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The purpose of this study was to develop and validate a population pharmacokinetic model for Z-endoxifen in patients with advanced solid tumors and to identify clinical variables that influence pharmacokinetic parameters. Z-endoxifen-HCl was administered orally once a day on a 28-day cycle (±3 days) over 11 dose levels ranging from 20 to 360 mg. A total of 1256 Z-endoxifen plasma concentration samples from 80 patients were analyzed using nonlinear mixed-effects modeling to develop a population pharmacokinetic model for Z-endoxifen. A 2-compartment model with oral depot and linear elimination adequately described the data. The estimated apparent total clearance, apparent central volume of distribution, and apparent peripheral volume of distribution were 4.89 L/h, 323 L, and 39.7 L, respectively, with weight-effect exponents of 0.75, 1, and 1, respectively. This model was used to explore the effects of clinical and demographic variables on Z-endoxifen pharmacokinetics. Weight, race on clearance, and aspartate aminotransferase on the absorption rate constant were identified as significant covariates in the final model. This novel population pharmacokinetic model provides insight regarding factors that may affect the pharmacokinetics of Z-endoxifen and may assist in the design of future clinical trials., (© 2022, The American College of Clinical Pharmacology.)

Purpose: NSC 161128, a phenylurea thiocarbamate, displays activity against the NCI60 anti-cancer cell line panel and xenograft models. The metabolite N-methyl-N'-phenylurea (M10) has been detected in animal plasma; however, detection and quantification of other putative NSC 161128 metabolites have not been undertaken. The purpose of this study was to characterize the pharmacokinetics and metabolism of NSC 161128 in mice and under in vitro conditions., Methods: An LC-MS/MS assay was developed to evaluate stability and in vitro metabolism of NSC 161128 in liver microsomes and S9 fractions. Single-dose pharmacokinetic profiles for NSC 161128 and its metabolite M10 were obtained following intraperitoneal (I.P.) administration., Results: A sensitive and specific positive ionization LC-MS/MS method for measuring NSC 161128 and its metabolites was developed. HPLC separation was achieved under gradient elution using an aqueous methanol mobile phase containing 0.05% formic acid and 0.05% ammonium hydroxide. The assay was linear over the range 1.0-1000 ng/mL. NSC 161128 was stable in aqueous solution and tissue culture media, but not in plasma, where rapid degradation of NSC 161128 to the metabolite M10 was observed. Following I.P. administration of a 200 mg/kg dose to male CD1 mice, the peak plasma concentration of NSC 161128 was 255 ng/mL after 5 min with a plasma half-life of 138 min. Potential bioactivation of NSC 161128 was explored using mouse S9., Conclusions: An analytical LC-MS/MS method was successfully developed for the detection and quantification of NSC 161128 and its metabolites. These results increase the understanding of NSC 161128 pharmacokinetic and metabolic properties., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High-dose methotrexate (HDMTX) pharmacokinetics (PKs), including the best estimated glomerular filtration rate (eGFR) equation that reflects methotrexate (MTX) clearance, requires investigation. This prospective, observational, single-center study evaluated adult patients with lymphoma treated with HDMTX. Samples were collected at predefined time points up to 96 h postinfusion. MTX and 7-hydroxy-MTX PKs were estimated by standard noncompartmental analysis. Linear regression determined which serum creatinine- or cystatin C-based eGFR equation best predicted MTX clearance. The 80 included patients had a median (interquartile range [IQR]) age of 68.6 years (IQR 59.2-75.6), 54 (67.5%) were men, and 74 (92.5%) were White. The median (IQR) dose of MTX was 7.6 (IQR 4.8-11.3) grams. Median clearance was similar across three dosing levels at 4.5-5.6 L/h and was consistent with linear PKs. Liver function, weight, age, sex, concomitant chemotherapy, and number of previous MTX doses did not impact clearance. MTX area under the curve (AUC) values varied over a fourfold range and appeared to increase in proportion to the dose. The eGFR cys (ml/min) equation most closely correlated with MTX clearance in both the entire cohort and after excluding outlier MTX clearance values (r = 0.31 and 0.51, respectively). HDMTX as a 4-h infusion displays high interpatient pharmacokinetic variability. Population PK modeling to optimize MTX AUC attainment requires further evaluation. The cystatin C-based eGFR equation most closely estimated MTX clearance and should be investigated for dosing and monitoring in adults requiring MTX as part of lymphoma management., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Objective: Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG., Methods: We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2-CDG for 12 months., Results: PMM enzyme activity increased post-epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2-deficient fibroblasts, 46% of which improved upon treatment. Total protein N-glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts' glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2-CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2-CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months., Interpretation: Epalrestat improved PMM enzyme activity, N-glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems-level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well-tolerated and led to significant clinical improvements in the first pediatric patient with PMM2-CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2-CDG. ANN NEUROL 20219999:n/a-n/a., (© 2021 American Neurological Association.)

