HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

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.

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.

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.

Differential expression of putative transbilayer amphipath transporters.

The aminophospholipid translocase transports phosphatidylserine and phosphatidylethanolamine from one side of a bilayer to another. Cloning of the gene encoding the enzyme identified a new subfamily of P-type ATPases, proposed to be amphipath transporters. As reported here, mammals express as many as 17 different genes from this subfamily. Phylogenetic analysis reveals the genes to be grouped into several distinct classes and subclasses. To gain information on the functions represented by these groups, Northern analysis and in situ hybridization were used to examine the pattern of expression of a panel of subfamily members in the mouse. The genes are differentially expressed in the respiratory, digestive, and urogenital systems, endocrine organs, the eye, teeth, and thymus. With one exception, all of the genes are highly expressed in the central nervous system (CNS); however, the pattern of expression within the CNS differs substantially from gene to gene. These results suggest that the genes are expressed in a tissue-specific manner, are not simply redundant, and may represent isoforms that transport a variety of different amphipaths.

The effect of bursectomy on natural xenoreactive antibodies and vascularized rat cardiac xenograft rejection in the chicken.

The clinical application of xenotransplantation between distantly related species is currently prevented by the occurrence of hyperacute rejection (HAR). Controversy exists over the importance of natural xenoreactive antibody (NAb)-mediated activation of the classical complement pathway vs. direct activation of the alternative C pathway in this process. In order to evaluate HAR of xenografts (Xgs) in the absence of NAb, this study utilized K strain leghorn chickens that were bursectomized (Bx) on day 17 in ovo (n = 18) to prevent B cell development and production of NAb. Aged-matched untreated siblings served as controls (n = 13). Based on pretransplant antibody levels, the Bx chickens were divided into two groups: totally Bx (Total Bx, n = 9) and partially Bx (Part Bx, n = 9). Chickens then underwent heterotopic cardiac xenotransplantation using PVG rats as donors, where the Xg was connected to the circulation of the chicken recipient utilizing cannulae. For the control group, Xg survival was 28 +/- 3 min (mean +/- SEM), while Part Bx prolonged survival to 80 +/- 15 min. Total Bx extended rat Xg survival to 102 +/- 11 min, with 5 of 9 Xgs functioning well at the time of termination of the study (90-120 min). Three chickens in the Total Bx group with rat cardiac Xgs that were functioning at 120 min were given a 1 ml i.v. injection of heat inactivated control chicken serum. This led to loss of Xg function within 10 min, confirming the important role for NAb in HAR in this species combination. Histologic examination of the Xgs following perfusion revealed significant arterial endothelial injury in the control and Part Bx groups but not in the Total Bx group. Conversely, Xgs from the Total Bx group showed marked venous congestion, which was not seen in the other two groups. This study demonstrates that: (1) Bx effectively eliminates NAb; (2) Xg survival is significantly prolonged in the absence of NAb in this rat-to-chicken xenogeneic combination; (3) the presence of NAb is associated with arterial endothelial injury; and (4) in the absence of NAb, marked venous congestion and injury occurs, which is possibly mediated by alternative C pathway activation or other humoral mechanisms.

Epitopes for chicken monoclonal antibodies in spleens of selected Japanese quail lines.

A line of Japanese quail selected for high plasma cholesterol is highly susceptible to diet-induced atherosclerosis. Lymphocyte epitopes recognized by mouse anti-chicken monoclonal antibodies (c-mAb), TCR-1, TCR-2, TCR-3. CD-3, CD-4, CD-8, and BU-1a/b were reacted with spleens from quail selected for high (HL) and low (LL) plasma total cholesterol and their nonselected controls (CL). Cross reactivity to c-mAb and effect of line and gender were immunohistochemically evaluated. Chicken spleens were positive controls. Quail were immunologically stimulated with either sheep red blood cells (SRBC) or Brucella abortus 2 weeks before spleens were removed. Quail spleen epitopes of all lines recognized TCR-3 and CD-8 c-mAb, but no other c-mAb. Number of reacting cells and staining intensity to the TCR-3 c-mAb were greater in the HL than in the LL regardless of the stimulating Ag or dose used. For the CD-8 c-mAb, there were no differences among lines in birds receiving SRBC. In B. abortus-immunized birds, sex x line interactions indicated that males of the HL and CL had lower responses than females but LL males were not different than females. TCR-3 and CD8 c-mAb may be useful in studying immunological mechanisms for atherosclerosis in Japanese quail.

Treatment of presumed arrhythmogenic right ventricular dysplasia in an adolescent.

Familial arrhythmogenic right ventricular dysplasia is a rare cardiomyopathy that is usually diagnosed on postmortem examination or on presentation with progressive congestive heart failure. We present a patient in whom an automatic implantable cardioverter-defibrillator was inserted prophylactically. A review of the condition and possible therapies is included.

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.

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.

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.

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.

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.

The preparedness of pediatricians for emergencies in the office. What is broken, should we care, and how can we fix it?

