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Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions.

Purpose:: The aim of this study was to determine the incidence, risk factors, and proper management for asymptomatic cholelithiasis in children undergoing bone marrowtransplantation (BMT). Methods:: The authors reviewed retrospectively the records of 575 children who underwent bone marrow transplantation at a University bone marrow transplantation unit (BMT) unit between February 1991 and October 1999. Of these patients, 235 underwent abdominal ultrasonography for evaluation of jaundice, sepsis, abdominal pain, or metastasis. To identify risk factors for cholelithiasis, the authors stratified the patients based on their disease and treatment regimen. Finally, the authors analyzed the natural history and management of BMT children with cholelithiasis. Results:: The authors identified 20 cases of cholelithiasis (8.5%) in the 235 BMT patients who underwent ultrasonography. Children who underwent BMT to treat bone marrow failure showed a significantly increased risk of cholelithiasis compared with children treated for malignancy (27% v 7.4%; P < .01). Most children (85%) with gallstones did not require surgical intervention. Specifically, 9 (45%) died from their primary disease, 5 (25%) showed sonographic resolution of their gallstones, and 3 (15%) underwent follow-up nonoperatively with persistent cholelithiasis. Three of the 20 patients with gallstones (15%) had signs of acute cholecystitis and underwent surgery. There were no surgical complications or deaths in the operative group. Conclusions:: Cholelithiasis occurs at a high incidence in pediatric bone marrow transplant patients. Children undergoing BMT for bone marrow failure are at higher risk of having gallstones than those being treated for malignancy. Finally, these data support a strategy of nonoperative management for asymptomatic cholelithiasis in this highly selected group of patients.

Following our initial observation of in vivo conversion of CD7+, CD4-, CD8- acute lymphoblastic leukemia (ALL) cells from lymphoid to myeloid lineages (Proc Natl Acad Sci (USA) 81:253, 1984) we have studied eight additional cases of ALL with this leukemic cell phenotype. The CD7+, CD4-, CD8- phenotype was associated with a distinct clinical entity with those affected predominantly male (either less than 35 years or greater than 65 years of age), with frequent mediastinal and/or thymic masses, skin and CNS disease, high peripheral WBC counts, and bone marrow blasts that were morphologically L1 or not ascribable to a specific lineage. These patients did not respond to conventional chemotherapeutic regimens for either acute lymphoid or myeloid leukemias. No common karyotype or T-cell gene rearrangement pattern could be defined. Importantly, seven of eight patient's leukemic cells studied were capable of multilineage (myeloid, erythroid, monocytoid, megakaryocytoid, and lymphoid) differentiation in vitro. Data is presented suggesting that CD7+, CD4-, CD8- leukemias, in many instances, are leukemias of immature hematopoietic cells. The development of novel therapeutic approaches to this form of leukemia will be necessary to alter its poor prognosis.

We report six patients with progressive primary tumors of the brain who had prolonged periods with stable contrast-enhancing CT lesions following initial responses to chemotherapy. Chemotherapy was discontinued after 21 to 36 months, despite the persistence of apparent disease in each patient. PET using (F-18) fluorodeoxyglucose was performed in three patients, revealing hypometabolic lesions. All six patients are alive and well, with no clinical or radiographic evidence of progressive disease at 24 to 57 months following termination of treatment. The usual criteria for terminating phase II chemotherapy in patients with a recurrent brain tumor are evidence of progressive disease or unacceptable toxicity. However, chemotherapeutic success mandates that these criteria be expanded to include patients whose response following the initiation of phase II treatment is followed by prolonged (greater than 1 year) radiographic and clinical stability. Complete response, ie, disappearance of all evidence of disease, is unusual in patients with recurrent primary brain tumors, even with highly effective therapy. Continued improvement in the therapy of patients with these tumors will allow wider application of these criteria.

