A randomized trial exploring the biomarker effects of neoadjuvant sequential treatment with exemestane and anastrozole in post-menopausal women with hormone receptor-positive breast cancer.

Several adjuvant endocrine strategies exist for postmenopausal women with breast cancer. This study compared the effect of two sequences of aromatase inhibitor use [steroidal (exemestane) and non-steroidal (anastrozole)] on serological and pathological biomarkers when given in the neoadjuvant setting to postmenopausal women with breast cancer. Thirty women were assigned to receive exemestane 25 mg or anastrozole 1 mg each given for 8 weeks in a randomized sequence. The effect of this treatment on serum estrone sulfate and estradiol levels, as well as tumor changes in the proliferation biomarker Ki67 were evaluated at baseline, 8 weeks and 16 weeks. WHO clinical response criteria, patient preference, and quality of life were also assessed. Assessable data was available from 28 patients. There were no differences in concentration changes of serum estradiol or Ki67 between patients in the two arms. Overall clinical response rate was 68% (19/28 assessable patients) and clinical benefit was 93% (26/28 assessable patients). There was no significant difference in toxicity or quality of life scores. The majority of patients expressed a personal preference for anastrozole over exemestane. Results suggest that the order of steroidal and non-steroidal aromatase inhibitors has little effect on outcome. The majority of patients express clear preferences for drug treatments.

Vitamin D insufficiency in a large female SLE cohort.

The objective of this study was to determine the vitamin D status and its relationship with disease and therapy features and with bone mineral density in women with systemic lupus erythematosus. Non-pregnant systemic lupus erythematosus women with dual-energy X-ray absorptiometry and vitamin D measurements performed between May 1 2005 and August 31 2006 were studied. In each patient, the lowest T-score of the first dual-energy X-ray absorptiometry scan during the study period was used. In postmenopausal women, a T-score > or = 1.0 standard deviation was considered normal, between -1.0 and -2.5 standard deviations osteopenia and < or = 2.5 standard deviations osteoporosis; in premenopausal women a T-score > or = 2.5 standard deviations was normal and < or = 2.5 standard deviations defined as reduced bone density. 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels were determined at the time of dual-energy X-ray absorptiometry. A 25-hydroxyvitamin D level of <80 nmol/L was defined as sub-optimal and a level <40 nmol/L as deficient. Demographic and clinical variables were investigated for association with vitamin D levels by univariate and multivariate analyses. One-hundred and twenty-four systemic lupus erythematosus women had dual-energy X-ray absorptiometry scans and vitamin D assays performed during the study period. Sub-optimal 25-hydroxyvitamin D levels were found in 82 (66.7%) and deficient 25-hydroxyvitamin D levels in 22 (17.9%) patients. The disease-related features examined at the time of vitamin D assays or bone mineral density showed no correlation with vitamin D levels by univariate analyses. Neither 25-hydroxyvitamin D nor 1,25-dihydroxyvitamin D was associated with bone mineral density status among these patients. A multivariate logistic regression model identified season, cumulative glucocorticoid exposure, and serum creatinine as being associated with 25-hydroxyvitamin D levels, whereas ethnicity, glucocorticoid exposure, and serum creatinine were associated with 1,25-dihydroxyvitamin D levels. In conclusion, sub-optimal vitamin D status is common in women with systemic lupus erythematosus and is related to season, cumulative glucocorticoid dose, and serum creatinine.

Heterogeneous disease modeling for Hardy-Weinberg disequilibrium in case-control studies: application to renal stones and calcium-sensing receptor polymorphisms.