OBJECTIVES: To determine the frequency of emergencies in pediatric practices, assess office emergency preparedness, and explore practitioner's reasons for levels of preparedness. DESIGN AND SETTING: Telephone survey of pediatric offices in Fairfield County, Connecticut. SAMPLE: Fifty-one (98%) of 52 offices participated, representing 481 staff. RESULTS: More than 2400 emergencies are seen each year in the pediatric offices, with a median of 24 emergencies per practice annually. Forty-two (82%) of the practices averaged at least one emergency per month, 13 (25%) experienced more than 50 emergencies annually, and seven (14%) experienced more than 100 emergencies annually. Of all eligible staff, 14% were certified in basic life support and 17% in pediatric advanced life support. The following emergency equipment was missing from offices: oxygen, 14 (27%); intravenous catheters, 14 (27%); bag-valve-mask, 15 (29%); nebulizers, 17 (33%); epinephrine 1:10 000, 27 (53%); and intravenous fluids, 28 (55%). Thirty-seven (73%) of offices had the minimum recommended equipment and training for status asthmaticus management; only 17 (33%) of offices had similar preparation for each of the six other emergencies. High-level emergency preparedness was rare. The perceptions of office pediatricians are that office emergencies are rare, and emergency preparedness is difficult to achieve because practices are too busy and the expense and time commitment are too great. CONCLUSIONS: Emergencies are common in pediatric practices, but problems in the emergency preparedness of practices persist despite previous studies with similar findings, and publication of guidelines. Perceptions of practitioners suggest that not enough effort has been devoted to increasing practitioner awareness of these problems. Greater awareness might result by emphasizing practical instruction in residency and continuing medical education courses, and by publication of guideline summaries.

Association of elevated lymph node cell release of histamine and tumor necrosis factor with genetic predisposition to limb edema formation in dogs infected with Brugia pahangi.

Brugia pahangi infection in the canine rear limb results in marked lymphatic duct and popliteal lymph node pathologic changes. Limb edema is variably associated with infection and does not correlate well with duct or node lesions. To understand the mechanisms of limb edema, lymph node cells were collected by sequential biopsy following infection and examined for production of inflammatory mediators. Lymph node cells from a litter of dogs selectively bred with a high incidence of edema formation (82%) demonstrated spontaneously released histamine and prostaglandin E2 levels higher than those of closely related nonedema-forming dogs (0-20%) and/or control dogs. These edema-forming dogs also showed elevated release of tumor necrosis factor-alpha when cells were cultured with Brugia antigen. Toluidine blue staining of infected lymph node sections revealed that the edema-forming dogs had higher numbers of mast cells than infected lymph nodes of nonedema-forming dogs.

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.

Diclofenac and ketorolac in the treatment of pain after photorefractive keratectomy.

BACKGROUND: Photorefractive keratectomy (PRK) can produce significant ocular pain. Topical diclofenac, a non-steroidal anti-inflammatory drug (NSAID), is effective in the reduction of this pain. This study compares a second NSAID, ketorolac, to diclofenac. METHODS: This prospective matched-control study involved 102 eyes of 102 patients. Prior to PRK, patients were randomly assigned to receive ketorolac or diclofenac drops. At the first postoperative visit, a standardized questionnaire was used to assess the patient's average and peak levels of discomfort. In addition, the quantities of acetaminophen and codeine consumed were recorded. RESULTS: The overall level of discomfort was 1.53 +/- 0.64 for diclofenac and 1.88 +/- 0.55 for ketorolac (scale: 0 to 4) (P = 0.004). The diclofenac group reported a peak discomfort level of 2.0 +/- 0.75 and the ketorolac group reported 2.3 +/- 0.62 (P > 0.05). The diclofenac group consumed 2000 +/- 1150 mg of acetaminophen and 92 +/- 54 mg of codeine whereas the ketorolac group consumed 2150 +/- 940 of acetaminophen and 98 +/- 50 mg of codeine (P > 0.05). CONCLUSIONS: The differences in levels of peak discomfort, acetaminophen ingestion, and codeine ingestion, were not statistically significant. As compared to ketorolac, diclofenac resulted in a statistically significant lower mean overall discomfort.

Traumatic dislocation of the atlanto-occipital articulation (AOA) with short-term survival. With a radiographic method of measuring the AOA.

Three patients with traumatic atlanto-occipital articulation (AOA) dislocation are presented, and an additional 10 well documented cases are reviewed from the literature. Medulla oblongata and/or spinal cord deficits, and evidence of cranial nerve injuries were noted in eight patients. Angiographic evidence of vertebral occlusion or narrowing was demonstrated in four patients. One patient had systemic hypertension, presumably from bilateral traumatic ninth nerve injuries. Five patients ultimately died. A new method of measuring the AOA is introduced.

Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus.

A single tube fluorogenic RT-PCR-based 'TaqMan' assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan-PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5' untranslated region (5' UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT-PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT-PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values (C(T)) versus copy number. Single tube TaqMan-PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.