Summary Hematopoietic recovery in 115 patients with metastatic breast cancer or metastatic melanoma, enrolled in phase-I studies of recombinant growth factors while undergoing treatment with high-dose chemotherapy with autologous bone marrow support, was examined with assays of bone marrow progenitor cells and peripheral blood progenitor cells, and by evaluation of peripheral blood counts. Groups of patients receiving hematopoietic cytokine support [with interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or monocyte CSF (M-CSF)] post marrow infusion were compared with contemporaneous control patients not receiving growth factor support. Patients receiving GM-CSF demonstrated statistically significant increases in the growth of granulocyte/macrophage colony-forming units (CFU-GM) in the bone marrow and peripheral blood compared with control patients. The effect of GM-CSF was dose dependent in the early period post marrow infusion (day +6) with bone marrow CFU-GM colonies at doses 8–16 µg/kg/ day 34 times those measured in controls. Significant increases in bone marrow multipotential progenitor cells (CFU-GEMM) were seen in patients receiving GMCSF day + 21 post marrow infusion. Patients receiving IL-1 demonstrated significant increases in bone marrow CFU-GM at day +21, maximal at dosages of 24–32 ng/kg/day. There were no significant increases in burst forming unit-erythroid (BFU-E) among any study group. Patients receiving G-CSF had significantly increased absolute neutrophil counts (ANC) and total white blood cell counts (WBC) by day +11 post transplant compared with control patients. Patients receiving GM-CSF demonstrated significantly increased WBC (greater than 2000/mm3) at day +11 and ANC greater than 500/mm3 at day +16. Optimal dose of GCSF and GM-CSF to stimulate neutrophil recovery post transplant was 4–8 µg/kg/day and 8–16 µg/kg/day, respectively. Platelet recovery did not differ among the six study groups. These data demonstrate accelerated myeloid recovery after high-dose chemotherapy and autologous bone marrow support in patients receiving either G-CSF or GM-CSF. Moreover, GM-CSF and IL-1 stimulate myelopoiesis at the level of bone marrow CFU-GM, while G-CSF causes earlier neutrophil recovery peripherally.

Individuals infected by the human immunodeficiency virus type 1 (HIV-1) demonstrate progressive depletion and qualitative dysfunction of the helper T4 (CD4+) cell population. Mechanisms proposed for attrition of CD4+ T cells include direct cytopathicity of these mature cells following infection as well as infection of early T-lymphocyte progenitors. The latter mechanism could lead to failure to regenerate mature functioning CD4+ T cells. The present study determines the susceptibility of thymocytes at various stages of maturity to infection with HIV-1. Various normal thymocyte populations were inoculated with HIV-1, including unfractionated (UF), CD3- CD4- CD8- ["triple negative" (TN)], CD4+ CD8+ ["double positive" (DP)] thymocytes, and thymocyte populations obtained by limited dilution cloning. Cultures were studied for the presence of HIV-1 DNA by polymerase chain reaction in addition to examination for reverse transcriptase activity. We determined that transformed T-cell and thymocyte cell lines completely lacking CD4 were not susceptible to infection by HIV-1, whereas all of the following lines were: UF thymocytes (70-90% CD4hi+); DP thymocytes (99% CD4hi+); TN thymocytes (0% CD4hi+); and TCR alpha beta +, TCR gamma delta +, or CD16+ CD3- (natural killer) thymocyte clones expressing variable levels of CD4 and representing the progeny of TN thymocytes. [TCR alpha beta + and TCR gamma delta + refer to the chains of the T-cell antigen receptor (TCR), and CD4hi refers to a strong rightward shift (greater than 30 linear channels) of the CD4 curve on flow cytometric analysis compared with control.] Monoclonal antibodies (mAbs) to CD4 (T4a epitope) but not to CD3 (T3) were capable of blocking infection of mature and immature CD4hi+ thymocytes. Moreover, anti-CD4(T4a) mAbs also inhibited infection of CD4hi- TN thymocytes, indicating that these T-cell precursors--despite their apparent "triple negativity" (CD3- CD4hi- CD8-)--expressed sufficient CD4 molecules to become infected. Cell sorter analysis with a panel of CD4 mAbs demonstrated a mean shift of the mean fluorescence channel (MFC) with CD4 mAbs on TN thymocytes of 6 +/- 4 MFC units. Thus, intrathymic T-cell precursors and their progeny representing many stages of T-cell ontogeny are susceptible to infection by HIV-1, including early TN thymocytes, which express very low levels of CD4. Infection of multiple stages and multiple subsets of the T-cell lineage in man, mediated via the CD4 molecule, may explain the inability of the T-cell pool to regenerate in the setting of progressive HIV infection.

