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The Pediatric Hydroxyurea Phase 3 Clinical Trial (BABY HUG) was a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial of hydroxyurea in infants (beginning at 9-18 months of age) with sickle cell anemia. An important secondary objective of this study was to compare clinical events between the hydroxyurea and placebo groups. One hundred and ninety-three subjects were randomized to hydroxyurea (20 mg/kg/d) or placebo; there were 374 patient-years of on-study observation. Hydroxyurea was associated with statistically significantly lower rates of initial and recurrent episodes of pain, dactylitis, acute chest syndrome, and hospitalization; even infants who were asymptomatic at enrollment had less dactylitis as well as fewer hospitalizations and transfusions if treated with hydroxyurea. Despite expected mild myelosuppression, hydroxyurea was not associated with an increased risk of bacteremia or serious infection. These data provide important safety and efficacy information for clinicians considering hydroxyurea therapy for very young children with sickle cell anemia. This clinical trial is registered with the National Institutes of Health (NCT00006400, www.clinicaltrials.gov).

Youth infected with HIV at birth often have sleep disturbances, neurocognitive deficits, and abnormal psychosocial function which are associated with and possibly resulted from elevated blood cytokine levels that may lead to a decreased quality of life. To identify molecular pathways that might be associated with these disorders, we evaluated 38 HIV-infected and 35 uninfected subjects over 18-months for intracellular cytokine levels, sleep patterns and duration of sleep, and neurodevelopmental abilities. HIV infection was significantly associated with alterations of intracellular pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12), sleep factors (total time asleep and daytime sleep patterns), and neurocognitive factors (parent and patient reported problems with socio-emotional, behavioral, and executive functions; working memory-mental fatigue; verbal memory; and sustained concentration and vigilance. By better defining the relationships between HIV infection, sleep disturbances, and poor psychosocial behavior and neurocognition, it may be possible to provide targeted pharmacologic and procedural interventions to improve these debilitating conditions.

Background The laboratory and clinical benefits of hydroxyurea therapy for children with sickle cell anemia (SCA) are well recognized, but treatment in young patients is limited in part by concerns about long-term genotoxicity, and specifically possible carcinogenicity.

The purpose of this study is to perform and evaluate baseline abdominal ultrasound in infants with sickle cell anemia who participated in the BABY HUG multiinstitutional randomized placebo-controlled trial of hydroxyurea therapy and to examine the potential relationships among ultrasound results and clinical, nuclear medicine, and laboratory data.After local institutional review board approval and with informed guardian consent, 116 girls and 87 boys (age range, 7.5-18 months) with sickle cell anemia underwent standardized abdominal sonography at 14 institutions. Imaging was centrally reviewed by one radiologist who assessed and measured the spleen, kidneys, gallbladder, and common bile duct. Baseline physical assessment of spleen size, serum alanine aminotransferase and bilirubin levels, (99m)Tc sulfur colloid liver-spleen scans, and (99m)Tc diethylenetriaminepentaacetic acid clearance glomerular filtration rates (GFRs) were obtained. Analysis of variance and the Student test were performed to compare sonographic findings to published results in healthy children and to clinical and laboratory findings.The mean (± SD) spleen volume (108 ± 47 mL) was significantly greater than published normal control values (30 ± 14 mL; p0.0001). There was no correlation between spleen volume and function assessed by liver-spleen scan. The mean GFR (125 ± 34 mL/min/1.73 m(2)) was elevated compared with control GFRs (92 ± 18 mL/min/1.73 m(2)). Renal volumes (right kidney, 29 ± 8 mL; left kidney, 31 ± 9 mL) were significantly greater than control volumes (right kidney, 27 ± 3 mL; left kidney, 27 ± 3 mL; p0.0001) and were positively correlated with GFR (p = 0.0009). Five percent of patients had sonographic biliary abnormalities (sludge, n = 6; dilated common bile duct, n = 2; and cholelithiasis and thickened gallbladder wall, n = 1 each). There was no correlation between biliary sonographic findings and laboratory results.In infants with sickle cell anemia, sonographic spleen volume does not reflect function, but increased renal volume correlates with GFR and is consistent with hyperfiltration. Sonographic biliary abnormalities can occur early in life, while remaining clinically silent.