Renal stone formation due to hypercalciuria is a relatively common disorder with clear evidence for genetic predisposition, but cryptic phenotypic heterogeneity has hampered identification of candidate genes. The R990G single-nucleotide polymorphism (SNP) of the calcium sensing receptor (CASR) gene has been associated with hypercalciuria in stone formers and shows the appropriate functional phenotype in cell culture. In our preliminary association analysis of a case-control cohort, however, we observed significant Hardy-Weinberg disequilibrium (HWD) for the cases (n= 223), but not controls (n= 676) at the R990G locus, pointing us toward the general disease model incorporating HWD. Because there is an adjacent CASR SNP, A986S, which is in negative linkage disequilibrium with R990G, we extended the general disease model to enable testing of a two-site hypothesis. In our data set, there is no lack of fit (P= .345) for the single-locus model for the R990G genotype, and likelihood ratio testing favors a recessive effect with an eight-fold increase in risk (P < .001) for GG homozygotes, relative to wild-type, based on a population prevalence of 2%. Addition of the A986S genotype provides no additional information either by itself or when included in our two-site model.

Replication of associations between LRP5 and ESRRA variants and bone density in premenopausal women.

UNLABELLED: Replication is a critical step to validate positive genetic associations. In this study, we tested two previously reported positive associations. The low density lipoprotein receptor-related protein 5 (LRP5) Val667Met and lumbar spine bone density are replicated. This result is in line with results from large consortiums such as Genomos. However, the estrogen-related receptor alpha (ESRRA) repeat in the promoter is not replicated although the polymorphism studied was functional and could have been a causative variant. INTRODUCTION: We sought to validate associations previously reported between LRP5 V667M polymorphism and lumbar spine (LS, p = 0.013) and femoral neck (FN, p = 0.0002) bone mineral density (BMD), and between ESRRA 23 base pair repeat polymorphism and LS BMD (p = 0.0036) in a sample of premenopausal Caucasian women using an independent sample. METHODS: For the replication sample, we recruited 673 premenopausal women from the Toronto metropolitan area. All women were Caucasian and had BMD measured. LRP5 V667M was genotyped by allele-specific PCR and ESRRA repeats by sizing of PCR products on agarose gels. RESULTS: We reproduced the same association as we reported previously between LRP5 V667M and LS BMD (p = 0.015) but not with FN BMD (p = 0.254). The combined data from the two populations indicate an effect size of 0.28SD for LS BMD (p = 0.00048) and an effect size of 0.26 SD for FN BMD (p = 0.00037). In contrast, the association we reported earlier between ESRRA repeats and LS BMD was not replicated in the sample from Toronto (p = 0.645). CONCLUSIONS: The association between LRP5 V667M and LS BMD is confirmed but not that between ESRRA repeats and LS BMD. This result indicates that it is imperative to validate any positive association in an independent sample.

Cardiovascular disease-risk factors in middle-aged osteopaenic women treated with calcium alone or combined to three nutrients essential to artery and bone collagen.

BACKGROUND: Recent research suggests that cardiovascular disease (CVD) and bone loss are functionally interwoven. This study examined the concomitant effects of a nutritional treatment of osteopaenia on CVD-risk factors. METHODS: A 1-year placebo-controlled trial was conducted on middle-aged women with normal (group A) or low (groups B and C) bone mineral density. Subjects (n = 20 per group) took daily either a placebo, calcium carbonate alone or combined to a vitamin (C and B(6))-proline capsule, respectively. Urinary pyridoxic acid (used to assess treatment compliance), plasma homocysteine, serum lipids and lipoproteins were measured before and after nutritional intervention. RESULTS: Groups were comparable at baseline in most parameters of interest. No changes occurred in groups A and B. The 4%, 7% and 25% reductions of total cholesterol, LDL and triglycerides, and 14% elevation of HDL were all significant in group C. A trend toward reduction was observed for homocysteine in this group. CONCLUSIONS: Vitamins C (500 mg) and B(6) (75 mg) combined with proline had consistent beneficial effects on CVD-risk factors, whereas calcium alone did not. This study also underlined the importance of considering vitamin B(6) status as a potential CVD risk factor.

Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations have been identified in the coding region of the extracellular calcium-sensing receptor (CASR) gene and cause human autosomal dominant hypo- and hypercalcemic disorders. The functional effects of several of these mutations have been characterized in either Xenopus laevis oocytes or in human embryonic kidney (HEK293) cells. All of the mutations that have been examined to date, however, cause single putative amino acid substitutions. In this report, we studied a mutant CASR with an Alu-repetitive element inserted at codon 876, which was identified in affected members of families with the hypercalcemic disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), to understand how this insertion affects CASR function. After cloning of the Alu-repetitive element into the wild-type CASR cDNA, we transiently expressed the mutant receptor in HEK293 cells. Expression of mutant and wild-type receptors was assessed by Western analysis, and the effects of the mutation on extracellular calcium (Ca2+(o)) and gadolinium (Gd3+(o)) elicited increases in the cytosolic calcium concentration (Ca2+(i)) were examined in fura-2-loaded cells using dual wavelength fluorimetry. The insertion resulted in truncated receptor species that had molecular masses some 30 kD less than that of the wild-type CASR and exhibited no Ca2+(i) responses to either Ca2+(o) or Gd3+(o). A similar result was observed with a mutated CASR truncated at residue 876. However, the Alu mutant receptor had no impact on the function of the coexpressed wild-type receptor. Interestingly, the Alu mutant receptor demonstrated decreased cell surface expression relative to the wild-type receptor, whereas the CASR (A877stop) mutant exhibited increased cell surface expression. Thus, like the missense mutations that have been characterized to date in families with FHH, the Alu insertion in this family is a loss-of-function mutation that produces hypercalcemia by reducing the number of normally functional CASRs on the surface of parathyroid and kidney cells. In vitro transcription of exon 7 of the CASR containing the Alu sequence yielded the full-length mutant product and an additional shorter product that was truncated due to stalling of the polymerase at the poly(T) tract. In vitro translation of the mutant transcript yielded three truncated protein products representing termination in all three reading frames at stop codons within the Alu insertion. Thus sequences within the Alu contribute to slippage or frameshift mutagenesis during transcription and/or translation.

Absence of hereditary p53 mutations in 10 familial leukemia pedigrees.

Germline p53 mutations have been identified in the Li-Fraumeni syndrome but the role of such mutations in familial leukemia is not established. The p53 gene was examined by single-strand conformation polymorphism analysis of exons 4-8 in 10 families with multiple members affected with leukemia. The diagnoses included acute and chronic leukemias and Hodgkin's disease. Identified in two families were p53 mutations that were nonhereditary. These included a 2-bp deletion in exon 6 found in the lymphoblast DNA of one child whose sibling, cousin, and several adult relatives had acute leukemia. The other nonhereditary p53 mutation was a transition at codon 248 (CGG to CAG, arginine to glutamine) found in the lymphoblasts of a patient with a preleukemic syndrome and acute lymphoblastic leukemia (ALL) whose brother is a long-term survivor of ALL. Thus, p53 mutations were found to occur in two families but both were nonhereditary. Moreover, in the remaining eight families no p53 mutation was identified in the regions of p53 where most mutations have been found in other cancers. Although p53 mutations sometimes may be present, they do not appear to be a primary event responsible for hereditary susceptibility to familial leukemia. This study suggests involvement of other genes or mechanisms.

Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia.

The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.

Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype.

Neonatal severe hyperparathyroidism is a rare life-threatening disorder characterized by very high serum calcium concentrations (> 15 mg/dl). Many cases have occurred in families with familial hypocalciuric hypercalcemia, a benign condition transmitted as a dominant trait. Among several hypothesized relationships between the two syndromes is the suggestion that neonatal severe hyperparathyroidism is the homozygous form of familial hypocalciuric hypercalcemia. To test this hypothesis, we refined the map location of the gene responsible for familial hypocalciuric hypercalcemia on chromosome 3q. Analyses in 11 families defined marker loci closely linked to the gene responsible for familial hypocalciuric hypercalcemia. These loci were then analyzed in four families with parental consanguinity and offspring with neonatal severe hyperparathyroidism. Each individual who was homozygous for loci that are closely linked to the gene responsible for familial hypocalciuric hypercalcemia had neonatal severe hyperparathyroidism. The calculated odds of linkage between these disorders of > 350,000:1 (lod score = 5.56). We conclude that dosage of the gene defect accounts for these widely disparate clinical phenotypes; a single defective allele causes familial hypocalciuric hypercalcemia, while two defective alleles causes neonatal severe hyperparathyroidism.