To study the role that epithelial cells of the thymic microenvironment play in promoting activation of immature CD7+, CD2+, CD4-, CD8- (double-negative) human thymocytes, we have isolated thymocyte subsets from normal postnatal thymus and have cocultured autologous double-negative thymocytes with pure populations of thymic epithelial (TE) cells. We report that TE cells directly activate double-negative thymocytes to proliferate and that TE cells enhance the ability of double-negative thymocytes to proliferate in response to stimulation with exogenous interleukin 2. Activated double-negative thymocytes that proliferated in vitro in the presence of TE cells and interleukin 2 remained double-negative after 23 days in culture. Moreover, TE-cell culture supernatants in the absence of intact TE cells contain interleukin 1, interleukin 3, and granulocyte/macrophage-colony-stimulating factor activity for human bone marrow cells and can activate double-negative thymocytes to proliferate. Antibodies against interleukin 1 and against granulocyte/macrophage-colony-stimulating factor inhibited TE-cell-induced thymocyte activation. These data indicate that one role of TE cells in vivo may be to activate double-negative thymocytes to proliferate.

We analyzed a t(1;14)(p32;q11) chromosomal translocation in a human lymphohemopoietic stem cell line derived from a patient with acute T-lymphoblastic leukemia. The chromosomal joining on the 1p+ chromosome occurred at the T-cell receptor delta diversity (D delta 2) segment, and the reciprocal chromosomal joining on the 14q- chromosome occurred at the T-cell delta diversity segment D delta 1. The involvement of delta diversity segments at the translocation junctions suggests that the translocation occurred during an attempt at D delta 1-D delta 2 joining in a stem cell. The segment of chromosome 1 at band p32, adjacent to the chromosomal breakpoint, encodes a transcriptional unit designated TCL5 (T-cell leukemia/lymphoma 5). The differential expression of the TCL5 RNA transcripts in this lymphohemopoietic stem cell line relative to several other T- and B-cell lines suggests that TCL5 gene expression is an integral event in the pathogenesis of the T-cell leukemia. Rearrangement of the TCL5 locus in a human melanoma cell line carrying a del(1p32) further implies that the TCL5 gene may play a role in malignant transformation.

We have studied a leukemic stem-cell line, DU.528, that is able to differentiate into myeloid and lymphoid cells. The leukemic cells have a translocation between chromosomes 1 and 14, t(1;14)(p33;q11), which we have molecularly cloned and sequenced. Initial screening used joining (J)-segment probes from the T-cell receptor (TCR) alpha- and delta-chain loci. In apparent concert with the translocation, a deletion has occurred between delta-chain diversity (D)-region genes D delta 1 and D delta 2. D delta 2 was observed on derivative chromosome 1 [der(1)] and D delta 1 on der(14) with a deletion of the intervening 10 kilobases of germ-line DNA. The nature of the D delta 1-D delta 2 deletional event implicates a lymphoid recombinase in the mechanism of the translocation. As a consequence of the translocation, an unusual fusion transcript was generated. Probes from chromosome 1 detected a previously unreported transcript in RNA from both the cell line and the patient. A chromosome 14 probe identified the same transcript, thus confirming a fusion transcript derived from both chromosomes 1 and 14. This translocation may identify a gene for which we propose the name SCL (stem-cell leukemia) that is important for hemopoietic development and oncogenesis and that has been disrupted or altered in this stem-cell line.

The signals and cellular interactions required for hematopoietic stem-cell commitment to the T lineage are unknown, yet are central to understanding the early stages of normal T-cell development. To study the differentiative capacity of T-cell precursors, we isolated CD4-, CD8-, surface(s) CD3- thymocytes from postnatal human thymuses and determined their capacity to differentiate into lymphoid and nonlymphoid lineages in vitro. We found that CD4-, CD8-, sCD3- thymocytes, which differentiated in the presence of T-cell conditioned medium plus interleukin 2 into T cells expressing the gamma delta receptor for antigen, were capable of differentiating into myeloid or erythroid lineages in the presence of either 5637 bladder carcinoma cell line conditioned medium plus recombinant human erythropoietin or human thymic epithelial cell conditioned medium. Thymic epithelial cell conditioned medium was as effective as 5637 supernatant plus erythropoietin in inducing myeloerythroid differentiation in the CD4-, CD8-, sCD3- thymocytes. Sixty-eight +/- 14% of CD4-, CD8-, sCD3- thymocytes underwent nonlymphoid differentiation within 4 days in culture with 5637 supernatant plus erythropoietin. Twenty-six +/- 4% of freshly isolated CD4-, CD8-, sCD3- cells were CD34+, and clonal granulocyte/macrophage, granulocyte/erythrocyte/monocyte/megakaryocyte, and T-cell progenitors were found in both CD34+ and CD34- subsets of CD4-, CD8-, sCD3- thymocytes. Thus, cells within the human CD4-, CD8-, sCD3- thymocyte subset can give rise to gamma delta+ T cells as well as to cells of myeloerythroid lineages. Moreover, CD34+, CD4-, CD8-, sCD3- cells can give rise to clonal T-cell progenitors as well as to clonal myeloid progenitors.

