresis, she was pale and hypotensive, complaining of respiratory discomfort, nausea and weakness. Intravenous saline and calcium gluconate were given, with partial improvement. Apheresis was stopped after 5 h and she received another dose of G-CSF, because the number of CD34 + cells collected had been insufficient. Returning in the next morning, she complained of dyspnea, pleuritic pain and 'tightness in the chest'. Hematocrit was 33% and WBC 74.7 × 10 9 /l. Minutes later, she fainted and was admitted to the emergency room with severe hypotension, pallor, pulmonary rales, hypoxemia, cyanosis and confusion. A CT scan showed ascites, pericardial fluid and bilateral pleural effusion (Figures 1 and 2). Pericardiocentesis yielded clear fluid with 65 cells/mm 3 (77% neutrophils), 4.2 g/dl protein and LDH 512 U/l. Pleural fluid had the same characteristics. Her hemodynamic status improved with i.v. saline and dopamine. She remained confused and agitated for the next 12 h, normalizing her mental status gradually thereafter. A CT scan of the head was normal. Her weight was 8 kg above baseline. Her previously normal alanine aminotransferase peaked at 568 U/l (normal: Ͻ37 U/l). Bilirubin and alkaline phosphatase were unchanged. Hypoalbuminemia (3....

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

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