Reduction in kidney function decline and risk of severe clinical events in agalsidase beta-treated Fabry disease patients: a matched analysis from the Fabry Registry.

Background: Patients with Fabry disease (FD, α-galactosidase A deficiency or absence) accumulate glycosphingolipids, leading to progressive dysfunction of kidneys, heart and nervous system. Generalizable real-world outcomes following agalsidase beta treatment initiation outside trials are limited. We investigated the associations of long-term agalsidase beta treatment with estimated glomerular filtration rate (eGFR) changes over time and the risk of developing a composite clinical event in a matched analysis of treated and untreated patients with FD. Methods: Agalsidase beta-treated adult patients (aged ≥16 years) from the Fabry Registry and adult untreated patients from a natural history cohort were matched 1:1 and X:X (with one occurrence and multiple occurrences of each untreated patient, respectively) by sex, phenotype, age and (for eGFR slope analysis) baseline eGFR. Outcomes included eGFR slope over 5 years and composite clinical event risk (cardiovascular, cerebrovascular or renal event, or death) over 10+ years. As a surrogate indicator of therapeutic response in paediatric patients, the percentage experiencing normalization in plasma globotriaosylceramide (GL-3) from treatment initiation was assessed in patients aged 2 to <16 years. Results: Overall, eGFR slopes for 1:1-matched untreated and treated adult patients [122 pairs (72.1% male)] were -3.19 and -1.47 mL/min/1.73 m2/year, respectively (reduction in rate of decline = 53.9%, P = .007), and for X:X-matched [122 untreated/950 treated (59.4% male)] were -3.29 and -1.56 mL/min/1.73 m2/year, respectively (reduction in rate of decline = 52.6%, P < .001). Agalsidase beta treatment was associated with lower risk of clinical events, with hazard ratios of 0.41 (P = .003) and 0.67 (P = .008) for 1:1-matched and X:X-matched analyses, respectively. Plasma GL-3 declined markedly in paediatric patients and normalized in most within 6 months of treatment initiation. Conclusion: Agalsidase beta treatment preserves kidney function and delays progression to severe clinical events among adult patients with FD. Plasma GL-3 levels analysed in paediatric patients showed normalization of elevated pre-treatment levels in most patients.

At three years, patients with acute lymphoblastic leukaemia are still at risk for relapse. Results of the international MRC UKALLXII/ECOG E2993 trial.

Late relapse [>3 years from complete remission (CR)] in acute lymphoblastic leukaemia (ALL), is unusual. Data from the MRC UKALLXII/ECOG E2993 trial are presented to evaluate the incidence and characteristics of late relapse in adult ALL. Of 1,909 patients, 1,752 (92%) achieved CR and among these 757 (43·2%) relapsed; 691 (91·3%) within three years and 66 (8·7%) beyond. Among these 66 patients, median time to relapse was 47 (37-144) months. Relapse beyond three years occurred in 3·8% of all who achieved CR. The cumulative risk of relapse was 40%, 43% and 45% at three, five and ten years respectively. Out of the 1 752 patients who achieved CR, 11·7% underwent autologous and 40·6% allogeneic transplant, while in CR1. Of the autologous patients, 43·2% relapsed early and 3·4% relapsed late. However, among the allogeneic patients, 13·2% relapsed early and only 1·3% late. The five-year overall survival from relapse was 5·8% and 20% in the early and late relapse patients respectively. In conclusion, late relapse in adults with ALL is not uncommon, and is associated with better outcome after relapse compared to early relapse.

Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

BACKGROUND: Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. METHODS: This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 108 vector genomes (vg); cohort 2, 2 × 109 vg; cohort 3, 6 × 109 vg; and cohort 4, 2 × 1010 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 1010 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. FINDINGS: 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 1010 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 1010 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to a mean of 53·2 ng/mL at week 52 (SD 17·1). At baseline, four of these five patients were negative for anti-AAV2 serum antibodies and the fifth had a very low titre (1:100) of anti-AAV2 antibodies, whereas four of the five non-expressers of sFLT01 had titres of 1:400 or greater. In 11 of 19 patients with intraretinal or subretinal fluid at baseline judged to be reversible, six showed substantial fluid reduction and improvement in vision, whereas five showed no fluid reduction. One patient in cohort 5 showed a large decrease in vision between weeks 26 and 52 that was not thought to be vector-related. INTERPRETATION: Intravitreous injection of AAV2-sFLT01 seemed to be safe and well tolerated at all doses. Additional studies are needed to identify sources of variability in expression and anti-permeability activity, including the potential effect of baseline anti-AAV2 serum antibodies. FUNDING: Sanofi Genzyme, Framingham, MA, USA.