The human CD7 molecule is a 40-kDa member of the immunoglobulin gene superfamily that is expressed on T-lymphoid and myeloid precursors in fetal liver and bone marrow. CD7 is also expressed on T lymphocytes in multiple stages of T-cell development, including a major subset of mature peripheral T cells. In this paper we report the isolation and characterization of the human CD7 gene and 5' flanking region. Sequence analysis revealed that the CD7 gene comprises four exons that span 3.5 kilobases. The 5' flanking region (506 base pairs) has a high G + C content and no "TATA" or "CCAAT" elements. DNase I sensitivity analysis of chromatin from the CD7+ progenitor cell leukemia line, DU528, and the CD7-, CD4+, CD8+, TCR alpha beta + T-cell line, DU980 (where TCR is the T-cell receptor), revealed two distinct hypersensitive sites 5' of the CD7 gene. Hypersensitive site 1, present in the DU980 T-cell line, was located 4.5 kilobases upstream of the presumed CD7 transcription initiation site. Only DNase I hypersensitive site 2, which mapped to the promoter region, was found in the DU528 line. Comparison of the organization of the CD7 gene with that of other members of the immunoglobulin gene superfamily revealed that the human CD7 gene most closely resembles the murine Thy-1 gene. Both CD7 and Thy-1 are encoded by small genes with four exons, contain TATA-less promoters, and have a similar functional organization. These structural similarities suggest that human CD7 and murine Thy-1 may be functional homologues.

We have established the DU.528 cell line from the pretreatment leukemia cells of a patient who underwent a T lymphoblastic-to-promyelocytic phenotype conversion during treatment with the adenosine deaminase inhibitor, deoxycoformycin. The cell line and clones obtained from it by limiting dilution have the same karyotype previously found in the patient's pretreatment T lymphoblasts and post-deoxycoformycin treatment promyelocytes. DU.528 cells in continuous culture for greater than 2 yr display a predominant undifferentiated T lymphoblastoid phenotype. These cells spontaneously generate progeny of at least three lineages, T lymphoid, granulocytic/monocytic, and erythroid. The surface marker most consistently expressed by DU.528 cells in the undifferentiated state is the 3A1 antigen, which has been found on prothymocytes in the embryonic thymus. Some undifferentiated DU.528 cells also expressed the IL-2 receptor, but no other T cell differentiation antigens. Exposure of DU.528 cells to a variety of agents induced myeloid maturation; adenosine and deoxyadenosine, in the presence of deoxycoformycin, induced expression of myeloid differentiation antigens. Our results suggest that DU.528 is a lymphohematopoietic stem cell line and support the hypothesis that differentiation of pluripotent stem cells may be altered by genetic deficiency of adenosine deaminase. DU.528 cells may provide a useful model for examining factors that regulate stem cell proliferation and differentiation.

