The impact of T-cell depletion techniques on the outcome after haploidentical hematopoietic SCT.

Several T-cell depletion (TCD) techniques are used for haploidentical hematopoietic SCT (HSCT), but direct comparisons are rare. We therefore studied the effect of in vitro TCD with graft engineering (CD34 selection or CD3/CD19 depletion, 74%) or in vivo TCD using alemtuzumab (26%) on outcome, immune reconstitution and infections after haploidentical HSCT. We performed a retrospective multicenter analysis of 72 haploidentical HSCT in Switzerland. Sixty-seven patients (93%) had neutrophil engraftment. The 1-year OS, TRM and relapse incidence were 48 (36-60)%, 20 (11-33)% and 42 (31-57)%, respectively, without differences among the TCD groups. In vivo TCD caused more profound lymphocyte suppression early after HSCT, whereas immune recovery beyond the second month was comparable between the two groups. Despite anti-infective prophylaxis, most patients experienced post-transplant infectious complications (94%). Patients with in vivo TCD had a higher incidence of CMV reactivations (54% vs 28%, P=0.015), but this did not result in a higher TRM. In conclusion, TCD by graft engineering or alemtuzumab are equally effective for haploidentical HSCT.

Do NK cells contribute to the pathophysiology of transplant-associated thrombotic microangiopathy?

Transplant-associated thrombotic microangiopathy (TA-TMA) is a life-threatening complication caused by the aggregation of platelets exposed to the thrombogenic subendothelial matrix of injured endothelial cells. Here, we present a case of a patient transplanted for idiopathic aplastic anemia with a T-cell depleted hematopoietic stem cell graft from an HLA-C mismatched unrelated donor. At day 7 posttransplant, she suffered from acute renal failure with hematuria. The presence of numerous schistocytes, an increased level of lactate dehydrogenase and a renal biopsy with multiple vascular injuries confirmed the diagnosis of severe TA-TMA. At day 14, she developed graft versus host disease and died 7 months posttransplantation of multiorgan failure. At day 15, we observed a sizable population of natural killer (NK) cells in the peripheral blood, the number of which reached 0.8 G/L at 4 months posttransplant. Most NK cells lacked inhibitory killer immunoglobulin-like receptors (KIR) specific for the KIR-ligands expressed in the patient. NK cells were also abundantly present in pericardial and pleural fluids and had invaded the kidney, where they colocalized with the renal vasculopathy. Because there are several mechanisms through which NK cells and platelets can activate each other reciprocally, it is conceivable that NK cells contribute to TA-TMA and its progression.

No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients.

BACKGROUND: Outcomes of Ewing tumor (ET) patients treated with allogeneic stem cell transplantation (allo-SCT) were compared regarding the use of reduced-intensity conditioning (RIC) and high-intensity conditioning (HIC) regimens as well as human leukocyte antigen (HLA)-matched and HLA-mismatched grafts. PATIENTS AND METHODS: We retrospectively analyzed data of 87 ET patients from the European Group for Blood and Marrow Transplantation, Pediatric Registry for Stem Cell Transplantations, Asia Pacific Blood and Marrow Transplantation and MetaEICESS registries treated with allo-SCT. Fifty patients received RIC (group A) and 37 patients received HIC (group B). Twenty-four patients received HLA-mismatched grafts and 63 received HLA-matched grafts. RESULTS: Median overall survival was 7.9 months [±1.24, 95% confidence interval (CI) 5.44-10.31] for group A and 4.4 months (±1.06, 95% CI 2.29-6.43) for group B patients (P = 1.3). Death of complications (DOC) occurred in 4 of 50 (0.08) and death of disease (DOD) in 33 of 50 (0.66) group A and in 16 of 37 (0.43) and 17 of 37 (0.46) group B patients, respectively. DOC incidence was decreased (P < 0.01) and DOD/relapse increased (P < 0.01) in group A compared with group B. HLA mismatch was not generally associated with graft-versus-Ewing tumor effect (GvETE). CONCLUSIONS: There was no improvement of survival with RIC compared with HIC due to increased DOD/relapse incidence after RIC despite less DOC incidence. This implicates general absence of a clinically relevant GvETE with current protocols.

Bronchoalveolar cytology for diagnosing pulmonary GVHD after bone marrow transplant in children.