We evaluated spleen function in 193 children with sickle cell anemia 8 to 18 months of age by 99mTc sulfur-colloid liver-spleen scan and correlated results with clinical and laboratory parameters, including 2 splenic biomarkers: pitted cell counts (PIT) and quantitative Howell-Jolly bodies (HJB) enumerated by flow cytometry. Loss of splenic function began before 12 months of age in 86% of infants in association with lower total or fetal hemoglobin and higher white blood cell or reticulocyte counts, reinforcing the need for early diagnosis and diligent preventive care. PIT and HJB correlated well with each other and liver-spleen scan results. Previously described biomarker threshold values did define patients with abnormal splenic function, but our data suggest that normal spleen function is better predicted by PIT of ≤ 1.2% or HJB ≤ 55/106 red blood cells and absent function by PIT ≥ 4.5% or HJB ≥ 665/106. HJB is methodologically advantageous compared with PIT, but both are valid biomarkers of splenic function. This trial was registered at www.clinicaltrials.gov as #NCT00006400.

Evidence of the laboratory benefits of hydroxyurea and its clinical efficacy in reducing acute vaso-occlusive events in adults and children with sickle cell anemia has accumulated for more than 15 years. A definitive clinical trial showing that hydroxyurea can also prevent organ damage might support widespread use of the drug at an early age. BABY HUG is a randomized, double-blind placebo-controlled trial to test whether treating young children ages 9–17 months at entry with a liquid preparation of hydroxyurea (20 mg/kg/day for 2 years) can decrease organ damage in the kidneys and spleen by at least 50%. Creation of BABY HUG entailed unique challenges and opportunities. Although protection of brain function might be considered a more compelling endpoint, preservation of spleen and renal function has clinical relevance, and significant treatment effects might be discernable within the mandated sample size of 200. Concerns about unanticipated severe toxicity and burdensome testing and monitoring requirements were addressed in part by an internal Feasibility and Safety Pilot Study, the successful completion of which was required prior to enrolling a larger number of children on the protocol. Concerns over recruitment of potentially vulnerable subjects were allayed by inclusion of a research subject advocate, or ombudsman. Finally, maintenance of blinding of research personnel was aided by inclusion of an unblinded primary endpoint person, charged with transmitting endpoint data and monitoring blood work locally for toxicity (ClinicalTrials.gov number, NCT00006400). Pediatr Blood Cancer 2010;54:250–255. © 2009 Wiley-Liss, Inc.

Background GB virus type C (GBV-C) viraemia is associated with a beneficial outcome in HIV-infected individuals in several though not all studies. GBV-C viraemia was examined in a matched case–control study of 133 HIV-infected pregnant women who transmitted HIV to their infants (‘cases’) and 266 non-transmitting controls. Methods HIV-infected children and controls were pair-matched for high-risk delivery, race and year of delivery. GBV-C status was determined in maternal plasma samples obtained at or within 3 months of delivery. Results Pregnant women with GBV-C viraemia (11% of those studied) had lower HIV RNA levels (P=0.01) and higher CD4 percentages (P=0.006) than women without GBV-C. A trend towards decreased mother-to-child transmission in the multivariate analysis was observed among GBV-C viraemic women delivering after highly active antiretroviral therapy (HAART) became available [odds ratio (OR) 0.30, 95% confidence interval (CI) 0.08–1.05; P=0.06], but not in women delivering prior to the widespread use of HAART. Conclusions GBV-C viraemia was associated with a beneficial effect on CD4 percentage and HIV RNA level in these pregnant women, and was also associated with a trend towards reduced risk of mother-to-child HIV transmission among women after HAART became available. Further studies with larger or multiple cohorts are necessary to assess possible benefits in this population.