The application of scaffold-based stem cell transplantation to enhance peripheral nerve regeneration has great potential. Recently, the neuroregenerative potential of tacrolimus (a U.S. Food and Drug Administration-approved immunosuppressant) has been explored. In this study, a fibrin gel-based drug delivery system for sustained and localized tacrolimus release was combined with rat adipose-derived mesenchymal stem cells (MSC) to investigate cell viability in vitro. Tacrolimus was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres and suspended in fibrin hydrogel, using concentrations of 0.01 and 100 ng/ml. Drug release over time was measured. MSCs were cultured in drug-released media collected at various days to mimic systemic exposure. MSCs were combined with (i) hydrogel only, (ii) empty PLGA microspheres in the hydrogel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus PLGA microspheres in the hydrogel. Stem cell presence and viability were evaluated. A sustained release of 100 ng/ml tacrolimus microspheres was observed for up to 35 days. Stem cell presence was confirmed and cell viability was observed up to 7 days, with no significant differences between groups. This study suggests that combined delivery of 100 ng/ml tacrolimus and MSCs in fibrin hydrogel does not result in cytotoxic effects and could be used to enhance peripheral nerve regeneration., (© 2021 Wiley Periodicals LLC.)

Background: Differential responses to tamoxifen may be due to inter-patient variability in tamoxifen metabolism into pharmacologically active Z-endoxifen. Z-endoxifen administration was anticipated to bypass these variations, increasing active drug levels, and potentially benefitting patients responding sub-optimally to tamoxifen., Materials and Methods: Patients with treatment-refractory gynecologic malignancies, desmoid tumors, or hormone receptor-positive solid tumors took oral Z-endoxifen daily with a 3+3 phase 1 dose escalation format over 8 dose levels (DLs). Safety, pharmacokinetics/pharmacodynamics, and clinical outcomes were evaluated., Results: Thirty-four of 40 patients were evaluable. No maximum tolerated dose was established. DL8, 360 mg/day, was used for the expansion phase and is higher than doses administered in any previous study; it also yielded higher plasma Z-endoxifen concentrations. Three patients had partial responses and 8 had prolonged stable disease (≥ 6 cycles); 44.4% (8/18) of patients at dose levels 6-8 achieved one of these outcomes. Six patients who progressed after tamoxifen therapy experienced partial response or stable disease for ≥ 6 cycles with Z-endoxifen; one with desmoid tumor remains on study after 62 cycles (nearly 5 years)., Conclusions: Evidence of antitumor activity and prolonged stable disease are achieved with Z-endoxifen despite prior tamoxifen therapy, supporting further study of Z-endoxifen, particularly in patients with desmoid tumors., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Takebe et al.)