BACKGROUND: Cytological composition of bronchoalveolar lavage (BAL) fluid in pediatric bone marrow transplant (BMT) recipients with pulmonary complications has not been comprehensively described and BAL specific markers of pulmonary GVHD are lacking. The aim of this retrospective study was to assess the role of BAL in the diagnosis of pulmonary GVHD by comparing BAL cytological findings between pediatric allogenic BMT patients with pulmonary complications and oncology children receiving chemotherapy alone. METHODS: Retrospective analysis of BAL specimens for cytology, total and differential cell counts and presence of infections. RESULTS: Seventeen BMT and 13 chemotherapy BAL were analyzed. BAL total cell count was increased but similar between groups (96.9 x 10(4) vs. 98.2 x 10(4), P = NS). BAL cellular composition differed considerably between groups with a significantly higher number of lymphocytes (18% vs. 6.25%, P = 0.03) and a significantly lower number of neutrophils (25.9% vs. 58%, P = 0.02) in BMT BAL specimens. Atypical epithelial cells were significantly more frequent (75% vs. 30.8%, P = 0.027), and significantly more severe (P = 0.01) in BMT patients. The presence and severity of atypia was not associated with infection or pneumotoxic drug exposure (P = NS). CONCLUSION: BAL cytology differs significantly between BMT and chemotherapy patients. The presence BAL lymphocytosis and severe epithelial cell atypia concomitantly to respiratory symptoms and GVHD in other organs may suggest the diagnosis of pulmonary GHVD. Prospective studies assessing the reliability of this finding combined with markers such as epithelial cell apoptosis and increased cytokines are needed.

Obesity in long-term survivors of childhood acute lymphoblastic leukemia.

BACKGROUND: Childhood acute lymphoblastic leukemia (ALL) with current cure rates reaching 80% emphasizes the necessity to determine treatment related long-term effects. The present study examines the prevalence of and the risk factors for overweight and obesity in a cohort of ALL survivors treated and living in the French speaking part of Switzerland. METHODS: In this retrospective two-center study, height and weight of 54 patients diagnosed with ALL in first complete remission and treated with chemotherapy only were recorded at specified time points during treatment and off-therapy. Body mass index (BMI) and its age- and gender-adjusted standard deviation score (BMI-SDS) were calculated for the patients and their parents separately. Overweight and obesity were defined by a threshold of BMI-SDS >1.645 and BMI-SDS >1.96, respectively. RESULTS: At last follow-up, 16 (30%) of the 54 survivors were overweight and 10 (18%) were obese. The off-treatment period was most at risk with 11 of the 16 becoming overweight and 9 of the 10 becoming obese during that period. Overweight/obesity at diagnosis and abnormal maternal BMI were significantly associated with abnormal weight at follow-up, while age at diagnosis, gender, cumulative dose of steroids and paternal BMI showed no association. CONCLUSIONS: Consistent with published evidence from other regions of the developed and developing world, there is a significant prevalence of obesity in young ALL survivors in the French speaking part of Switzerland. Factors significantly associated with this late effect were mostly related to the familial background rather than to the treatment components.

Successful treatment of neonatal rhesus hemolytic anemia with high doses of recombinant human erythropoietin.

The authors report the use of high-dose recombinant erythropoietin (r-HuEPO) in a full-term newborn baby with severe postnatal rhesus hemolytic anemia (RHA). Hemoglobin (Hb) value and reticulocyte count at day 13 of life were 59 g/L and 234 x 10(9)/L, respectively. Three days after the r-HuEPO (870 U/kg/d) administration, reticulocyte count had increased more than 4-fold and Hb rose to 73 g/L. r-HuEPO was gradually decreased after 18 days of treatment. No major side effect was observed. In selected cases of severe anemia due to hemolytic disorders, transfusions may be avoided by the use of high doses of r-HuEPO.

Sources of variation in patient response to radiation treatment.

PURPOSE: To investigate sources of variation in radiosensitivity displayed by cancer patients and blood donors using the leukocyte apoptosis assay. METHODS AND MATERIALS: Probes were obtained from 105 healthy blood donors, 48 cancer patients displaying normal sensitivity to radiotherapy, 12 cancer patients displaying hypersensitivity to radiotherapy, 12 Ataxia telangiectasia blood donors, and 4 additional individuals with genetic diseases of potentially modified radiosensitivity; 2 neurofibromatosis (NF) donors, a Nijmegen breakage syndrome (NBS) donor, and an Immunodeficiency, Chromosome fragility, Facial anomaly syndrome (ICF) donor. Heparinized blood was diluted in medium, irradiated, and left to incubate for 48 h. CD4 and CD8 T-lymphocyte DNA was stained with propidium iodide and the cells were analyzed by flow cytometry. RESULTS: Radiation-induced apoptosis depended on age and cell type. Cohorts of hypersensitive cancer patients, NBS and AT donors displayed compromised apoptotic response. An asymmetric apoptotic response of T-lymphocytes was observed in an ICF donor and a cryptic hypersensitivity donor. Two NF donors displayed no abnormal sensitivity to radiotherapy but compromised apoptotic T-cell response to X-rays. CONCLUSION: Our studies reveal 4 physiologic sources of variation in radiation response-2 are genetic: cryptic hypersensitivity and hereditary disease, and 2 are epigenetic: cell type and donor age. They emphasize the important role of proteins involved in the recognition and repair of DNA double-strand breaks in determining the response of individuals to radiotherapy.