The recently completed BABY HUG trial investigated the safety and efficacy of hydroxyurea in infants with sickle cell anemia (SCA). To investigate the effects of known genetic modifiers, genomic DNA on 190 randomized subjects were analyzed for alpha thalassemia, beta-globin haplotype, polymorphisms affecting endogenous fetal hemoglobin (HbF) levels (XmnI, BCL11A, and HBS1L-MYB), UGT1A1 promoter polymorphisms, and the common G6PD A− mutation. At study entry, infants with alpha thalassemia trait had significantly lower mean corpuscular volume, total bilirubin, and absolute reticulocyte count. Beta-globin haplotypes associated with milder disease had significantly higher hemoglobin and %HbF. BCL11A and XmnI polymorphisms had significant effects on baseline HbF, while UGT1A1 promoter polymorphisms significantly influenced baseline serum bilirubin. At study exit, subjects randomized to placebo still exhibited laboratory effects of alpha thalassemia and other modifiers, while those assigned hydroxyurea had treatment effects that exceeded most genetic influences. The pain phenotype was influenced by HbF modifiers in both treatment groups. These data document that genetic polymorphisms do modify laboratory and clinical phenotypes even in very young patients with SCA. The hydroxyurea effects are more potent, however, indicating that treatment criteria should not be limited to certain genetic subsets, and supporting the use of hydroxyurea for all young patients with SCA. Am. J. Hematol. 88:571–576, 2013. © 2013 Wiley Periodicals, Inc.

The laboratory and clinical benefits of hydroxyurea therapy for children with sickle cell anemia (SCA) are well recognized, but treatment in young patients is limited in part by concerns about long-term genotoxicity, and specifically possible carcinogenicity.The Pediatric Hydroxyurea Phase III Clinical Trial (BABY HUG) was a multicenter double-blinded placebo-controlled randomized clinical trial (NCT00006400) testing whether hydroxyurea could prevent chronic organ damage in very young patients with SCA. An important secondary objective was the measurement of acquired genotoxicity using three laboratory assays: chromosomal karyotype, illegitimate VDJ recombination events, and micronucleated reticulocyte formation.Our data indicate that hydroxyurea treatment was not associated with any significant increases in genotoxicity compared to placebo treatment.These data provide additional support to the safety profile of hydroxyurea for young patients with SCA, and suggest that genotoxicity in this patient population is low.

Sickle-cell anaemia is associated with substantial morbidity from acute complications and organ dysfunction beginning in the first year of life. Hydroxycarbamide substantially reduces episodes of pain and acute chest syndrome, admissions to hospital, and transfusions in adults with sickle-cell anaemia. We assessed the effect of hydroxycarbamide therapy on organ dysfunction and clinical complications, and examined laboratory findings and toxic effects.This randomised trial was undertaken in 13 centres in the USA between October, 2003, and September, 2009. Eligible participants had haemoglobin SS (HbSS) or haemoglobin Sβ(0)thalassaemia, were aged 9-18 months at randomisation, and were not selected for clinical severity. Participants received liquid hydroxycarbamide, 20 mg/kg per day, or placebo for 2 years. Randomisation assignments were generated by the medical coordinating centre by a pre-decided schedule. Identical appearing and tasting formulations were used for hydroxycarbamide and placebo. Patients, caregivers, and coordinating centre staff were masked to treatment allocation. Primary study endpoints were splenic function (qualitative uptake on (99)Tc spleen scan) and renal function (glomerular filtration rate by (99m)Tc-DTPA clearance). Additional assessments included blood counts, fetal haemoglobin concentration, chemistry profiles, spleen function biomarkers, urine osmolality, neurodevelopment, transcranial Doppler ultrasonography, growth, and mutagenicity. Study visits occurred every 2-4 weeks. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00006400.96 patients received hydroxycarbamide and 97 placebo, of whom 83 patients in the hydroxycarbamide group and 84 in the placebo group completed the study. Significant differences were not seen between groups for the primary endpoints (19 of 70 patients with decreased spleen function at exit in the hydroxycarbamide group vs 28 of 74 patients in the placebo group, p=0·21; and a difference in the mean increase in DTPA glomerular filtration rate in the hydroxycarbamide group versus the placebo group of 2 mL/min per 1·73 m(2), p=0·84). Hydroxycarbamide significantly decreased pain (177 events in 62 patients vs 375 events in 75 patients in the placebo group, p=0·002) and dactylitis (24 events in 14 patients vs 123 events in 42 patients in the placebo group, p0·0001), with some evidence for decreased acute chest syndrome, hospitalisation rates, and transfusion. Hydroxyurea increased haemoglobin and fetal haemoglobin, and decreased white blood-cell count. Toxicity was limited to mild-to-moderate neutropenia.On the basis of the safety and efficacy data from this trial, hydroxycarbamide can now be considered for all very young children with sickle-cell anaemia.The US National Heart, Lung, and Blood Institute; and the National Institute of Child Health and Human Development.

