Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

HSP90 inhibition leads to degradation of the TYK2 kinase and apoptotic cell death in T-cell acute lymphoblastic leukemia.

We previously found that tyrosine kinase 2 (TYK2) signaling through its downstream effector phospho-STAT1 acts to upregulate BCL2, which in turn mediates aberrant survival of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we show that pharmacologic inhibition of heat shock protein 90 (HSP90) with a small-molecule inhibitor, NVP-AUY922 (AUY922), leads to rapid degradation of TYK2 and apoptosis in T-ALL cells. STAT1 protein levels were not affected by AUY922 treatment, but phospho-STAT1 (Tyr-701) levels rapidly became undetectable, consistent with a block in signaling downstream of TYK2. BCL2 expression was downregulated after AUY922 treatment, and although this effect was necessary for AUY922-induced apoptosis, it was not sufficient because many T-ALL cell lines were resistant to ABT-199, a specific inhibitor of BCL2. Unlike ABT-199, AUY922 also upregulated the proapoptotic proteins BIM and BAD, whose increased expression was required for AUY922-induced apoptosis. Thus, the potent cytotoxicity of AUY922 involves the synergistic combination of BCL2 downregulation coupled with upregulation of the proapoptotic proteins BIM and BAD. This two-pronged assault on the mitochondrial apoptotic machinery identifies HSP90 inhibitors as promising drugs for targeting the TYK2-mediated prosurvival signaling axis in T-ALL cells.

Identification of additional IDH mutations associated with oncometabolite R(-)-2-hydroxyglutarate production.

Mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or its mitochondrial homolog IDH2 can lead to R(-)-2-hydroxyglutarate (2HG) production. To date, mutations in three active site arginine residues, IDH1 R132, IDH2 R172 and IDH2 R140, have been shown to result in the neomorphic production of 2HG. Here we report on three additional 2HG-producing IDH1 mutations: IDH1 R100, which is affected in adult glioma, IDH1 G97, which is mutated in colon cancer cell lines and pediatric glioblastoma, and IDH1 Y139. All these new mutants stereospecifically produced 2HG's (R) enantiomer. In contrast, we find that the IDH1 SNPs V71I and V178I, as well as a number of other single-sample reports of IDH non-synonymous mutation, did not elevate cellular 2HG levels in cells and retained the wild-type ability for isocitrate-dependent NADPH production. Finally, we report the existence of additional rare, but recurring mutations found in lymphoma and thyroid cancer, which while failing to elevate 2HG nonetheless displayed loss of function, indicating a possible tumorigenic mechanism for a non-2HG-producing subset of IDH mutations in some malignancies. These data broaden our understanding of how IDH mutations may contribute to cancer through either neomorphic R(-)-2HG production or reduced wild-type enzymatic activity, and highlight the potential value of metabolite screening in identifying IDH-mutated tumors associated with elevated oncometabolite levels.

Long-term follow-up of patients treated with daclizumab for steroid-refractory acute graft-vs-host disease.

Daclizumab has been shown to have activity in acute GVHD, but appears to be associated with an increased risk of infection. To investigate further the long-term effects of daclizumab, we performed a retrospective review of 57 patients who underwent an allogeneic hematopoietic stem cell transplant from January 1993 through June 2000 and were treated with daclizumab for steroid-refractory acute GVHD. The median number of daclizumab doses given was 5 (range 1-22). GVHD was assessed at baseline, days 15, 29 and 43. By day 43, 54% patients had an improvement in their overall GVHD score, including 76% patients aged < or =18. Opportunistic infections developed in 95% patients. Forty-three patients (75%) died following treatment with daclizumab. The causes of death included active GVHD and infection (79%), active GVHD (5%), chronic GVHD (2%) and relapse (14%). Patients with grade 3-4 GVHD had a significantly shorter median survival than patients with grade 1-2 GVHD (2.0 vs 5.1 months, P=0.001). Daclizumab has no infusion-related toxicity, is active in steroid-refractory GVHD, especially among pediatric patients, but is associated with significant morbidity and mortality due to infectious complications. Careful patient selection and aggressive prophylaxis against viral and fungal infections are recommended.

Preemptive diagnosis and treatment of Epstein-Barr virus-associated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: an approach in development.

Hematopoietic stem cell transplant (HSCT) recipients are at risk for Epstein-Barr virus (EBV)-associated, post transplant lymphoproliferative disorder (PTLD). Studies have suggested that early treatment may improve the outcome of patients with PTLD. Thus, significant attention has been focused on PCR-based approaches for preemptive (i.e., prior to clinical presentation) diagnosis. Reports from several transplant centers have demonstrated that HSCT recipients with PTLD generally have higher concentrations of EBV DNA in the peripheral blood than patients without PTLD. However, the PCR values of patients with PTLD typically span multiple orders of magnitude and overlap significantly with values from patients without PTLD. Thus, questions remain about the sensitivity and predictive value of these assays. Preemptive strategies using rituximab and/or EBV-specific cytotoxic T lymphocytes have been evaluated in patients with elevated EBV viral loads. We review the current literature, discuss our institutional experience and identify several areas of future research that could improve the diagnosis and treatment of this life-threatening disorder in HSCT recipients.