Fatal myocardial aspergillosis in an immunosuppressed child.

A girl with resistant acute myeloid leukemia (AML) had a stem cell transplantation. Preceding transplantation, she had recurrent pneumonitis. No causative agent was identified. Despite several antibiotics including high-dose liposomal amphotericin-B, pulmonary infection progressed. Aspergillosis, always considered, could not be documented. She died from cardiac arrest on the second day after transplantation, with no forewarning of previous heart disease. Pericardial and myocardial aspergillosis was an autopsy finding. Pericardial and myocardial aspergillosis, rare manifestations of systemic aspergillosis, should be considered in any immunocompromised patient with long-lasting pulmonary infection, even in the absence of specific cardiac findings.

Trimethoprim-sulfamethoxazole salvage for refractory listeriosis during maintenance chemotherapy for acute lymphoblastic leukemia.

A 5-year-old boy with acute lymphoblastic leukemia (ALL) and intolerance to oral trimethoprim-sulfamethoxazole (TMP/SMX) had Listeria monocytogenes bacteremia and meningitis develop during maintenance chemotherapy. Despite prompt administration of IV amoxicillin/gentamicin and microbiologic clearance of the bloodstream, the patient had no response to therapy after a course of 7 days. Intravenous TMP/SMX (10 mg/kg per day of TMP) was added to the antibiotic regimen after desensitization. Fever and meningeal signs rapidly resolved, and the patient was ultimately cured. Amoxicillin and gentamicin, although highly active and synergistic in vitro against L. monocytogenes, have limited intracellular penetration and activity. In contrast, TMP/SMX has bactericidal extracellular and intracellular activity against Listeria and excellent central nervous system penetration, and thus may be effective for the treatment of refractory listeriosis.

Successful treatment of invasive aspergillosis in chronic granulomatous disease by bone marrow transplantation, granulocyte colony-stimulating factor-mobilized granulocytes, and liposomal amphotericin-B.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency with complete absence or malfunction of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the phagocytic cells. Life-threatening infections especially with aspergillus are common despite optimal antimicrobial therapy. Bone marrow transplantation (BMT) is contraindicated during invasive aspergillosis in any disease setting. We report an 8-year-old patient with CGD who underwent HLA-genoidentical BMT during invasive multifocal aspergillus nidulans infection, nonresponsive to treatment with amphotericin-B and gamma-interferon. During the first 10 days post-BMT, the patient received granulocyte colony-stimulating factor (G-CSF)-mobilized, 25 Gy irradiated granulocytes from healthy volunteers plus G-CSF beginning on day 3 to prolong the viability of the transfused granulocytes. This was confirmed in vitro by apoptosis assays and in vivo by finding nitroblue tetrazolium (NBT)-positive granulocytes in peripheral blood 12 and 36 hours after the transfusions. Clinical and biological signs of infection began to disappear on day 7 post-BMT. Positron emission tomography with F18-fluorodeoxyglucose (FDG-PET) and computed tomography (CT) scans at 3 months post-BMT showed complete disappearance of infectious foci. At 2 years post-BMT, the patient is well with full immune reconstitution and no sign of aspergillus infection. Our results show that HLA-identical BMT may be successful during invasive, noncontrollable aspergillus infection, provided that supportive therapy is optimal.

Blockade of acute grade IV skin and eye graft-versus-host disease by anti-interleukin-2 receptor monoclonal antibody in genoidentical bone marrow transplantation setting.

A six-year-old boy with homozygous beta-thalassemia in the favorable class 1 risk group received a bone marrow transplant, from his histocompatible sister. He developed grade IV skin and eye graft-versus-host disease (GVHD) following varicella zoster reactivation. Despite the appropriate prophylactic use of cyclosporin A (CsA), methotrexate (MTX), and prompt treatment with high-dose steroids, GVHD progressed resulting in total body epidermal necrolysis. Anti-IL-2 receptor monoclonal antibodies (anti-IL-2R moAb) in combination with steroids were administered to selectively block the activated T cells. After 27 days of daily administration, followed by 17 doses of alternate-day therapy with anti-IL-2R moAb, the severe skin and eye GVHD resolved. The patient, at two years posttransplant, has full engraftment and immune reconstitution without chronic GVHD (cGVHD). In conclusion, we suggest that in the HLA-genoidentical bone marrow transplantation setting, very severe and steroid-resistant GVHD can be controlled through the use of anti-IL-2 receptor antibodies which specifically block the activated IL-2 receptor expressing T cells.