Evidence of the laboratory benefits of hydroxyurea and its clinical efficacy in reducing acute vaso-occlusive events in adults and children with sickle cell anemia has accumulated for more than 15 years. A definitive clinical trial showing that hydroxyurea can also prevent organ damage might support widespread use of the drug at an early age. BABY HUG is a randomized, double-blind placebo-controlled trial to test whether treating young children ages 9-17 months at entry with a liquid preparation of hydroxyurea (20 mg/kg/day for 2 years) can decrease organ damage in the kidneys and spleen by at least 50%. Creation of BABY HUG entailed unique challenges and opportunities. Although protection of brain function might be considered a more compelling endpoint, preservation of spleen and renal function has clinical relevance, and significant treatment effects might be discernable within the mandated sample size of 200. Concerns about unanticipated severe toxicity and burdensome testing and monitoring requirements were addressed in part by an internal Feasibility and Safety Pilot Study, the successful completion of which was required prior to enrolling a larger number of children on the protocol. Concerns over recruitment of potentially vulnerable subjects were allayed by inclusion of a research subject advocate, or ombudsman. Finally, maintenance of blinding of research personnel was aided by inclusion of an unblinded primary endpoint person, charged with transmitting endpoint data and monitoring blood work locally for toxicity (ClinicalTrials.gov number, NCT00006400).

Abstract 8 The laboratory and clinical benefits of hydroxyurea therapy for children with sickle cell anemia (SCA) are well recognized, but treatment in young patients is limited in part by concerns about long-term genotoxicity, and specifically possible carcinogenicity. To date, few prospective data have been available to assess the mutagenic and carcinogenic potential of hydroxyurea in young patients with SCA. The Pediatric Hydroxyurea Phase III Clinical Trial (BABY HUG) was a multi-center double-blinded placebo-controlled randomized clinical trial (NCT00006400) testing whether hydroxyurea could prevent chronic organ damage in very young patients with SCA. BABY HUG was conducted across 14 centers and was approved by the local institutional review boards of all participating centers. A total of 193 infants (mean 13.6 months) with SCA (HbSS or HbS/ß°-thalassemia) were randomized to receive hydroxyurea (fixed dose of 20 mg/kg/day) or placebo for two years. An important secondary objective of the study was the in vivo measurement of acquired genotoxicity using three laboratory assays: chromosomal karyotype including quantitation of chromosomal breaks, chromatid breaks, and fusion events; illegitimate VDJ recombination events representing inversion events on chromosome 7 with juxtaposition of T-cell receptor Vg and Jb and gene loci; and micronucleated reticulocyte formation signifying aberrant erythroid production. Subjects in both the hydroxyurea and placebo groups had significantly increased numbers of total chromosome breaks and similar numbers of chromatid breaks at study exit compared to study entry. However, at study exit, subjects with hydroxyurea exposure had similar numbers of chromosome and chromatid breaks as subjects receiving placebo (0.5 ± 1.4 chromosome breaks per 100 metaphases vs. 0.4 ± 2.5, p=NS; 0.6 ± 1.1 chromatid breaks per 100 metaphases vs.0.8 ± 3.4, p=NS). There were no changes in the number of illegitimate VDJ recombination events observed, comparing study entry and exit samples either in the hydroxyurea or the placebo treatment group. At study exit, subjects treated with hydroxyurea had similar numbers of illegitimate VDJ recombination events as subjects receiving placebo (0.7 ± 0.5 events per μg of DNA versus 0.7 ± 0.4 events, p=NS). Subjects treated with hydroxyurea had a similar number of early reticulocytes containing micronuclei at study exit compared to subjects receiving placebo (0.3 ± 0.2% versus 0.3 ± 0.2%, p=NS). Together, these data indicate that hydroxyurea treatment in very young patients with SCA was not associated with any significant increases in genotoxicity compared to placebo treatment. These data provide evidence of cytogenetic stability in this susceptible population of young children and contribute to a growing body of evidence to suggest that in vivo genotoxicity of hydroxyurea in patients with SCA appears to be low. Disclosures: Dertinger: Litron Laboratories: Employment.