Two hematopoietic colony-stimulating factors, granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), have been shown to accelerate leukocyte and neutrophil recovery after high-dose chemotherapy and autologous bone marrow (BM) support. Despite their use, a prolonged period of absolute leukopenia persists during which infections and other complications of transplantation occur. We collected large numbers of peripheral blood (PB) progenitors after CSF administration using either G-CSF or GM-CSF and tested their ability to affect hematopoietic reconstitution and resource utilization in patients undergoing high-dose chemotherapy and autologous BM support. Patients with breast cancer or melanoma undergoing high-dose chemotherapy and autologous BM support were studied in sequential nonrandomized trials. After identical high-dose chemotherapy, patients received either BM alone, with no CSF; BM with either G-CSF or GM-CSF; or BM with G-CSF or GM-CSF and G-CSF or GM-CSF primed peripheral blood progenitor cells (PBPC). Hematopoietic reconstitution, as well as resource utilization, was monitored in these patients. The use of CSF- primed PBPC led to a highly significant reduction in the duration of leukopenia with a white blood cell (WBC) count under 100 and 200 cells/mL, and neutrophil count under 100 and 200 cells/mL with both GM- and G-CSF primed PB progenitor cells, compared with the use of the CSF with BM or with historical controls using BM alone. In addition, the use of CSF-primed PBPC resulted in a significant reduction in median number of antibiotics used, days in the Bone Marrow Transplant Unit, and hospital resources used. Patients receiving G-CSF primed PBPC also experienced a reduction in the median number of days in the hospital, red blood cell (RBC) transfusions, platelet transfusions, days on antibiotics, and discounted hospital charges. Phenotypic analysis of the CSF-primed PBPC indicated the presence of cells bearing antigens associated with both early and late hematopoietic progenitor cells. The use of CSF-primed PBPC can significantly improve hematopoietic recovery after high-dose chemotherapy and autologous BM support. In addition, the use of G-CSF-primed PBPC was associated with a significant reduction in hospital resource utilization, and a reduction in hospital charges.

Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 x 10(9)/L) due to these diseases were enrolled in this multicenter phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony- stimulating factor (filgrastim) (3.45 to 11.50 micrograms/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of = or = 1.5 x 10(9)/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently; adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.

Selective failure of lymphoid development occurs in genetic deficiency of adenosine deaminase (ADA). We examined the in vivo effects of a potent inhibitor of ADA, 2'-deoxycoformycin, which was used to treat a patient with refractory acute leukemia. Unexpectedly, within 7 days of starting treatment, the leukemic phenotype underwent complete conversion from T lymphoblastic to promyelocytic, with kinetics that suggested a precursor-product relationship between the two cell populations. Pretreatment T lymphoblasts and posttreatment promyelocytes had the same abnormal karyotype. Upon culture in vitro, the former transformed spontaneously over several weeks into mature myeloid cells. We conclude that the leukemia arose from a multipotent stem cell capable of both lymphoid and myeloid differentiation. Effects of ADA inhibition on leukemia cells during treatment included expansion of the deoxyadenosine nucleotide pool and accumulation of S-adenosylhomocysteine, a potent inhibitor of S-adenosylmethionine-dependent methylation. The influence of these changes on the leukemic phenotype is discussed in terms of (i) selective cytotoxicity to T lymphoblasts, which accumulated deoxyadenosine nucleotides more efficiently than did the patient's promyelocytes during in vitro incubation with deoxycoformycin plus deoxyadenosine, and (ii) induction of an altered program of differentiation.

During early fetal development, T cell precursors home from fetal yolk sac and liver to the epithelial thymic rudiment. From cells that initially colonize the thymus arise mature T cells that populate T cell zones of the peripheral lymphoid system. Whereas colonization of the thymus occurs late in the final third of gestation in the mouse, in birds and humans the thymus is colonized by hematopoietic stem cell precursors during the first third of gestation. Using a large series of early human fetal tissues and a panel of monoclonal antibodies that includes markers of early T cells (CD7, CD45), we have studied the immunohistologic location and differentiation capacity of CD45+, CD7+ cells in human fetal tissues. We found that before T cell precursor colonization of the thymus (7-8 wk of gestation), CD7+ cells were present in yolk sac, neck, upper thorax, and fetal liver, and were concentrated in mesenchyme throughout the upper thorax and neck areas. By 9.5 wk of gestation, CD7+ cells were no longer present in upper thorax mesenchyme but rather were localized in the lymphoid thymus and scattered throughout fetal liver. CD7+, CD2-, CD3-, CD8-, CD4-, WT31- cells in thorax and fetal liver, when stimulated for 10-15 d with T cell-conditioned media and rIL-2, expressed CD2, CD3, CD4, CD8, and WT31 markers of the T cell lineage. Moreover, CD7+ cells isolated from fetal liver contained all cells in this tissue capable of forming CFU-T colonies in vitro. These data demonstrate that T cell precursors in early human fetal tissues can be identified using a mAb against the CD7 antigen. Moreover, the localization of CD7+ T cell precursors to fetal upper thorax and neck areas at 7-8.5 wk of fetal gestation provides strong evidence for a developmentally regulated period in man in which T cell precursors migrate to the epithelial thymic rudiment.