Prevention of methotrexate cytotoxicity by asparaginase inhibition of methotrexate polyglutamate formation.

Escherichia coli asparaginase (Asnase) pretreatment of Asnase-sensitive L5178Y cells in vitro is thought to antagonize methotrexate (MTX) cytotoxicity through nonspecific inhibition of protein synthesis and MTX uptake. We have reexamined the mechanism of this interaction in view of recent data demonstrating the importance of MTX metabolism to polyglutamate derivatives (MTXPGs) in the cytotoxic effects of the antifolate. After a 3-hr exposure to 0.5 microM MTX, 67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2-5), and cloning efficiency in drug-free medium was only 7% of untreated control. After a 3-hr pretreatment with E. coli Asnase (0.1 unit/ml), [3H]thymidine incorporation dropped by 29%, MTXPG formation during subsequent MTX exposure decreased by more than one-half (MTX-Glu2 unchanged; MTX-Glu3 and 4 decreased to 51.7 and 18.5% of levels achieved in cells not pretreated with Asnase; no MTX-Glu5 formed), and cloning efficiency increased to 71% of untreated control. This effect was not due to decreased MTX uptake into L5178Y cells or to decreased intracellular free L-glutamate or L-glutamine levels. A 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an essential amino acid for cell growth, prior to MTX exposure inhibited [3H]thymidine incorporation by 37%, decreased subsequent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels. Decreased MTXPG formation was responsible for the prevention of MTX cytotoxicity seen after both pretreatments. Unmetabolized MTX rapidly left L5178Y cells after removal of extracellular MTX. Consequently, lower levels of unbound intracellular drug, a prerequisite of drug activity, were maintained in pretreated than in control cells after passage in drug-free medium. Asnase pretreatment protects L5178Y cells from the cytotoxic effects of MTX, possibly through inhibition of cell growth which nonspecifically decreases MTXPG formation.

Plasma and cerebrospinal fluid pharmacokinetic study of topotecan in nonhuman primates.

Topotecan, a water soluble semisynthetic analogue of camptothecin, is a topoisomerase I inhibitor that has recently entered phase II clinical trials. Topotecan has shown significant preclinical activity in refractory murine tumors and in human tumor xenograft models. In addition, objective antineoplastic activity has been observed in recent adult phase I clinical trials. Topotecan is unstable in solution and is rapidly and spontaneously converted to a less active open ring form which predominates at physiological pH. This study was undertaken to better define the pharmacokinetic behavior of this highly unstable compound in both plasma and cerebrospinal fluid (CSF) and to measure the degree of CSF penetration of this novel antineoplastic agent. Three nonhuman primates with indwelling Ommaya reservoirs received 10 mg/m2 i.v. topotecan administered as a 10-min infusion. Frequent plasma and CSF samples were obtained and immediately extracted and assayed with a reverse phase high performance liquid chromatography assay to quantitate the concentration of topotecan (lactone). Samples were then acidified and reinjected to quantitate total drug (lactone ring plus open ring). Peak plasma concentrations of topotecan ranged from 0.27 to 0.45 microM. Plasma disappearance of the lactone ring was biexponential with a distribution half-life (t1/2 alpha) of 22 +/- 5 min and an elimination half-life (t1/2 beta) of 1.3 +/- 0.1 h. Total body clearance of topotecan was 72.1 +/- 15.8 liters/h/m2. The volume of distribution at steady state was 88.6 +/- 33.2 liters/m2. Peak CSF concentrations of topotecan occurred at 30 min following drug administration and ranged from 0.044 to 0.074 microM. CSF disappearance paralleled that in plasma. The mean ratio of the area under the CSF concentration-time curve to that in plasma was 0.32 (range, 0.29 to 0.37). The mean CSF penetration of topotecan exceeds 30%, which is significantly greater than the penetration of most structurally similar chemotherapeutic agents. The impact of chemotherapy on the survival of patients with primary or metastatic central nervous system malignancies is very limited. Therefore, this novel antineoplastic agent is an excellent candidate for further study in patients with high risk or refractory central nervous system tumors.

Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and time course of peripheral blood mononuclear cell O6-methylguanine-DNA methyltransferase inhibition in the nonhuman primate.

O6-Benzylguanine (O6BG) enhances the cytotoxicity of the nitrosoureas by irreversibly binding and inhibiting the DNA repair enzyme O6-methyl-guanine-DNA methyltransferase (MGMT). The plasma and cerebrospinal fluid (CSF) pharmacokinetics of O6BG and its active metabolite, O6-benzyl-8-oxoguanine, were studied in a nonhuman primate model after 200 mg/m2 had been injected i.v. The parent drug and the metabolite were measured with a reverse-phase HPLC assay. A pharmacokinetic model incorporating separate compartments for O6BG and the O6-benzyl-8-oxoguanine metabolite, first-order conversion of O6BG to the metabolite, and additional first-order elimination rate constants for each compound, was simultaneously fitted to the parent drug and metabolite plasma concentration time data. Elimination of O6BG from plasma was rapid; it had a half-life of 1.6 h and a clearance of 68 ml/min/m2. On the basis of the pharmacokinetic model, essentially all of the O6BG was converted to O6-benzyl-8-oxoguanine. The plasma pharmacokinetic profile of the metabolite differed considerably from that the parent drug. The half-life (14 h) was 10-fold longer and the area under the curve (2420 microM/h) was 11-fold higher than that of O6BG (212 microM/h). The clearance rate of O6-benzyl-8-oxoguanine was 6.4 ml/min/m2. The CSF:plasma ratio was 4.3% for O6BG and 36% for O6-benzyl-8-oxoguanine, and the metabolite area under the curve was 90-fold higher than that of O6BG in CSF. The excellent CSF penetration of the active metabolite provides a rationale for the use of O6BG as a chemosensitizing agent for brain tumors. We also studied the duration of MGMT inhibition in peripheral blood mononuclear cells. By 2 h after a 200 mg/m2 dose of O6BG, > 98% of MGMT activity was suppressed, and > 95% suppression of enzyme activity persisted at 18 and 48 h after the dose. By 2 weeks after the treatment, MGMT levels had returned to baseline. Persistent high concentrations of the active metabolite appear to provide a pharmacological explanation for the prolonged suppression of MGMT activity.

Modulation of the cytotoxic effect of cyclopentenylcytosine by its primary metabolite, cyclopentenyluridine.

Cyclopentenylcytosine (CPE-C), a synthetic cytidine analogue with significant preclinical antitumor activity against both solid tumor xenografts and 1-beta-D-arabinofuranosylcytosine resistant murine leukemia cell lines, will soon enter phase I clinical trials. Unlike 1-beta-D-arabinofuranosylcytosine which is activated by deoxycytidine kinase, the enzyme responsible for the phosphorylation of CPE-C is uridine/cytidine kinase. Preclinical pharmacokinetic studies of CPE-C in nonhuman primates revealed that the primary route of elimination in this species was deamination to cyclopentenyluridine (CPE-U), an inhibitor of uridine/cytidine kinase. Since CPE-C is likely to be deaminated in humans, we investigated the modulating effect of CPE-U on the in vitro cytotoxicity of CPE-C in Molt-4 lymphoblasts. Concurrent exposure of cells to cytotoxic concentrations of CPE-C and 50 microM CPE-U resulted in the rescue of 50% of cells and exposure to CPE-U concentrations in excess of 100 microM resulted in the rescue of greater than 90% of cells. Progressive attenuation of the rescue effect was observed with delayed administration of CPE-U and no cells were rescued when addition of CPE-C was delayed for more than 2 h. At the intracellular level it was observed that the formation of the cytotoxic metabolite, cyclopentenylcytosine triphosphate, was blocked by increasing concentrations of CPE-U presumably secondary to inhibition of uridine/cytidine kinase by CPE-U. Although CPE-U can modulate the cytotoxic effects of CPE-C in vitro, the minimum CPE-U levels that are required for modulation coupled with the available preclinical pharmacokinetic data from nonhuman primates suggests that this modulation is not likely to impact on the antitumor effects of CPE-C in humans.