Abstract 171 Background: The BABY HUG trial was a multi-center double-blinded randomized comparison of hydroxyurea (HU) versus placebo in infants (mean age 13.6 mo. at entry) with HbSS/Sβ° thalassemia who were followed for 2 years [Lancet 377(2011):1663–1672; Clinical Trials #NCT00006400]. Hydroxyurea therapy was associated with less pain, dactylitis, acute chest syndrome (ACS), hospitalization (HSN), and transfusion (TX) and with improved hematologic values; toxicity was limited to mild-moderate neutropenia. On the basis of the safety and efficacy data from this trial, it was concluded that hydroxyurea therapy can be considered for all very young children with sickle cell anemia (SCA). With anticipated broader use of hydroxyurea in this population, we examined estimated medical costs of care (based on Medicaid reimbursement) in treated versus placebo subjects. Methods: The BABY HUG database (C-TASC, Baltimore, MD) was utilized to compare inpatient events in subjects receiving hydroxyurea with those receiving placebo. Unit costs were estimated from the 2009 Thomson Reuters MarketScan® Multi-state Medicaid Database for children with HbSS (ICD-9 codes 282.61 or 282.62), ages 1–3 years. Inpatient costs included emergency department (ED) costs for admissions from an ED in about 80% of the 748 admissions in the database. Inpatient cost estimates were based on length of stay (LOS) modified by a diagnosis of ACS or splenic sequestration (SpS) or a procedure code for a TX. Outpatient expenses were estimated based on the schedule required for BABY HUG (and recommended for clinical use) and a “standard” schedule for 1–3 year-olds with SCA based on management protocols at 3 pediatric sickle cell centers in the US. Results: 96 subjects were randomized to hydroxyurea (83 completed the trial); 97 received placebo (84 completed the trial). In the full study, there were 232 hospitalizations (for any cause) in those receiving hydroxyurea and 324 in those on placebo; inpatient data were captured for only the final 77% of admissions (between 2/06 and 9/09). The LOS for subjects receiving hydroxyurea (mean 3.7, median 3, range 1–9 days) did not differ from those receiving placebo (3.6, 3, 1–13). Estimated inpatient and outpatient costs are shown in Tables 1 and 2. When inpatient and outpatient expenses were combined, the annual cost for 1–3 year-old children with SCA was $11,345 on hydroxyurea and $14,815 on placebo, a difference of $3,470. Discussion/Conclusion: Despite increased outpatient care expenses from clinic visits, laboratory monitoring, and hydroxyurea, savings on inpatient care resulted in an overall reduction in estimated annual per patient expenditure of approximately 23%. A limitation of our analysis was the dependence on MarketScan Medicaid data in lieu of the availability of specific expenses of the subjects participating in the BABY HUG study. Medicaid data may understate costs of care; based on prior analyses, we estimate that costs to private payers may be 20–30% greater than Medicaid reimbursements. We conclude that increased use of hydroxyurea treatment in children with SCA can lead to significant medical cost savings. Disclosures: Off Label Use: Hydroxyurea is not indicated for treatment of children with sickle cell disease. Use of this medication was for clinical indications and not mandated by this observational study.