Immune tolerance induction for laronidase treatment in mucopolysaccharidosis I.

Enzyme replacement therapy (ERT) can produce anti-drug antibody (ADA) responses that reduce efficacy or lead to hypersensitivity reactions. Six patients with severe mucopolysaccharidosis type I (MPS I/Hurler syndrome) who did not receive hematopoietic stem cell transplantation underwent an immunosuppression regimen prior to initiating ERT with laronidase. The primary endpoint for immune tolerance induction was the number of patients with an ADA titer ≤ 3200 after 24 weeks of laronidase at the labeled dose. Cyclosporine levels were measured weekly and doses adjusted to maintain trough levels above 400 mg/mL. A 6-week (Cohort 1) or 12-week (Cohort 2) immune tolerance induction period with cyclosporine (initial dose: 15 or 20 mg/kg/day), azathioprine (initial dose: 2.5 or 5 mg/kg/day) and low-dose laronidase infusions (0.058-0.29 mg/kg/week) was followed by an immune-challenge period with laronidase infusions at the labeled dose (0.58 mg/kg/week) for 24 weeks. Anti-laronidase IgG titers were determined following treatment. There were 147 treatment-emergent adverse events reported, most of which were mild and not related to the study treatment. While there was no evidence of immune tolerance in 3 of 3 patients in Cohort 1, there were some indications of immune tolerance induction in 2 of 3 patients in Cohort 2. Patients with lower ADA titers showed greater reductions in urinary glycosaminoglycan excretion. Routine monitoring of plasma cyclosporine parent-compound levels by high pressure liquid chromatography proved difficult for clinical practice. The evolving clinical management of MPS I and a better understanding of the clinical impact of laronidase-related immunogenicity require reassessment of immune modulation strategies in patients with MPS I receiving laronidase treatment. CLINICAL TRIAL REGISTRATION: NCT00741338.

Transient low-dose methotrexate generates B regulatory cells that mediate antigen-specific tolerance to alglucosidase alfa.

Biologic drugs, including enzyme-replacement therapies, can elicit anti-drug Abs (ADA) that may interfere with drug efficacy and impact patient safety. In an effort to control ADA, we focused on identifying regimens of immune tolerance induction that may be readily available for clinical use. Data generated in both wild-type mice and a Pompe disease mouse model demonstrate that single-cycle, low-dose methotrexate can be as effective as three cycles of methotrexate in providing a long-lived reduction in alglucosidase alfa-specific ADA. In addition, we show that methotrexate induces Ag-specific tolerance as mice generate similar Ab responses to an irrelevant Ag regardless of prior methotrexate treatment. Methotrexate-induced immune tolerance does not seem to involve cell depletion, but rather a specific expansion of IL-10- and TGF-ß-secreting B cells that express Foxp3, suggesting an induction of regulatory B cells. The mechanism of immune tolerance induction appears to be IL-10 dependent, as methotrexate does not induce immune tolerance in IL-10 knockout mice. Splenic B cells from animals that have been tolerized to alglucosidase alfa with methotrexate can transfer tolerance to naive hosts. We hypothesize that methotrexate induction treatment concomitant with initial exposure to the biotherapeutic can induce Ag-specific immune tolerance in mice through a mechanism that appears to involve the induction of regulatory B cells.

Levels of teen dating violence and substance use in an urban emergency department.

OBJECTIVE: Teen dating violence (TDV) is associated with multiple sequelae including substance use. The objective of this study was to determine the prevalence and association between levels of dating violence and substance use among urban adolescents presenting at a pediatric emergency department (ED). METHODS: As part of standard practice, 282 adolescents were screened for relationship status, producing 135 dating violence screens. Scales from the Revised Conflict Tactics Scale and the Youth Risk Behavior Survey were used to capture variables of interest. Logistic regression was performed to test the influence of levels of dating violence on substance use, while controlling for gender, race, age, sexual orientation, and psychiatric symptoms. RESULTS: Over one-quarter of those teens (27.3%) within a current relationship reported experiencing any dating violence, 26.1% experienced psychological violence, and 11.9% experienced physical violence. Teens experiencing psychological violence were at twice the risk for any substance use (alcohol, marijuana, and tobacco use) and specifically for alcohol and marijuana, whereas no increased risk was found for teens experiencing physical violence. CONCLUSIONS: This study contributes to the understanding of TDV within the context of high-risk, urban adolescents presenting at a pediatric ED. Identifying levels of TDV and understanding the association with substance use can provide an important foundation for prevention and early intervention for urban youth.