The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.

3'-Azido-2',3'-deoxythymidine and carbovir [racemic and (-) enantiomer] were evaluated individually and in combination for antiviral activity against human immunodeficiency virus type 1 replication and cytotoxicity in vitro. The combination of these drugs synergistically inhibited human immunodeficiency virus type 1 replication in C3 and Jurkat cells and in human peripheral blood mononuclear cells, although the same combination also produced synergistic cytotoxicity in human peripheral blood mononuclear cells.

Reticular dysgenesis is a rare congenital disorder characterized by severe combined immunodeficiency and profound neutropenia, curable to date, only by bone marrow transplantation. This report describes the association of bilateral sensorineural deafness with this disease. (J Pediatr 1999;135:387-9)

Introduction: Severe VOD is an important regimen-related toxicity of hematopoietic SCT characterized by MOF and high mortality (>80% at d+100 post SCT). DF is a novel polydisperse oliogonucleotide with anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. DF appears to modulate endothelial cell (EC) injury in sVOD, with encouraging complete response (CR) rates, survival at d+100 post SCT and minimal toxicity reported. Methods: A multi-center, phase II trial was conducted in which patients (pts) stratified for age (<18y) and cyclophosphamide-based conditioning were randomized to 25 mg/kg/d or 40 mg/kg/d of DF. VOD diagnosis was made by Baltimore criteria, with severity determined by MOF or Bearman model (>30% risk). Study endpoints included CR, d+100 survival post SCT and toxicity. CR was defined as bilirubin <2 mg/dL and resolution of VOD-related MOF, with pts having ≥3d of DF evaluable for response. Abdominal ultrasound (US) with Doppler was required at study entry, d14 of DF, and completion of therapy, with blood collected serially to examine markers of EC stress. Results: 150 pts were treated with DF, with 75 pts on each treatment arm: 129 pts underwent allo- and 21 auto- SCT. Median age was 36y (6m–63y). At DF start, median bilirubin was 5.0 mg/dL; median weight gain 12%; ascites was present in 77%; RUQ pain in 71%; hepatomegaly in 69%; abnormal portal flow in 44%; and MOF in 99% (at study entry, 7% were dialysis-dependent (DD), 7% ventilator-dependent (VD); 28% developed DD and 23% VD during treatment). Median duration of DF therapy was 19d (1–82d). Of 150 pts treated, 141 were evaluable for response: 65 pts achieved CR (46%) and 62 survived to d+100 (41%), including pts with prior DD and VD. Results for CR and d+100 survival have been analyzed by dose level according to age, prior SCT and Mylotarg use. No significant difference in CR and d+100 survival was found between the 2 doses, although in pediatric pts receiving 25 mg/kg/d, CR and d+100 survival was 67% (p=0.06). Toxicity was limited, but more attributable G3/4 events, including bleeding and hypotension, were recorded in pts on 40 mg/kg/d, although the difference was not significant (p=0.11). Preliminary US review demonstrated improvement in portal flow abnormalities with DF therapy. EC stress markers showed reduction in median levels of PAI-1 and nitric oxide with increase in Protein C in pts achieving CR (p<0.05): median serum thrombomodulin, TFPI and soluble tissue factor increased in pts without response (p<0.05). Correlation with additional markers is ongoing. Similarly, multivariate analysis of factors predicting CR and d+100 survival is in process. Conclusion: DF treatment of sVOD/MOF results in a 46% CR rate and 41% d+100 survival post SCT: 25 mg/kg/d is the preferred dose. A phase 3 pivotal trial to confirm safety and efficacy is underway, comparing a prospective cohort of 80 DF-treated pts with sVOD/MOF to a matched historical group at 30 SCT centers across North America.

Introduction: Severe VOD is an important regimen-related toxicity of hematopoietic SCT characterized by MOF and high mortality (>80% at d+100 post SCT). DF is a novel polydisperse oliogonucleotide with anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. DF appears to modulate endothelial cell (EC) injury in sVOD, with encouraging complete response (CR) rates, survival at d+100 post SCT and minimal toxicity reported.