A pharmacokinetic model of topotecan clearance from plasma and cerebrospinal fluid.

We present a physiological pharmacokinetic model that describes the plasma and cerebrospinal fluid (CSF) concentrations of topotecan [(S)-9-dimethylaminomethyl-10-hydroxyamptothecin hydrochloride, SK&F 104864-A, NSC 609699] following i.v. and intraventricular administrations in monkeys. The model consists of three physical spaces: the CSF, the plasma, and a body compartment. The model incorporates such processes as reversible conversion of topotecan lactone to an inactive hydroxy acid form, microvascular exchange between CSF and plasma, bulk CSF flow, exchange between plasma and body compartments, and elimination of drug from the plasma compartment. Several parameters in the model were obtained from published literature on the physiology of the monkey. The model was then fit to the plasma and CSF data to deduce the other parameters. Calculated clearances of topotecan lactone and total drug from the CSF after intraventricular injection were 3.9 and 2.2 ml/h, respectively. Clearances of topotecan lactone and total drug from the plasma following a 10-min infusion were 26.3 liters/h/m2 and 17.8 liters/h/m2, respectively. The calculated ratios of the area under the concentration curve in the CSF following i.v. infusion to the area under the concentration curve in plasma were 0.11 and 0.19 for topotecan and total drug, respectively, indicating significant CSF penetration. The volume of distribution was 0.77 liters/kg, which represents distribution in a volume approximating total body water. The forward and reverse rate constants for the lactone-to-hydroxy acid conversion were 1.0 and 0.29 h-1, respectively. Comparison of the clearances (normalized to body surface area) with values reported for mice and humans shows reasonable similarity across species. This pharmacokinetic model may help guide future development and refinement of clinical protocols, especially in the treatment of diseases of the central nervous system.

Pediatric phase I trial and pharmacokinetic study of topotecan administered as a 24-hour continuous infusion.

Topotecan, a water-soluble semisynthetic analogue of camptothecin, is the first topoisomerase I inhibitor to undergo evaluation in pediatric patients with refractory malignancies. A phase I and pharmacokinetic study was performed to determine the maximum tolerated dose (MTD) and dose-limiting toxicities, the incidence and severity of other toxicities, and the pharmacokinetics of topotecan in children. Twenty-nine patients received 42 courses of i.v. topotecan administered as a 24-h continuous infusion every 21 days at doses ranging from 2.0 to 7.5 mg/m2. Dose-related hematological toxicity was the dose-limiting toxicity. Leukopenia, neutropenia, and thrombocytopenia occurred sporadically at the 3.0- to 5.5-mg/m2 dose levels, but at 7.5 mg/m2 4 of 5 patients experienced dose-limiting thrombocytopenia (grade 4) and 2 of 5 had dose-limiting neutropenia (grade 4). No other dose-limiting toxicities were observed. Nausea and vomiting were mild and occurred in < 20 and 10% of patients, respectively. Grade 2 hematuria occurred in one patient. No objective responses were observed. Pharmacokinetic studies revealed a linear relationship between the steady-state topotecan concentration and dose. The mean steady-state concentration at the MTD was 18.2 +/- 3.7 nmol/liter and the total body clearance was 28.3 +/- 6.5 liters/h/m2. Elimination was biexponential with a t1/2 alpha of 14.4 +/- 1.8 min and a t1/2 beta of 2.9 +/- 1.1 h. The recommended starting dose for phase II pediatric trials is 5.5 mg/m2. Although this dose exceeds the MTD identified in heavily pretreated adult patients receiving topotecan on the same schedule, it is less than the MTD for minimally pretreated adult patients. Therefore, dose escalation to 7.5 mg/m2 in phase II pediatric trials should be considered for patients who tolerate treatment well at the 5.5-mg/m2 dose.