High rates of infection and colonization by nontuberculous mycobacteria after allogeneic hematopoietic stem cell transplantation.

Nontuberculous mycobacteria (NTM) are essentially ubiquitous and can infect both immunocompetent and immunocompromised hosts. However, NTM infection is surprisingly uncommon in reports from allogeneic hematopoietic stem cell transplant (alloSCT) centers that do not routinely perform allograft T-cell depletion. We reviewed medical records for all adult patients who underwent alloSCT at our center between January 1993 and December 2001. American Thoracic Society and Centers for Disease Control and Prevention guidelines Were used to define definite, probable, and possible NTM infection. Of 571 patients, 36 of 372 (9.7%) T-cell depleted and 14 of 199 (7.0%) conventional alloSCT recipients (P=0.26) had a positive culture for NTM after alloSCT. Of the 50 patients with NTM infection, 16 had definite infection and 34 had probable or possible infection. Rates of NTM infection were 5 to 20-fold higher than rates reported by other centers. Of the 16 definite infections, nine were caused by Mycobacterium haemophilum. Two patients had disseminated M. avium complex (MAC) infection and one had a vascular catheter infected by MAC. Three patients died from complications of NTM infection. Patients with probable or possible NTM infection had markedly different epidemiology, risk factors, site and species of NTM infection, and prognosis than patients with definite NTM infection.

A comparison of verbal fluency tasks in schizophrenic patients and normal controls.

Previous studies have reported significant impairment on verbal fluency tasks (semantic and letter) among schizophrenic subjects. However, the possibility of specific categorical deficits has not been adequately investigated. Nor have the effects of task duration, the stability between testing sessions, and the relationship between intelligence and performance on fluency been thoroughly studied. We performed a series of 3 min fluency tasks (semantic/syntactic and letter) to determine whether duration specific or category-specific differences exist between schizophrenic subjects and normal controls. Each subject was tested at three different times as a means of estimating word pool and assessing the stability of fluency output. Subjects were asked to generate exemplars from each of four semantic/syntactic categories (animals, tools, common nouns and verbs) and three letters (G, E and T). Data from 13 schizophrenic subjects and 15 sex-, age- and pre-morbid-IQ-matched control subjects revealed that patients' overall performance on both the semantic and letter fluency tasks was impaired. While differential impairment on specific semantic categories was noted between groups, no differential effects relating to task duration or testing session were present. Further, by comparing the number of novel words produced in the three testing sessions, we found the groups to be equivalent, a finding we take to suggest that schizophrenic patients' lexicon is intact. Covarying current IQ eliminated the group difference robustly for letter fluency, while only marginally for semantic fluency. Our data revealed the presence of impairment in semantic and letter fluency tasks in schizophrenic patients consistent with previous reports, and also that patients were differentially impaired on semantic categories.

Risk for tuberculosis infection among internally displaced persons in the Republic of Georgia.

OBJECTIVE: To determine the prevalence of tuberculosis (TB) infection and disease among internally displaced persons residing in Tbilisi, Republic of Georgia. DESIGN: Residents of eight refugee hostels were screened for TB infection using a tuberculin skin test (TST) and a symptom questionnaire. Participation was voluntary. TST-positive individuals were referred for chest radiography. Subjects with cough, fever, or night sweats of > 2 weeks duration provided sputum for acid-fast bacilli (AFB) microscopy and culture. RESULTS: Of approximately 4000 potential subjects (internally displaced persons), 988 (24.7%) participated in the screening program. Of these 988, 931 (94.2%) who had a TST placed returned at 48-72 hours to have the skin test examined; 447 (48.0%) were TST-positive (> or = 10 mm induration). In multivariate analysis, risk factors for a positive TST included male sex, ever having received BCG, history of close contact with a case of active tuberculosis, and living in one specific refugee hostel. Risk for a positive TST was greater among subjects > 20 years old, but there was no difference between age groups over the age of 20 years. Five patients with active TB were identified through the screening program, giving a case rate of 537 per 100,000 population. CONCLUSION: Tuberculosis infection and disease were common in this group of internally displaced persons. Screening was a useful mechanism of case finding among this high prevalence population.

Control of influenza A on a bone marrow transplant unit.

In January 1998, an outbreak of influenza A occurred on our adult bone marrow transplant unit. Aggressive infection control measures were instituted to halt further nosocomial spread. A new, more rigorous approach was implemented for the 1998/99 influenza season and was extremely effective in preventing nosocomial influenza at our institution.

Rotavirus outbreak on a pediatric oncology floor: possible association with toys.