Abstract 2134 BABY HUG randomly assigned children with sickle cell anemia ages 9 to 18 months at entry to a two-year double-masked treatment course of 20 mg/kg/d of hydroxyurea (HU) or placebo (PBO). The primary goal of this clinical trial was to determine if treatment with HU could reduce organ damage or dysfunction (spleen function assessed by 99mTc-sulfur colloid and renal function by 99mTc-DTPA clearance) by 50%. Children were seen at least monthly for blood and clinical evaluations. [Lancet 2011; 377(9778):1663–1672.] Between March 23, 2006 and June 1, 2006, the BABY HUG study was placed on “clinical hold” due to the omission of an expiration date on the treatment label for one lot of the study treatments. During the clinical hold, there were 94 children on active study treatment (47 HU, 47 PBO); four and six children, respectively, had finished their two-year follow up. Subjects continued regular visits and clinical event monitoring but received no study medication. To examine the impact of the clinical hold on toxicity and clinical events, the BABY HUG study period was divided into three intervals: Before, During, and After the clinical hold. Each period was offset by a one-month delay from the actual onset and end date to allow for wash-out and wash-in periods surrounding the clinical hold. The clinical hold produced both hold effects (effects produced by the hold that were consistent in the two treatment groups) and treatment-hold interaction effects (hold effects that were different by treatment). The interaction effects are presented below. The table presents the rate or prevalence of selected clinical events during each interval. The rate of pain, one of the clinical events shown to benefit from treatment with HU, was comparable to the PBO rate during the clinical hold but was significantly lower in the pre- and post-hold intervals. The prevalence of splenomegaly (defined by palpation ≥2 cm below the costal margin), which was slightly greater in the HU group than in the PBO group during the pre- and post-hold intervals, increased to a four-fold difference during the clinical hold. There was no difference between the groups in the rate of splenic sequestration in any interval.Clinical Events Before, During and After Clinical Hold+Before HoldDuring HoldAfter HoldHU 55.1*PBO 50.3*HU 8.3*PBO 7.8*HU 125.1*PBO 126.8*AENo.RateNo.RateNo.RateNo.RateNo.RateNo.RateDactylitis59.13467.6112.0338.51814.48667.8Pain3461.772143.114168.715192.3129103.1288227.1Splenomegaly^3054.42039.814168.7338.58366.36551.6Lab ResultsMeanMeanMeanMeanMeanMeanRetic Ct (k/mm3)191.5329.4300.5327.3237.0335.4Hgb (gm/dL)10.078.699.098.739.288.69MCV (fL)86.8481.9082.8279.8585.0080.00+Rates in bold are significantly different at p < 0.05 within each time period*Person Years of Exposure^Prevalence Hematologic changes showing a treatment-period interaction included a significantly larger difference in absolute reticulocyte count between the HU and placebo group During the Hold as compared to Before and After. The HU-induced increase in Hb and MCV was significantly diminished During the Hold. All measurements moved back towards their pre-hold differences after the hold was lifted. The clinical hold in BABY HUG provided an unforeseen opportunity to study the clinical and laboratory impact of cessation of HU therapy for a brief time period. No serious clinical effects or toxicities occurred during the clinical hold, perhaps due to its limited time span. Parents and physicians should be aware that stopping HU therapy may result in increased episodes of pain or splenic enlargement. Disclosures: Off Label Use: Hydroxyurea which is not approved for use in children with sickle cell disease. Casella:Adventrix: Honoraria.

With exponential cultures of C3H/10T1/2 cells, we have investigated the effect of X-ray dose protraction on oncogenic cell transformation in the dose range 0.25-2 Gy. Within a particular experiment a constant exposure time was used. In different experiments exposure time varied between 1 and 5h. Cell transformation was analysed using the linear-quadratic relation, gamma (D) = alpha 1D + alpha 2D2, between transformation frequency per surviving cell and X-ray dose. Based on values of the linear coefficients, we developed an empirical formula for relating slopes of dose induction curves obtained at high or reduced dose rate condition. Our estimate of repair half-time for cell transformation with 95 per cent confidence limits is 2.4 (1.8, 3.0) h.

This paper develops a simple statistical model, the weighted hazard model, which incorporates the toxicological idea of DNA repair and its role in chemical carcinogenesis. We restrict attention to a small segment of DNA that migrates in and out of the high risk states; it is shown that random hazard functions play an important role in the distributional properties of the time to first detectable tumor. Included in the many shapes of the weighted hazard model is one that has a shape in the low dose region similar to that of the probit model, a model that many toxicologists favor. The analyses of two data sets are presented and interpreted, and suggestions for further research are given.

To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

To enable decision makers to become more aware of the levels of confidence that can be placed in the results of particular computerized decision models, means of establishing levels of confidence for such models are discussed.