Thiopurine methyltransferase genotype-phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia.

AIMS: In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. METHODS: We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. RESULTS: Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. CONCLUSIONS: In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.

Cathelicidin antimicrobial peptide expression is not induced or required for bacterial clearance during salmonella enterica infection of human monocyte-derived macrophages.

Salmonella enterica serovar Typhimurium is able to resist antimicrobial peptide killing by induction of the PhoP-PhoQ and PmrA-PmrB two-component systems and the lipopolysaccharide (LPS) modifications they mediate. Murine cathelin-related antimicrobial peptide (CRAMP) has been reported to inhibit S. Typhimurium growth in vitro and in vivo. We hypothesize that infection of human monocyte-derived macrophages (MDMs) with Salmonella enterica serovar Typhi and S. Typhimurium will induce human cathelicidin antimicrobial peptide (CAMP) production, and exposure to LL-37 (processed, active form of CAMP/hCAP18) will lead to upregulation of PmrAB-mediated LPS modifications and increased survival in vivo. Unlike in mouse macrophages, in which CRAMP is upregulated during infection, camp gene expression was not induced in human MDMs infected with S. Typhi or S. Typhimurium. Upon infection, intracellular levels of ΔphoPQ, ΔpmrAB, and PhoP(c) S. Typhi decreased over time but were not further inhibited by the vitamin D(3)-induced increase in camp expression. MDMs infected with wild-type (WT) S. Typhi or S. Typhimurium released similar levels of proinflammatory cytokines; however, the LPS modification mutant strains dramatically differed in MDM-elicited cytokine levels. Overall, these findings indicate that camp is not induced during Salmonella infection of MDMs nor is key to Salmonella intracellular clearance. However, the cytokine responses from MDMs infected with WT or LPS modification mutant strains differ significantly, indicating a role for LPS modifications in altering the host inflammatory response. Our findings also suggest that S. Typhi and S. Typhimurium elicit different proinflammatory responses from MDMs, despite being capable of adding similar modifications to their LPS structures.

Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo.

Salmonella enterica serovar Typhimurium uses two-component regulatory systems (TCRSs) to respond to environmental stimuli. Upon infection, the TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals detected in the lumen of the intestine and within host cells. TCRS-mediated gene expression leads to upregulation of genes involved in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide (CAMP) resistance. This research expands on previous studies which have shown that CAMPs can activate Salmonella TCRSs in vitro. The focus of this work was to determine if CAMPs can act as environmental signals for PhoPQ- and PmrAB-mediated gene expression in vitro, during infection of macrophages and in a mouse model of infection. Monitoring of PhoPQ and PmrAB activation using recombinase-based in vivo expression technology (RIVET), alkaline phosphtase and ß-galactosidase reporter fusion constructs demonstrated that S. Typhimurium PhoQ can sense CAMPs in vitro. In mouse macrophages, the cathelecidin CRAMP does not activate the PhoPQ regulon. Acidification of the Salmonella-containing vacuole activates PhoP- and PmrA-regulated loci but blocking acidification still does not reveal a role for CRAMP in TCRS activation in mouse macrophages. However, assays performed in susceptible wild type (WT), CRAMP knockout (KO), and matrilysin (a metalloproteinase necessary for activating murine α-defensins) KO mice suggest CRAMP, but not α-defensins, serve as a putative direct TCRS activation signal in the mouse intestine. These studies provide a better understanding of the in vivo environments that result in activation of these virulence-associated TCRSs.

Liver and skin histopathology in adults with acid sphingomyelinase deficiency (Niemann-Pick disease type B).