Greater omentum flaps and granulocyte transfusions as combined therapy of liver abscess in chronic granulomatous disease.

We report the case of a 17-year-old boy with gp91phax-deficient chronic granulomatous disease who developed a liver abscess due to Staphylococcus aureus. Despite treatment with appropriate antibiotics and gamma interferon for three months as well as incision and drainage, the abscess persisted unchanged in size. After surgical debridement, the abscess cavity was filled with two pedunculated greater omentum flaps as a direct feeder road of granulocytes to the infectious focus. An average of 48.5 x 10(9) granulocytes a day harvested from G-CSF-prestimulated donors were transfused for a total of 8 days without side effects. Ultrasound 3 months later showed no residual abscess. Combination of greater omentum flaps and transfusion of G-CSF-prestimulated granulocytes may be the optimal treatment for liver abscesses refractory to conventional therapy.

Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes.

PURPOSE: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. METHODS AND MATERIALS: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. RESULTS: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r = 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r = 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen-ebb. Antigen-ebb was clearly observed in both cell types, and correlated significantly with cytotoxicity. A trend was observed between radiosensitivity and donor age, but there was no correlation for gender. Blood from a 4-year-old girl presenting with ICF demonstrated compromised radiation-induced cytotoxicity in her CD4 T-lymphocytes, and an 11-year-old boy presenting with AT demonstrated compromised radiation-induced cytotoxicity in both his CD4 and CD8 T-lymphocytes. CONCLUSION: We conclude that the assay provides a rapid means of determining radiosensitivity, can discriminate differences in radiation-induced cytotoxicity between individuals, and can be used as a rapid screen for genetically hypersensitive patients. Antigen-ebb offers interesting possibilities for molecular biological investigations, permitting characterization and isolation of abnormal but vital cells in the absence of clastogenic agents.

Adenosine deaminase deficiency in adults.

Adenosine deaminase (ADA) deficiency typically causes severe combined immunodeficiency (SCID) in infants. We report metabolic, immunologic, and genetic findings in two ADA-deficient adults with distinct phenotypes. Patient no. 1 (39 years of age) had combined immunodeficiency. She had frequent infections, lymphopenia, and recurrent hepatitis as a child but did relatively well in her second and third decades. Then she developed chronic sinopulmonary infections, including tuberculosis, and hepatobiliary disease; she died of viral leukoencephalopathy at 40 years of age. Patient no. 2, a healthy 28-year-old man with normal immune function, was identified after his niece died of SCID. Both patients lacked erythrocyte ADA activity but had only modestly elevated deoxyadenosine nucleotides. Both were heteroallelic for missense mutations: patient no. 1, G216R and P126Q (novel); patient no. 2, R101Q and A215T. Three of these mutations eliminated ADA activity, but A215T reduced activity by only 85%. Owing to a single nucleotide change in the middle of exon 7, A215T also appeared to induce exon 7 skipping. ADA deficiency is treatable and should be considered in older patients with unexplained lymphopenia and immune deficiency, who may also manifest autoimmunity or unexplained hepatobiliary disease. Metabolic status and genotype may help in assessing prognosis of more mildly affected patients.

Bone marrow transplantation in 26 patients with Wiskott-Aldrich syndrome from a single center.

We retrospectively analyzed the outcome of bone marrow transplantation (BMT) performed in 26 patients with Wiskott-Aldrich syndrome (WAS) in one center. Twenty-eight transplantation procedures were performed. Ten unselected patients received unmanipulated marrow from a donor with genetically identical human leukocyte antigen (HLA). Eight patients were cured and survive 1.5 to 16.5 years after BMT. One patient successfully received a T-cell-depleted marrow from a matched unrelated donor. Sixteen patients were selected to receive a related HLA partially incompatible BMT because of the occurrence of life-threatening complications from the WAS (i.e., refractory thrombocytopenia, autoimmunity including vasculitis and sepsis). All but one received T-cell-depleted marrow after a conditioning regimen of busulfan and cyclophosphamide. One patient had two BMTs. Engraftment occurred in 12 of 17 attempts. The addition of monoclonal antibodies to lymphocyte function-associated antigen-1 and CD2 molecules appeared to improve engraftment. Six patients were long-term survivors, whereas others died of viral infections (n = 7), among which Epstein-Barr virus-induced B-lymphocyte proliferative disorder was predominant. Delay in development of full T- and B-cell functions accounted for severe infectious complications. These results confirm the excellent outcome of HLA genetically identical BMT in WAS, whereas BMT from HLA partially incompatible donors should be strictly restricted to patients with severe complications of WAS.