Background: The tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in endocrine-resistant estrogen receptor-positive (ER+) breast cancer. We compared the antitumor activity of Z-endoxifen with tamoxifen and letrozole in the letrozole-sensitive MCF7 aromatase expressing model (MCF7AC1), as well as with tamoxifen, fulvestrant, exemestane, and exemestane plus everolimus in a letrozole-resistant MCF7 model (MCF7LR)., Methods: MCF7AC1 tumor-bearing mice were randomized to control (no drug), letrozole (10 μg/day), tamoxifen (500 μg/day), or Z-endoxifen (25 and 75 mg/kg). Treatment in the letrozole arm was continued until resistance developed. MCF7LR tumor-bearing mice were then randomized to Z-endoxifen (50 mg/kg) or tamoxifen for 4 weeks and tumors harvested for microarray and immunohistochemistry analysis. The antitumor activity of Z-endoxifen in the MCF7LR tumors was further compared in a second in vivo study with exemestane, exemestane plus everolimus, and fulvestrant., Results: In the MCF7AC1 tumors, both Z-endoxifen doses were significantly superior to control and tamoxifen in reducing tumor volumes at 4 weeks. Additionally, the 75 mg/kg Z-endoxifen dose was additionally superior to letrozole. Prolonged letrozole exposure resulted in resistance at 25 weeks. In MCF7LR tumor-bearing mice, Z-endoxifen significantly reduced tumor volumes compared to tamoxifen, letrozole, and exemestane, with no significant differences compared to exemestane plus everolimus and fulvestrant. Additionally, compared to tamoxifen, Z-endoxifen markedly inhibited ERα target genes, Ki67 and Akt expression in vivo., Conclusion: In endocrine-sensitive and letrozole-resistant breast tumors, Z-endoxifen results in robust antitumor and antiestrogenic activity compared to tamoxifen and aromatase inhibitor monotherapy. These data support the ongoing development of Z-endoxifen.

Background: Vaginal diazepam is frequently used to treat pelvic floor tension myalgia and pelvic pain despite limited knowledge of systemic absorption., Aim: To determine the pharmacokinetic and adverse event profile of diazepam vaginal suppositories., Methods: We used a prospective pharmacokinetic design with repeated assessments of diazepam levels. Eight healthy volunteers were administered a 10-mg compounded vaginal diazepam suppository in the outpatient gynecologic clinic. Serum samples were collected at 0, 45, 90, 120, and 180 minutes; 8, 24, and 72 hours; and 1 week following administration of a 10-mg vaginal suppository. The occurrence of adverse events was assessed using the alternate step and tandem walk tests, the Brief Confusion Assessment Method, and numerical ratings. Plasma concentrations of diazepam and active long-acting metabolites were measured. Pharmacokinetic parameters were calculated by standard noncompartmental methods., Results: The mean peak diazepam concentration (C max ) of 31.0 ng/mL was detected at a mean time (T max ) of 3.1 hours after suppository placement. The bioavailability was found to be 70.5%, and the mean terminal elimination half-life was 82 hours. The plasma levels of temazepam and nordiazepam peaked at 0.8 ng/mL at 29 hours and 6.4 ng/mL at 132 hours, respectively. Fatigue was reported by 3 of 8 participants., Clinical Implications: Serum plasma concentrations of vaginally administered diazepam are low; however the half-life is prolonged., Strengths & Limitations: Strengths include use of inclusion and exclusion criteria aimed at mitigating clinical factors that could adversely impact diazepam absorption and metabolism, and the use of an ultrasensitive LC-MS/MS assay. Limitations included the lack of addressing the efficacy of vaginal diazepam in lieu of performing a pure pharmacokinetic study with healthy participants., Conclusion: Vaginal administration of diazepam results in lower peak serum plasma concentration, longer time to peak concentration, and lower bioavailability than standard oral use. Providers should be aware that with diazepam's long half-life, accumulating levels would occur with chronic daily doses, and steady-state levels would not be reached for up to 1 week. This profile would favor intermittent use to allow participation in physical therapy and intimacy. Larish AM, Dickson RR, Kudgus RA, et al. Vaginal Diazepam for Nonrelaxing Pelvic Floor Dysfunction: The Pharmacokinetic Profile. J Sex Med 2019;16;763-766., (Copyright © 2019. Published by Elsevier Inc.)