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin (SM) in multiple cell types, and occurs most prominently within the liver, spleen, and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial and each received a single dose of rhASM and underwent a repeat liver biopsy on day 14. Biopsies were evaluated for fibrosis, SM accumulation, and macrophage infiltration by light and electron microscopy. When present, fibrosis was periportal and pericellular, predominantly surrounding affected Kupffer cells. Two baseline biopsies exhibited frank cirrhosis. SM was localized to isolated Kupffer cells in mildly affected biopsies and was present in both Kupffer cells and hepatocytes in more severely affected cases. Morphometric quantification of SM storage in liver biopsies ranged from 4% to 44% of the microscopic field. Skin biopsies were also performed at baseline and day 14 to compare the SM distribution in a peripheral tissue with that of liver. SM storage was present at lower levels in multiple cell types of the skin, including dermal fibroblasts, macrophages, vascular endothelial cells, vascular smooth muscle cells, and Schwann cells. This phase 1 trial of rhASM in adults with ASMD provided a unique opportunity for a prospective assessment of hepatic and skin pathology in this rare disease and their potential usage as pharmacodynamic biomarkers.

Outcomes in older adults with acute lymphoblastic leukaemia (ALL): results from the international MRC UKALL XII/ECOG2993 trial.

Although the incidence rate of acute lymphoblastic leukaemia (ALL) is slightly higher in older than in younger adults, response rates to induction chemotherapy and survival rates are poorer. The contribution of disease-related versus treatment-related factors remains unclear. We analysed 100 older patients (aged 55-65 years) treated on the UKALLXII/ECOG2993 trial compared with 1814 younger patients (aged 14-54 years). Baseline characteristics, induction chemotherapy course, infections, drug reductions and survival outcomes were compared. There were more Philadelphia-positive (Ph+) patients in the older group (28% vs. 17%, P = 0·02), and a trend towards higher combined cytogenetic risk score (46% vs. 35%, P = 0·07). The complete remission rate in older patients was worse (73% vs. 93%, P < 0·0001) as was 5-year overall survival (21% vs. 41%, P < 0·0001) and event-free survival (EFS) (19% vs. 37%, P < 0·0001). Older patients had more infections during induction (81% vs. 70%, P = 0·05), and drug reductions (46% vs. 28%, P = 0·0009). Among older patients, Ph+ and cytogenetic risk category as well as infection during induction predicted for worse EFS. Poorer outcomes in these patients are partly due to cytogenetic risk, but there is significant morbidity and mortality during induction chemotherapy with frequent delays and drug reductions. New approaches, including better risk stratification and use of targeted therapies, could improve treatment for these patients.

Anti-α-galactosidase A antibody response to agalsidase beta treatment: data from the Fabry Registry.

Agalsidase beta, a form of recombinant human α-galactosidase A (αGAL), is approved for use as enzyme replacement therapy (ERT) for Fabry disease. An immunogenic response against a therapeutic protein could potentially impact its efficacy or safety. The development of anti-αGAL IgG antibodies was evaluated in 571 men and 251 women from the Fabry Registry who were treated with agalsidase beta. Most men developed antibodies (416 of 571, 73%), whereas most women did not (31 of 251, 12%). Women were also significantly more likely to tolerize than men; whereas 18 of 31 women tolerized (58%, 95%CI: 52%-64%), only 47 of 416 men tolerized during the observation period (11%, 95% CI: 8%-15%). Patients who eventually tolerized had lower median peak anti-αGAL IgG antibody titers than patients who remained seropositive at their most recent assessment (400 versus 3200 in men, 200 versus 400 in women, respectively). Patients with nonsense mutations in the GLA gene were more likely to develop anti-αGAL IgG antibodies than patients with missense mutations. Approximately 26% of men (151 of 571) reported infusion-associated reactions (IARs), compared to 11% of women (27 of 251). Men who developed anti-αGAL IgG antibodies were more likely to experience IARs compared to those who remained seronegative. Nine percent of seronegative men and women (34 of 375) reported IARs. The majority of IARs occurred during the first 6 to 12 months of agalsidase beta treatment and decreased over time, in both seroconverted and seronegative patients.

Murine antithymocyte globulin T-cell depletion is mediated predominantly by macrophages, but the Fas/FasL pathway selectively targets regulatory T cells.