Bone marrow transplantation in cartilage-hair hypoplasia: correction of the immunodeficiency but not of the chondrodysplasia.

UNLABELLED: We diagnosed cartilage-hair hypoplasia (CHH) in a female child with prenatal-onset short stature, metaphyseal chondrodysplasia, and severe combined immunodeficiency leading to recurrent, severe respiratory tract infections. The patient required several hospital admissions during her 1st year of life and failed to thrive in spite of antimicrobial therapy and hypercaloric nutrition. Bone marrow transplantation (BMT) from an HLA-identical sister was performed at age 16 months after conditioning with busulphan and cyclophosphamide, using 9 x 10(8) nucleated bone marrow cells/kg body weight. Graft-versus-host disease prophylaxis consisted of cyclosporine and methotrexate. The post-transplantation period was uneventful. She developed full and sustained chimerism as demonstrated by DNA analysis of granulocytes and mononucleated cells on days 44, 69 and 455 post BMT. Cellular immunity was completely reconstituted at 4 months, humoral immunity at 15 months post BMT. The patient is alive and well 24 months post BMT without medication, but the radiological osseous changes persist, and longitudinal growth remains markedly below the 10th percentile for CHH standards; her height at age 3 years 4 months is 66 cm. CONCLUSION: In this patient with unusually severe CHH, bone-marrow transplantation has fully corrected the immune deficiency but has had no influence on the course of the chondrodysplasia.

In vivo effects of GM-CSF and IL-3 on hematopoietic cell recovery in bone marrow and blood after autologous transplantation with mafosfamide-purged marrow in lymphoid malignancies.

This retrospective study evaluates the impact of GM-CSF and interleukin 3 (IL-3) on bone marrow (BM) and peripheral blood (PB) cell recovery following autologous bone marrow transplantation (ABMT) with mafosfamide-purged BM in patients with lymphoid malignancies compared with a control group receiving no colony-stimulating factor. GM-CSF was administered at 250 micrograms/m2/day (8 patients) as a continuous infusion from day of autologous BMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l for 7 days or until day 30, whichever was first. IL-3 was administered daily starting on the first day of transplant at a dose of 1 microgram/kg/day (6 patients) and 5 micrograms/kg/day (6 patients) for 30 days. CFU-GM and BFU-E were sequentially evaluated in BM and PB at days 7, 14, 21, 28, and 56 post-graft. The neutrophil recovery (ANC > 0.5 x 10(9)/l) was significantly faster in the GM-CSF group compared with IL-3 5 micrograms, IL-3 1 microgram and control group (respectively, days 15, 21, 22, 24) (p < 0.05 to p < 0.01). Similarly, leukocyte recovery was faster in the GM-CSF group compared with control and IL-3 1 microgram groups (p < 0.01 and p < 0.05). No difference was noticed between the two IL-3 groups. Although no difference was observed in platelet recoveries (> 50 x 10(9)/l), it appeared that the GM-CSF group required more units of platelets than either the IL-3 1 microgram or 5 micrograms groups (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of BCR/ABL translocation by polymerase chain reaction in leukemic progenitor cells (ALL-CFU) from patients with acute lymphoblastic leukemia (ALL).

In about 30% of cases, BCR/ABL transcripts are present in freshly isolated mononuclear cells from the bone marrow of patients with acute lymphoblastic leukemia (ALL) using the polymerase chain reaction (PCR) technique. We applied PCR to investigate the leukemic nature of ALL colony-forming unit (ALL-CFU) colonies grown in a clonogenic assay using a double feeder system. The high sensitivity of PCR enables us to detect 1 leukemic cell among 10(5) normal cells. Several controls were taken to assess contamination. In this report we studied three patients with ALL. In all of them, BCR/ABL transcripts were detected at initial diagnosis. We showed that the PCR technique could identify the leukemic nature of ALL-CFU progenitors. In addition, we demonstrated the nonleukemic nature of granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies using the same analysis. We conclude that the PCR technique is of great value in the identification of leukemic clones whenever specific molecular markers are present.