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor-positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Endoxifen, the primary active metabolite of tamoxifen, is currently being investigated as a novel endocrine therapy for the treatment of breast cancer. Tamoxifen is a selective estrogen receptor modulator that elicits potent anti-breast cancer effects. However, long-term use of tamoxifen also induces bone loss in premenopausal women and is associated with an increased risk of endometrial cancer in postmenopausal women. For these reasons, we have used a rat model system to comprehensively characterize the impact of endoxifen on the skeleton and uterus. Our results demonstrate that endoxifen elicits beneficial effects on bone in ovary-intact rats and protects against bone loss following ovariectomy. Endoxifen is also shown to reduce bone turnover in both ovary-intact and ovariectomized rats at the cellular and biochemical levels. With regard to the uterus, endoxifen decreased uterine weight but maintained luminal epithelial cell height in ovariectomized animals. Within luminal epithelial cells, endoxifen resulted in differential effects on the expression levels of estrogen receptors α and β as well as multiple other genes previously implicated in regulating epithelial cell proliferation and hypertrophy. These studies analyze the impact of extended endoxifen exposure on both bone and uterus using a Food and Drug Administration-recommended animal model. Although endoxifen is a more potent breast cancer agent than tamoxifen, the results of the present study demonstrate that endoxifen does not induce bone loss in ovary-intact rats and that it elicits partial agonistic effects on the uterus and skeleton in ovariectomized animals., (Copyright © 2017 Endocrine Society.)

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel's strain of Theiler's murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

Methane is an important greenhouse gas and tropospheric ozone precursor. Simultaneous observation of ethane with methane can help identify specific methane source types. Aerodyne Ethane-Mini spectrometers, employing recently available mid-infrared distributed feedback tunable diode lasers (DFB-TDL), provide 1 s ethane measurements with sub-ppb precision. In this work, an Ethane-Mini spectrometer has been integrated into two mobile sampling platforms, a ground vehicle and a small airplane, and used to measure ethane/methane enhancement ratios downwind of methane sources. Methane emissions with precisely known sources are shown to have ethane/methane enhancement ratios that differ greatly depending on the source type. Large differences between biogenic and thermogenic sources are observed. Variation within thermogenic sources are detected and tabulated. Methane emitters are classified by their expected ethane content. Categories include the following: biogenic (<0.2%), dry gas (1-6%), wet gas (>6%), pipeline grade natural gas (<15%), and processed natural gas liquids (>30%). Regional scale observations in the Dallas/Fort Worth area of Texas show two distinct ethane/methane enhancement ratios bridged by a transitional region. These results demonstrate the usefulness of continuous and fast ethane measurements in experimental studies of methane emissions, particularly in the oil and natural gas sector.

Major challenges in the development of drug delivery systems (DDSs) have been the short half-life, poor bioavailability, insufficient accumulation and penetration of the DDSs into the tumor tissue. Understanding the pharmacokinetic (PK) parameters of the DDS is essential to overcome these challenges. Herein we investigate how surface chemistry affects the PK profile and organ distribution of a gold nanoparticle-based DDS containing both a passive and active targeting moiety via two common routes of administration: intravenous and intraperitoneal injections. Using LC/MS/MS, ELISA and INAA we report the half-life, peak plasma concentrations, area under the curve, ability to cross the peritoneal barrier and biodistribution of the nanoconjugates. The results highlight the design criteria for fine-tuning the PK parameters of a targeted drug delivery system that exploits the benefits of both active and passive targeting.