BACKGROUND: Thymoglobulin is a T-cell-depleting polyclonal rabbit anti-human thymocyte antibody used clinically for immunosuppression in solid organ and hematopoietic stem-cell transplantation. By using a surrogate rabbit anti-mouse thymocyte globulin (mATG), we previously demonstrated that murine regulatory and memory T cells are preferentially spared from mATG depletion in vivo. The current studies were designed to determine whether different effector mechanisms are involved in differential depletion of T-cell subsets by mATG. METHODS: Complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and apoptotic mechanisms of depletion by mATG were evaluated in vitro and in vivo. RESULTS: In vitro, there was evidence of differential susceptibility of T-cell subsets by different effector mechanisms where naïve and CD4 effector memory T cells show reduced susceptibility to apoptosis, whereas regulatory T cells are less susceptible to mATG-mediated complement-dependent cytotoxicity and ADCC. However, mATG treatment of mice depleted of ADCC effector cell types (neutrophils, natural killer cells, or macrophages) or deficient in complement C5 or Fas demonstrated that mATG depletion of all T-cell subsets is mediated primarily by macrophages and that the role of neutrophils, natural killer cells, and complement is minimal in vivo. Interestingly, the Fas/FasL pathway does play a role in regulatory T-cell depletion, which is likely a result of increased basal expression of Fas on these cells. CONCLUSIONS: These data suggest that macrophages deplete most T cells by mATG in mice, but regulatory T cells are also uniquely susceptible to mATG-mediated Fas-dependent depletion.

Protecting against antimicrobial effectors in the phagosome allows SodCII to contribute to virulence in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium replicates in macrophages, where it is subjected to antimicrobial substances, including superoxide, antimicrobial peptides, and proteases. The bacterium produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both are expressed during infection, only SodCI contributes to virulence in the mouse by combating phagocytic superoxide. The differential contribution to virulence is at least partially due to inherent differences in the SodCI and SodCII proteins that are independent of enzymatic activity. SodCII is protease sensitive, and like other periplasmic proteins, it is released by osmotic shock. In contrast, SodCI is protease resistant and is retained within the periplasm after osmotic shock, a phenomenon that we term "tethering." We hypothesize that in the macrophage, antimicrobial peptides transiently disrupt the outer membrane. SodCII is released and/or phagocytic proteases gain access to the periplasm, and SodCII is degraded. SodCI is tethered within the periplasm and is protease resistant, thereby remaining to combat superoxide. Here we test aspects of this model. SodCII was released by the antimicrobial peptide polymyxin B or a mouse macrophage antimicrobial peptide (CRAMP), while SodCI remained tethered within the periplasm. A Salmonella pmrA constitutive mutant no longer released SodCII in vitro. Moreover, in the constitutive pmrA background, SodCII could contribute to survival of Salmonella during infection. SodCII also provided a virulence benefit in mice genetically defective in production of CRAMP. Thus, consistent with our model, protecting the outer membrane against antimicrobial peptides allows SodCII to contribute to virulence in vivo. These data also suggest direct in vivo cooperative interactions between macrophage antimicrobial effectors.

T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the large randomized prospective trial (UKALL XII/ECOG 2993).

The biology and outcome of adult T-cell acute lymphoblastic leukemia are poorly understood. We present here the clinical and biologic features of 356 patients treated uniformly on the prospective trial (UKALL XII/ECOG 2993) with the aim of describing the outcome and identifying prognostic factors. Complete remission was obtained in 94% of patients, and 48% survived 5 years. Positivity of blasts for CD1a and lack of expression of CD13 were associated with better survival (P = .01 and < .001, respectively). NOTCH1 and CDKN2A mutations were seen in 61% and 42% of those tested. Complex cytogenetic abnormalities were associated with poorer survival (19% vs 51% at 5 years, P = .006). Central nervous system involvement at diagnosis did not affect survival (47% vs 48%, P = not significant). For 99 patients randomized between autograft and chemotherapy, 5-year survival was 51% in each arm. Patients with a matched sibling donor had superior 5-year survival to those without donors (61% vs 46%, chi(2), P = .02); this was the result of less relapse (25% vs 51% at 5 years, P < .001). Only 8 of 123 relapsed patients survive. This study provides a baseline for trials of new drugs, such as nelarabine, and may allow risk-adapted therapy in patients with poor-prognosis T-cell ALL.

Conditional modeling of antibody titers using a zero-inflated poisson random effects model: application to Fabrazyme.