The dose-response relationship between extracellular concentration of cytosine arabinoside (ara-C) and intracellular formation of the putative active metabolites of ara-C [ara-C incorporation into DNA and intracellular pools of ara-C in triphosphate form (ara-CTP)] was investigated in blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL) by exposing these cells in vitro to 10, 100, or 1,000 nmol/L of ara-C. We studied 23 untreated patients who subsequently achieved complete remission (CR) with a regimen using daunorubicin and conventional doses of ara-C (ara-C-sensitive group), and 30 patients judged to be ara-C-resistant either by failing initial induction therapy (16 patients) or by having relapsed on an ara-C- containing maintenance regimen (14 patients). In both patient groups, ara-C incorporation into DNA and intracellular ara-CTP both displayed statistically significant increases in response to increasing extracellular concentrations of ara-C (P = .0001 in both cases), with the rate of increase of ara-CTP greater than that of ara-C incorporation. Moreover, blast cells from all patients, even those who were most clinically resistant to ara-C, were able to form ara-CTP and to incorporate ara-C into DNA. Each tenfold increment in extracellular ara-C concentration caused an 8.5-fold increase in ara-CTP, but only a 3.6-fold increase in ara-C incorporation into DNA. Thus, the efficiency of incorporation of ara-C into DNA (defined as the ratio of ara-C incorporation to ara-CTP pools) decreased by 58% with each tenfold increment in the extracellular concentration of ara-C (P less than .0001), presumably as a result of the inhibitory effect of ara-CTP on DNA polymerase. Using an analysis of covariance, modest differences were found in the levels of the ara-C metabolite variables in the ara-C- sensitive group as compared with the resistant group. However, because there was considerable overlap in ara-C metabolite formation among the patient groups, it was not possible to predict clinical outcome by these in vitro assessments of ara-C metabolism.

The cases of 51 patients with bronchial adenomas were reviewed. There were 43 bronchial carcinoids, 5 adenoid cystic carcinomas, 2 mixed tumors, and 1 mucoepidermoid carcinoma. The carcinoid group was divided into typical (31, 72%) and atypical (12, 28%) subgroups. Nine carcinoids (20%) were categorized as metastasizing adenomas; in this group, 7 lesions were atypical and 2 were typical. Thirty-two lobectomies, 7 bilobectomies, 8 pneumonectomies, 2 sleeve resections, and 2 tracheal resections were performed. Ten-year survival was 88% for patients with typical carcinoids and 59% for those with atypical carcinoids. In the group with adenoid cystic carcinoma, 1 patient died postoperatively, 1 had recurrence of the tumor, 2 were alive and free from disease 16 and 23 years later, and 1 died of heart disease at 11 years. The patient with mucoepidermoid carcinoma was alive without recurrence 15 years after operation. In conclusion, bronchial adenomas of the carcinoid type are potentially malignant. Their prognosis depends on the histology of the tumor, and on the presence of metastasis to the regional lymph nodes and distant organs.

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrugresistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrugresistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15–20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160–kD membrane species identified by Vinca alkaloid photoaffinity labeling. The cytogenetic alterations found in P388/LoSort were different from those found in P388/adr, confirming that P388/LoSort cells are unique, although phenotypically similar to P388/adr. These studies suggest that spontaneous mutation to a highly multidrug-resistant phenotype does exist in P388/S cells, but that the mutation frequency is low, with an estimated frequency of 2 × 10−8.

Forty-eight consecutive patients with myasthenia gravis (MG) attended by generalized weakness were treated by complete thymectomy, performed transsternally in 46 patients and through a left thoracotomy in two with thymomas. There were no operative deaths. A 12-year-old child with fulminating MG died of acute pneumonia shortly after hospital discharge. Of the remaining 47 evaluable patients, thymectomy resulted in complete remission in six, marked improvement with a reduced need for medication in 20, and mild improvement on the same dosage of medication in 18. Neither the age of the patient, nor the histopathology of the excised thymus, nor the postoperative change in acetylcholine receptor antibody titer were found to have a significant influence on the response to thymectomy. If the ten patients who were 20 years of age or younger were excluded, the patients with a shorter duration of MG achieved a better response to operation. The authors conclude that thymectomy is effective treatment for MG, regardless of the age of the patient or the type of thymic pathology.