Background: We report results of a phase I trial designed to estimate the maximum tolerated dose (MTD), describe dose-limiting toxicities (DLT), and characterize the pharmacokinetic profile of MK-2206, an AKT inhibitor, in children with refractory or recurrent malignancies., Procedure: MK-2206 was administered either every other day (Schedule 1), or once a week (Schedule 2) in a 28-day cycle. Dose escalations in increments of ∼30% were independently made in each part using the rolling-six design. Serial pharmacokinetic (PK) studies were obtained. Biological studies include analysis of PI3K/PTEN/AKT-cell signaling pathway in pre and post-therapy in PBMC and in tumors at diagnosis or recurrence., Results: Fifty patients (26 males, median age 12.6 years [range, 3.1-21.9]) with malignant glioma (16), ependymoma (4), hepatocellular carcinoma (3), gliomatosis cereberi (2), or other tumors (22) were enrolled; 40 were fully evaluable for toxicity (Schedule 1, n = 23; Schedule 2, n = 17). Schedule 1 DLTs included: grade 3 dehydration in 1/6 patients at 28 mg/m(2) ; grade 4 hyperglycemia and neutropenia in 1/6 patients at 45 mg/m(2) ; and grade 3 rash in 3/6 patients at dose level 4 (58 mg/m(2) ). Schedule 2 DLTs included: grade 3 alkaline phosphatase in 1/6 patients at 90 mg/m(2) ; grade 3 rash in 1/6 patients at 120 mg/m(2) ; and grade 3 rash in 2/6 patients at 155 mg/m(2) ., Conclusions: The recommended pediatric phase 2 dose of MK-2206 is 45 mg/m(2) /dose every other day or 120 mg/m(2) /dose weekly. PK appeared linear over the dose range studied., (© 2014 Wiley Periodicals, Inc.)

Background: Accumulating evidence shows evidence of efficacy with the combination of vorinostat and bortezomib in solid tumors. We previously examined a once-daily continuous dosing schedule of vorinostat in combination with bortezomib which was well tolerated in cycles 1 and 2; however, there was concern regarding the tolerability through multiple cycles. This study was conducted to evaluate an intermittent dosing schedule of vorinostat with bortezomib., Methods: Vorinostat was initially administered orally twice daily on days 1-14 with bortezomib IV on days 1, 4, 8, and 11 of a 21 day cycle. Two DLTs (elevated ALT and fatigue) were observed at dose level 1, thus the protocol was amended to administer vorinostat intermittently twice daily on days 1-4 and 8-11., Results: 29 patients were enrolled; 13 men and 16 women. Common cancer types included sarcoma, pancreatic, colorectal, GIST, and breast. The most common Grade 3-4 toxicities at any dose level included thrombocytopenia, fatigue, increased ALT, elevated INR, and diarrhea. DLTs in the intermittent dosing scheduled included thrombocytopenia and fatigue. The Cmax and AUC for the intermittent dosing regimen were similar to those observed in the daily dosing. In this heavily pretreated population, stable disease was observed in patients with sarcoma, colorectal adenocarcinoma and GIST., Conclusions: The MTD was established at vorinostat 300 mg BID on days 1-4 and 8-11 and bortezomib 1.3 mg/m(2) IV on days 1, 4, 8, and 11 of a 21 day cycle. Tolerability was not improved with the intermittent dosing schedule of vorinostat when compared to continuous dosing.

Purpose: We performed a UGT1A1 genotype-guided study to determine the maximum tolerated dose (MTD) and evaluate the toxicities and pharmacokinetics of the combination of capecitabine (CAP), oxaliplatin (OX), and irinotecan (IRIN)., Experimental Design: Patients were screened for UGT1A1 *28 genotype prior to treatment. The starting dose (mg/m(2)) was IRIN (150), OX (85) and CAP (400), days 2-15. Doses were escalated or de-escalated within each genotype group (*28/*28, *1/*28 and *1/*1). IRIN pharmacokinetics was performed at the MTD., Results: 50 patients were evaluable for toxicity [11 (*28/*28); 18 (*1/*28); 21 (*1/*1)]. UGT1A1 *28/*28 patients experienced hematologic dose limiting toxicity (DLT), requiring dose-de-escalation. The UGT1A1 *28/*28 recommended phase 2 dose (RP2D) was IRIN (75), OX (85), and CAP (400). In contrast, both UGT1A1 *1/*28 and *1/*1 tolerated higher doses of IRIN and non-hematologic toxicity was dose limiting for UGT1A1 *1/*1. The RP2D was IRIN (150), OX (85), and CAP (400) for UGT1A1*1/*28 and IRIN (150), OX (100), and CAP (1600) for UGT1A1 *1/*1. UGT1A1 *1/*28 and *1/*1 patients treated with IRIN (150) had similar AUCs for the active irinotecan metabolite, SN38 (366 +/- 278 and 350 +/- 159 ng/ml*hr, respectively). UGT1A1 *28/*28 patients (n = 3) treated with a lower IRIN dose (100) had non-significantly higher mean SN38 exposures (604 +/- 289 ng/ml*hr, p = 0.14). Antitumor activity was observed in all genotype groups., Conclusions: UGT1A1 genotype affects the dose and pharmacokinetics of the CAPIRINOX regimen and UGT1A1 genotype-guided dosing of CAPIRINOX is ongoing in a phase II study of small bowel cancer (NCT00433550).