Phase I and pharmacokinetic evaluation of thiotepa in the cerebrospinal fluid and plasma of pediatric patients: evidence for dose-dependent plasma clearance of thiotepa.

A Phase I trial of thiotepa (TT) administered as an i.v. bolus was performed in 19 children with refractory malignancies. The starting dose was 25 mg/m2 with escalations to 50, 65, and 75 mg/m2. Seven additional patients were treated with 8-h infusions at 50 or 65 mg/m2. The maximum tolerated bolus dose was 65 mg/m2. Reversible myelosuppression was the dose-limiting toxicity. The plasma and cerebrospinal fluid (CSF) pharmacokinetic parameters of TT and its major active metabolite tepa (TP) were also evaluated. When the bolus or infusion methods of TT administration were compared, there was little difference observed in any pharmacokinetic parameter for either TT or TP. The plasma disappearance of TT was rapid and biphasic with half-lives of 0.14 to 0.32 and 1.34 to 2.0 h. Dose-dependent pharmacokinetics was demonstrated by steadily declining plasma clearance with increasing TT dose. Clearance values declined from 28.6 liters/m2/h at the 25-mg/m2 dose to 11.9 liters/m2/h at the 75-mg/m2 dose. The half-life of TP was longer than that of TT and ranged between 4.3 and 5.6 h. There was evidence of the saturation of TP production. TT and TP both exhibited excellent penetration into the CSF, producing lumbar and ventricular concentrations which were nearly identical to simultaneous plasma concentrations. In one patient with a Rickham reservoir, the CSF:plasma area under the (concentration x time) curve ratios for TT and TP were 1.01 and 0.95, respectively. The above data indicate that TT can be safely administered to pediatric patients at doses higher than conventionally used. The favorable CSF penetration of TT and TP suggests that Phase II studies of TT be considered in patients with central nervous system tumors.

Sulfate transport in human placenta: further evidence for a sodium-independent mechanism.

Sulfate transport in isolated placental brush-border membrane vesicles has properties consistent with an anion exchange process. To ascertain the relevance of this finding to sulfate accumulation by the fetus and placenta in vivo, we examined sulfate transport in human placental tissue slices, comparing sulfate uptake with that of a non-metabolizable amino acid marker, alpha-aminoisobutyrate (AIB). In contrast to AIB, which was actively concentrated from physiological media, sulfate uptake by the placenta slice was concentrative only in the absence of sodium and at low pH. Uptake of sulfate reached a steady state after 60 min. It was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a specific inhibitor of anion transport, but not by ouabain. We found no evidence for Na(+)-dependent uptake of sulfate in incubated placental tissue. It seems unlikely that Na(+)-dependent sulfate transport by the placenta can be responsible for net sulfate accumulation by the human fetus.

Sulfate transport by mouse renal cortical slices does not represent uptake by brush-border membrane.