Introduction: Veno-Occlusive Disease (VOD) is a significant regimen-related toxicity of hematopoietic stem cell transplant (SCT), which when complicated by multi-system organ failure (MOF) has a case fatality rate >90%. We have treated 139 patients (pts) with VOD/MOF in a randomized, multicenter Phase II dose-finding study of Defibrotide (DF), a polydisperse oligonucleotide, with anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. Earlier studies of DF therapy for VOD/MOF have consistently shown manageable toxicity with promising complete response (CR) rates and d+100 survival post SCT. Methods: We reviewed pts to define status of renal, pulmonary, and CNS dysfunction at entry and on DF therapy. We also determined dialysis dependence (DD) and/or ventilator dependence (VD) at study entry or when newly acquired while on study. These subgroups were examined for response to treatment (CR vs no CR) and d+100 survival. Results: Of 139 pts treated, 97 had creatinine (creat) ≥ 2x admission value at study entry, of whom 42 pts had creat ≥ 3x admission value, and 12 were DD. 35 pts developed new DD on DF therapy. At entry, 69 pts had room air O2 saturation ≤ 90%, 10 were VD and 34 developed new VD on DF therapy. 33 pts were encephalopathic at study entry and 23 became encephalopathic during treatment. CR was achieved in 59/124 (48%) evaluable pts. By Kaplan Meier and on intent to treat, d+100 survival was 51/133 (38%). Pts were analyzed according to MOF (Table 1). In those with both renal and pulmonary dysfunction at study entry, 44% achieved CR and 39% d+100 survival. Pts with renal or pulmonary dysfunction at study entry, combined with encephalopathy, had a CR rate of 55% and d+100 survival of 45%. Those with MOF in all 3 systems achieved a CR rate of 50%, although d+100 survival was lower at 33%. The presence of DD at study entry or newly acquired on DF therapy reduced CR rates to 33 and 20% respectively, with a similar reduction in CR rates for VD at 20 and 25%. D+100 survival in both categories was also reduced. Conclusion: DF therapy achieved encouraging CR rates and d+100 survival in pts with MOF in up to three systems complicating VOD post SCT, and clinical benefit was seen even if pts were DD and/or VD. However, CR rates and d+100 survival were lower with DD and/or VD both at study entry and when newly acquired on DF therapy, which suggests that earlier intervention with DF may further improve outcome. Degree and Type of MOF CR D+100 survival *p <0.05 At Study Entry (%) Developed on DF Therapy (%) At Study Entry (%) Developed on DF Therapy (%) 2x Creat 23/50 (46) 7/12 (47) 22/55 (40) 4/12 (33) 3x Creat 25/42 (60) 15/32 (47) 22/42 (52) 15/34 (44) DD 4/12 (33) 7/35 (20)* 4/12 (33) 6/35 (17)* O2 Depend. 17/29 (59) 13/23 (57) 13/31 (42) 11/24 (46) VD 2/10 (20) 8/32 (25)* 2/10 (20) 7/34 (21)* Encephalopathy 16/30 (53) 8/23 (35) 13/33 (39) 8/23 (35) DD and VD 0/2 (0) 5/14 (36) 0/2 (0) 4/14 (29)

Introduction: Veno-Occlusive Disease (VOD) is a significant regimen-related toxicity of hematopoietic stem cell transplant (SCT), which when complicated by multi-system organ failure (MOF) has a case fatality rate >90%. We have treated 139 patients (pts) with VOD/MOF in a randomized, multicenter Phase II dose-finding study of Defibrotide (DF), a polydisperse oligonucleotide, with anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. Earlier studies of DF therapy for VOD/MOF have consistently shown manageable toxicity with promising complete response (CR) rates and d+100 survival post SCT.