Background: A phase I study to assess the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), pharmacokinetics (PK) and antitumor activity of vorinostat in combination with bortezomib in patients with advanced solid tumors., Methods: Patients received vorinostat orally once daily on days 1-14 and bortezomib intravenously on days 1, 4, 8 and 11 of a 21-day cycle. Starting dose (level 1) was vorinostat (400 mg) and bortezomib (0.7 mg/m(2)). Bortezomib dosing was increased using a standard phase I dose-escalation schema. PKs were evaluated during cycle 1., Results: Twenty-three patients received 57 cycles of treatment on four dose levels ranging from bortezomib 0.7 mg/m(2) to 1.5 mg/m(2). The MTD was established at vorinostat 400 mg daily and bortezomib 1.3 mg/m(2). DLTs consisted of grade 3 fatigue in three patients (1 mg/m(2),1.3 mg/m(2) and 1.5 mg/m(2)) and grade 3 hyponatremia in one patient (1.5 mg/m(2)). The most common grade 1/2 toxicities included nausea (60.9%), fatigue (34.8%), diaphoresis (34.8%), anorexia (30.4%) and constipation (26.1%). Objective partial responses were observed in one patient with NSCLC and in one patient with treatment-refractory soft tissue sarcoma. Bortezomib did not affect the PKs of vorinostat; however, the Cmax and AUC of the acid metabolite were significantly increased on day 2 compared with day 1., Conclusions: This combination was generally well-tolerated at doses that achieved clinical benefit. The MTD was established at vorinostat 400 mg daily × 14 days and bortezomib 1.3 mg/m(2) on days 1, 4, 8 and 11 of a 21-day cycle.