We measured uptake of isotopically 35S-labelled sulfate anion by slices and by brush border membrane vesicles prepared from mouse renal cortex to identify: (i) whether metabolic incorporation of anion influences net transport; (ii) which membrane is primarily exposed in the renal cortex slice. Slices accumulated sulfate without significant incorporation into metabolic pools. Net uptake of sulfate at 0.1 mM by the slice occurred against an electrochemical gradient as determined by measurement of free intracellular sulfate concentration, the isotopic distribution ratio at steady-state, and the distribution of lipophilic ions (TPP+ and SCN-). Carrier mediation of sulfate transport in the slice was confirmed by observing concentration-dependent saturation of net uptake and counter-transport stimulation of efflux. Anion uptake was Na+-independent, K+- and H+-stimulated, and inhibited by disulfonated stilbenes. Brush-border membrane vesicles accumulated sulfate by a saturable mechanism dependent on a Na+ gradient (outside greater than inside); others have shown that uptake of sulfate by brush-border membrane vesicles is insensitive to inhibition by disulfonated stilbenes. These findings indicate that different mechanisms serve sulfate transport in renal cortex slice and brush-border membrane vesicle preparations. They also imply that the slice exposes an epithelial surface different from the brush-border, presumably the basolateral membrane, or its equivalent, since sulfate transport by slices resembles that observed with isolated basolateral membrane vesicles.

Characterization of immunoglobulin and T-cell receptor gene patterns in B-cell precursor acute lymphoblastic leukemia of childhood.

Immunoglobulin (Ig) and T-cell receptor (TCR) genes were examined in the lymphoblasts of 70 children with immunophenotypically defined B-cell precursor acute lymphoblastic leukemia (ALL). The most frequent genes to rearrange were Ig heavy (H) chain (93%) and TCR delta (79%), followed by TCR gamma (49%), Ig kappa and/or lambda light (L) chain (46%), TCR alpha (46%), and TCR beta (29%). Thus, despite their putative "B-cell precursor" lineage, these leukemias manifest a remarkably high incidence of TCR gene rearrangements. While certain patterns predominate, there is considerable heterogeneity in Ig and TCR genotypes in this disease. No significant associations were found between Ig and TCR genotype and commonly used prognostic factors including age, sex, race, WBC, French-American-British (FAB) subtype, or cytogenetics. However, the lymphoblasts of three of six patients who failed to achieve initial remission had germline patterns of every Ig and TCR gene, a genotype not observed in the leukemic cells from any of the 64 patients who achieved complete remission (p2 = .0007). This study suggests that particular Ig and TCR genotypes may be of clinical relevance in childhood B-cell precursor ALL. The finding of rearranged TCR genes in a large proportion of cases raises fundamental questions about early lineage commitment and lymphocyte differentiation along B-cell and T-cell pathways.

Comparison of breast magnetic resonance imaging, mammography, and ultrasound for surveillance of women at high risk for hereditary breast cancer.

PURPOSE: Recommended surveillance for BRCA1 and BRCA2 mutation carriers includes regular mammography and clinical breast examination, although the effectiveness of these screening techniques in mutation carriers has not been established. The purpose of the present study was to compare breast magnetic resonance imaging (MRI) with ultrasound, mammography, and physical examination in women at high risk for hereditary breast cancer. PATIENTS AND METHODS: A total of 196 women, aged 26 to 59 years, with proven BRCA1 or BRCA2 mutations or strong family histories of breast or ovarian cancer underwent mammography, ultrasound, MRI, and clinical breast examination on a single day. A biopsy was performed when any of the four investigations was judged to be suspicious for malignancy. RESULTS: Six invasive breast cancers and one noninvasive breast cancer were detected among the 196 high-risk women. Five of the invasive cancers occurred in mutation carriers, and the sixth occurred in a woman with a previous history of breast cancer. The prevalence of invasive or noninvasive breast cancer in the 96 mutation carriers was 6.2%. All six invasive cancers were detected by MRI, all were 1.0 cm or less in diameter, and all were node-negative. In contrast, only three invasive cancers were detected by ultrasound, two by mammography, and two by physical examination. The addition of MRI to the more commonly available triad of mammography, ultrasound, and breast examination identified two additional invasive breast cancers that would otherwise have been missed. CONCLUSION: Breast MRI may be superior to mammography and ultrasound for the screening of women at high risk for hereditary breast cancer.