Introduction: DF, a polydisperse oligonucleotide, has anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. Studies have suggested that DF modulates endothelial injury in VOD. Methods: A phase II randomized study of two doses, 25 mg/kg/d [arm A] or 40 mg/kg/d [arm B], was carried out in pts with severe VOD. Correlation with markers of vascular injury was undertaken. Endpoints included CR rate, toxicity and mortality at d+100 post SCT. VOD diagnosis was by Baltimore criteria and severity by ≥30% risk on the Bearman model or MOF. Abdominal ultrasound (US) was required prior to enrollment, repeated during therapy and at completion of treatment.Pts with ≥ grade II GVHD were excluded. Treatment arms were stratified for age (<18y) and cyclophosphamide-based conditioning. Treatment was planned for ≥14d. Pts receiving ≤3d of therapy were inevaluable for response. CR was defined as a bilirubin <2 mg/dl and resolution of VOD-related MOF. Results: 102 pts have been treated; 51 pts on arm A and 51 pts on arm B: 87 pts underwent allo- and 15 auto-SCT. Median age was 33y (6 mos - 63y). At DF initiation, median bilirubin was 7.1 mg/dl; median wt gain 12%; ascites in 78%; RUQ pain in 65%; hepatomegaly in 71%; abnormal portal flow in 42%, and MOF in 97%. Median duration of therapy was 20d. There were no unexpected side effects and no treatment related deaths. In a subset of pts (n=14), central review of US was performed. Portal flow improved by wk 3 in pts with CR (n=8). Improvement was not seen in nonresponders (NR) (n=6), suggesting changes in portal flow may help predict outcome. Median values of markers of endothelial stress with DF treatment are summarized below. In pts with CR, median PAI-1 levels decreased and Protein C increased. Median NO declined regardless of response. Median thrombomodulin, tissue factor and TFPI increased in NR. CR was achieved in 50/93 pts (54%; 95% CI (43%,64%)), with survival to d+100 in 43/91 pts (47%; 95% CI (37%,58%)). No difference in outcome between the 2 dose arms was seen. An analysis using 38 controls matched for VOD severity confirmed a survival advantage (p=0.0004). Conclusion: US findings and trends in endothelial stress markers may help predict successful DF treatment. The CR rate (54%) and d+100 survival (47%) in this first prospective, randomized trial of DF in pts with severe VOD and MOF confirm the encouraging results of prior studies, and a case control analysis shows a highly significant survival benefit with DF treatment. *p<0.05 Arm A Arm B **p<0.001 CR NR CR NR Pre/Post Pre/Post Pre/Post Pre/Post PAI-1 (nl<40) 109/53** 84/92 87/64* 99/102 Protein C (nl>70) 35/50** 33/31 29/43* 26/31 NO (nl<58) 98/68* 119/91 84/67 108/57 Thrombomodulin (nl<40) 144/135 149/178* 149/140 139/283** TFPI (nl<111) 116/124 154/213* 132/138 150/196* Tissue Factor (nl<162) 195/164 176/278* 197/202 179/298**

Introduction:DF, a polydisperse oligonucleotide, has anti-thrombotic, anti-ischemic and thrombolytic properties, especially on microvasculature. Studies have suggested that DF modulates endothelial injury in VOD.

Selective lymphopenla and Immunodeficiency are known clinical consequences of ADA deficiency (ADA-D) which also causes biochemical abnormalities In RBC's, notably a marked accumulation of dATP. In reported cases, RBC ATP has been normal or elevated. Decreased RBC survival has not been described. We studied a 9 month old girl with ADA-D who, at diagnosis, also had a heinz body (+) HA, with retlculocytosls and ahaptogloblnemla. Her RBC's showed an increased tendency to lyse spontaneously in vitro, which was not corrected by addition of glucose. RBC ATP, measured on four occasions, was 0.5-0.81 umol/ml (controls=1.42±0.35), and the level of dATP 1.0-1.53 umol/ml (normally <0.002), with a ratio of ATP:dATP of 0.46-0.67. We have previously defined a mechanism by which accumulation of dATP causes ATP catabolism in ADA inhibited lymphoid cells in vitro. Moreover, in vivo treatment with deoxycoformycin (dCF), a potent ADA inhibitor, can result not only in dATP accumulation, but also in ATP depletion in RBC's, and in hemolysis. The occurrence in a patient with genetic ADA-D of both very low ATP:dATP ratio and hemolysls establishes that hemolysls is a consequence of ADA-D per se, and not of some effect of dCF unrelated to its inhibition of ADA. We conclude that ADA-D is among the inborn errors of metabolism, including pyruvate kinase deficiency and massively increased RBC ADA activity, that can cause HA due to ATP depletion.