Patients that are exposed to biotechnology-derived therapeutics often develop antibodies to the therapeutic, the magnitude of which is assessed by measuring antibody titers. A statistical approach for analyzing antibody titer data conditional on seroconversion is presented. The proposed method is to first transform the antibody titer data based on a geometric series using a common ratio of 2 and a scale factor of 50 and then analyze the exponent using a zero-inflated or hurdle model assuming a Poisson or negative binomial distribution with random effects to account for patient heterogeneity. Patient specific covariates can be used to model the probability of developing an antibody response, i.e., seroconversion, as well as the magnitude of the antibody titer itself. The method was illustrated using antibody titer data from 87 male seroconverted Fabry patients receiving Fabrazyme. Titers from five clinical trials were collected over 276 weeks of therapy with anti-Fabrazyme IgG titers ranging from 100 to 409,600 after exclusion of seronegative patients. The best model to explain seroconversion was a zero-inflated Poisson (ZIP) model where cumulative dose (under a constant dose regimen of dosing every 2 weeks) influenced the probability of seroconversion. There was an 80% chance of seroconversion when the cumulative dose reached 210 mg (90% confidence interval: 194-226 mg). No difference in antibody titers was noted between Japanese or Western patients. Once seroconverted, antibody titers did not remain constant but decreased in an exponential manner from an initial magnitude to a new lower steady-state value. The expected titer after the new steady-state titer had been achieved was 870 (90% CI: 630-1109). The half-life to the new steady-state value after seroconversion was 44 weeks (90% CI: 17-70 weeks). Time to seroconversion did not appear to be correlated with titer at the time of seroconversion. The method can be adequately used to model antibody titer data.

In vivo characterization of rabbit anti-mouse thymocyte globulin: a surrogate for rabbit anti-human thymocyte globulin.

BACKGROUND: Polyclonal rabbit anti-human thymocyte globulin (Thymoglobulin) is used clinically for immunosuppression in solid organ transplantation; however, it is difficult to fully characterize the effects of this agent in humans. METHODS: A surrogate rabbit anti-murine thymocyte globulin (mATG) was generated analogously to the commercial product Thymoglobulin and in vivo activities were evaluated, including pharmacokinetics, T-cell depletion, dose response and kinetics, depletion/sparing of T-cell subsets or other leukocyte populations, and depletion in different lymphoid organs. RESULTS: Within 1 day, T cells are depleted by mATG in the blood, spleen, lymph node, and bone marrow down to doses of 1 mg/kg. Although mATG binds and depletes thymocytes in vitro, there is no thymocyte depletion in vivo at any dose level, suggesting decreased antibody accessibility to the thymus. After two doses of mATG given 3 days apart, T-cell reconstitution begins as early as day 9 and returns to basal levels by day 21 and 29 for CD4 and CD8 T cells, respectively. There is also preferential depletion of naïve T cells that results in increased ratios of regulatory and memory T cells within 1 day after mATG administration. Depletion of natural killer-T cells, natural killer cells, plasma cells, and plasmablasts occurs, but is modest and more transient compared with T cells. B cells, macrophages, dendritic cells, hematopoetic stem cells, and bone marrow stromal cells seem resistant to mATG depletion. CONCLUSIONS: These studies characterize the depletive effects of mATG in normal mice and provide insight into mechanisms of action of Thymoglobulin.

WT1 mutations in T-ALL.

The molecular mechanisms involved in disease progression and relapse in T-cell acute lymphoblastic leukemia (T-ALL) are poorly understood. We used single nucleotide polymorphism array analysis to analyze paired diagnostic and relapsed T-ALL samples to identify recurrent genetic alterations in T-ALL. This analysis showed that diagnosis and relapsed cases have common genetic alterations, but also that relapsed samples frequently lose chromosomal markers present at diagnosis, suggesting that relapsed T-ALL emerges from an ancestral clone different from the major leukemic population at diagnosis. In addition, we identified deletions and associated mutations in the WT1 tumor suppressor gene in 2 of 9 samples. Subsequent analysis showed WT1 mutations in 28 of 211 (13.2%) of pediatric and 10 of 85 (11.7%) of adult T-ALL cases. WT1 mutations present in T-ALL are predominantly heterozygous frameshift mutations resulting in truncation of the C-terminal zinc finger domains of this transcription factor. WT1 mutations are most prominently found in T-ALL cases with aberrant rearrangements of the oncogenic TLX1, TLX3, and HOXA transcription factor oncogenes. Survival analysis demonstrated that WT1 mutations do not confer adverse prognosis in pediatric and adult T-ALL. Overall, these results identify the presence of WT1 mutations as a recurrent genetic alteration in T-ALL.

Homeostatic role of transforming growth factor-beta in the oral cavity and esophagus of mice and its expression by mast cells in these tissues.

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-beta antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-beta expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-beta1 and TGF-beta3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-beta1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-beta1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-beta that is associated with basal TGF-beta expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-beta in maintaining homeostasis within both oral and esophageal tissues.