BACKGROUND: Several outbreaks of rotavirus gastroenteritis have occurred in hospitals and day care centers. In the spring of 1997, an outbreak of rotavirus occurred on our pediatric unit. Aggressive infection control measures were instituted, and potential lapses in infection control were assessed. METHODS: Memorial Sloan-Kettering Cancer Center is a 434-bed cancer hospital in New York City. The pediatric unit is a 42-bed ward with both bone marrow transplant patients and non-bone marrow transplant oncology patients. Nosocomially acquired rotavirus was defined as diarrhea, vomiting, or gastrointestinal upset with onset 48 hours or more after hospital admission, accompanied by a positive enzyme immunoassay for rotavirus antigen. RESULTS: Between February 24 and April 4, 1997, 8 patients on the pediatric unit had nosocomial rotavirus. Aggressive infection control measures were instituted. Patients with rotavirus were cohorted and placed on contact precautions (strict handwashing, gloves, and gown). Investigation by the infection control team revealed that communal toys in the playroom were not being cleaned according to the weekly protocol. CONCLUSIONS: An outbreak of nosocomial rotavirus occurred on our pediatric oncology unit. Shared toys may have served as fomites in the transmission of rotavirus.

Seroconversion rates in healthcare workers using a latex agglutination assay after varicella virus vaccination.

OBJECTIVES: To determine the seroconversion rate after varicella immunization of healthcare workers (HCWs) and the effect of seroconversion rate on current cost-based recommendations for universal vaccination. METHODS: A voluntary vaccination program for HCWs was performed at a tertiary-care cancer center in New York City. A commercial latex agglutination assay was used to test postvaccination antibody response. Costs for vaccination and postvaccination serological testing were compared to potential costs of postexposure employee furloughs. RESULTS: Of 263 seronegative HCWs, 96 (36.5%) began the vaccine program. Thirty-nine HCWs received only one dose of vaccine. Seven returned for follow-up antibody testing, of whom 4 were seropositive. Of the 57 HCWs who received two doses, 38 returned for follow-up serology. Thirty-one (81.6%) HCWs were seropositive for varicella-zoster virus antibodies, and seven HCWs (18.4%) remained seronegative. Total cost of vaccination for all 263 seronegative HCWs was estimated and compared to the cost of varicella-related furloughs at our institution. CONCLUSIONS: We found a considerably lower rate of vaccine-induced seroconversion at our hospital compared to that of the published literature. Despite this finding, universal varicella vaccination remained an extremely cost-effective alternative to the furloughing of exposed, seronegative HCWs. Projected hospital savings exceeded $53,000 in the first year after vaccination alone.

Shigella infection as observed in the experimentally inoculated domestic pig, Sus scrofa domestica.

The domestic pig, Sus scrofa domestica, was investigated as a potential animal model for shigellosis. We examined the effects of pig age, pig breed and antibiotic pretreatment upon Shigella infection. Shigella dysenteriae, and Shigella flexneri (both virulent and avirulent strains) were utilized. Our results indicated that young (4-week-old), conventionally re ared, domestic pigs were routinely, but briefly, colonized (average=3.5+/-2.5 days) following oral or gavage administration ofS. flexneri, as determined by direct rectal cultures. The duration of S. dysenteriae colonization was significantly shorter. Inoculation of younger (2 days) or older (9 weeks) pigs with S. flexneri had no significant effect on infection duration. Similarly, infection of 4-week-old pigs with virulent and avirulent strains of S. flexneri had no effect upon the duration of infection, nor did the use of a swine-passaged S. flexneri isolate. Marked clinical, histopathological (gross and microscopic) and immunoIhistopathological signs of disease were absent in all infections. However, in instances where microscopic histopathological evidence was used to correctly identify infected pigs, tonsillar lesions were the consistently noted criteria. The tonsils are believed to be an important portal of entry for Salmonella choleraesuis, another member of the Enterobacteriaceae and a prevalent pig pathogen. Taken altogether, our results indicate that the domestic pig is unsuitable as a model for shigellosis.

Comparison of plasma prothrombin and factor VII and urine prothrombin F1 concentrations in patients on long-term warfarin therapy and those in the initial phase.

Control of warfarin anticoagulation during the initial phase of therapy is difficult and empirically based. Plasma and urine samples were obtained from normal controls, patients under stable anticoagulation, and patients in the initial phase of anticoagulation. Total plasma prothrombin, des-carboxy (non-adsorbable with barium chloride) prothrombin, and native (total minus non-adsorbable) prothrombin were quantitated using Echis carinatus venom activation. Functional plasma factor VII (VII) was measured using a one-stage clotting assay. Total and des-carboxy urine prothrombin F1 (F1) were measured by ELISA. All urine F1 in normals and both anticoagulated groups was adsorbed by barium chloride. Plasma des-carboxy prothrombin concentration was similar for the two anticoagulated groups and did not correlate with 1/INR. Native prothrombin correlated with 1/INR in both the stable (r = 0.76) and initial phase (r = 0.74) groups. For any given INR, the subjects on stable anticoagulation had lower native prothrombin concentrations than the initial phase patients. Functional factor VII concentration also correlated significantly with 1/INR in both the stable (r = 0.64) and initial phase (r = 0.76) patients. Unlike native prothrombin, VII concentrations did not vary between the two cohorts for any given INR. Previous studies indicate that native prothrombin is a superior predictor of both hemorrhagic and thromboembolic complications during warfarin therapy. Our findings indicate that VII, and not prothrombin, may be the predominant factor monitored by the INR. This further supports the need to reevaluate the usefulness of the INR in the monitoring of warfarin therapy during the initial phase.