Purpose: We conducted a pediatric phase I study to estimate the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetic properties of vorinostat, a histone deacetylase (HDAC) inhibitor, when given in combination with temozolomide in children with refractory or recurrent CNS malignancies., Patients and Methods: Vorinostat, followed by temozolomide approximately 1 hour later, was orally administered, once daily, for 5 consecutive days every 28 days at three dose levels using the rolling six design. Studies of histone accumulation in peripheral blood mononuclear cells were performed on Day 1 at 0, 6, and 24 hours after vorinostat dosing. Vorinostat pharmacokinetics (PK) and serum MGMT promoter status were also assessed., Results: Nineteen eligible patients were enrolled and 18 patients were evaluable for toxicity. There were no DLTs observed at dose level 1 or 2. DLTs occurred in four patients at dose level 3: thrombocytopenia (4), neutropenia (3), and leucopenia (1). Non-dose limiting grade 3 or 4 toxicities related to protocol therapy were also hematologic and included neutropenia, lymphopenia, thrombocytopenia, anemia, and leucopenia. Three patients exhibited stable disease and one patient had a partial response. There was no clear relationship between vorinostat dosage and drug exposure over the dose range studied. Accumulation of acetylated H3 histone in PBMC was observed after administration of vorinostat., Conclusion: Five-day cycles of vorinostat in combination with temozolomide are well tolerated in children with recurrent CNS malignancies with myelosuppression as the DLT. The recommended phase II combination doses are vorinostat, 300 mg/m(2) /day and temozolomide, 150 mg/m(2) /day., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: This phase I study was conducted to identify the maximum-tolerated dose (MTD) of alvocidib when combined with vorinostat in patients with relapsed, refractory, or poor prognosis acute leukemia, or refractory anemia with excess blasts-2. Secondary objectives included investigating the pharmacokinetic and pharmacodynamic effects of the combination., Experimental Design: Patients received vorinostat (200 mg orally, three times a day, for 14 days) on a 21-day cycle, combined with 2 different alvocidib administration schedules: a 1-hour intravenous infusion, daily × 5; or a 30-minute loading infusion followed by a 4-hour maintenance infusion, weekly × 2. The alvocidib dose was escalated using a standard 3+3 design., Results: Twenty-eight patients were enrolled and treated. The alvocidib MTD was 20 mg/m(2) (30-minute loading infusion) followed by 20 mg/m(2) (4-hour maintenance infusion) on days one and eight, in combination with vorinostat. The most frequently encountered toxicities were cytopenias, fatigue, hyperglycemia, hypokalemia, hypophosphatemia, and QT prolongation. Dose-limiting toxicities (DLT) were cardiac arrhythmia-atrial fibrillation and QT prolongation. No objective responses were achieved although 13 of 26 evaluable patients exhibited stable disease. Alvocidib seemed to alter vorinostat pharmacokinetics, whereas alvocidib pharmacokinetics were unaffected by vorinostat. Ex vivo exposure of leukemia cells to plasma obtained from patients after alvocidib treatment blocked vorinostat-mediated p21(CIP1) induction and downregulated Mcl-1 and p-RNA Pol II for some specimens, although parallel in vivo bone marrow responses were infrequent., Conclusions: Alvocidib combined with vorinostat is well tolerated. Although disease stabilization occurred in some heavily pretreated patients, objective responses were not obtained with these schedules., (©2013 AACR.)

Background: A pediatric Phase I trial was performed to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of vorinostat and bortezomib, in patients with solid tumors., Procedure: Oral vorinostat was administered on days 1-5 and 8-12 of a 21-day cycle (starting dose 180 mg/m(2) /day with dose escalations to 230 and 300 mg/m(2) /day). Bortezomib (1.3 mg/m(2) i.v.) was administered on days 1, 4, 8, and 11 of the same cycle. PK and correlative biology studies were performed during Cycle 1., Results: Twenty-three eligible patients [17 male, median age 12 years (range: 1-20)] were enrolled of whom 17 were fully evaluable for toxicity. Cycle 1 DLTs that occurred in 2/6 patients at dose level 3 (vorinostat 300 mg/m(2) /day) were Grade 2 sensory neuropathy that progressed to Grade 4 (n = 1) and Grade 3 nausea and anorexia (n = 1). No objective responses were observed. There was wide interpatient variability in vorinostat PK parameters. Bortezomib disposition was best described by a three-compartment model that demonstrated rapid distribution followed by prolonged elimination. We did not observe a decrease in nuclear factor-κB activity or Grp78 induction after bortezomib treatment in peripheral blood mononuclear cells from solid tumor patients., Conclusion: The recommended Phase 2 dose and schedule is vorinostat (230 mg/m(2) /day PO on days 1-5 and 8-12) in combination with bortezomib (1.3 mg/m(2) /day i.v. on days 1, 4, 8, and 11 of a 21-day cycle) in children with recurrent or refractory solid tumors., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: A phase I, dose-finding study of vorinostat in combination with temozolomide (TMZ) was conducted to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics in patients with high-grade glioma (HGG)., Experimental Design: This phase I, dose-finding, investigational study was conducted in two parts. Part 1 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m(2)/day for 5 days every 28 days. Part 2 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m(2)/day for 5 days of the first cycle and 200 mg/m(2)/day for 5 days of the subsequent 28-day cycles., Results: In part 1, the MTD of vorinostat administered on days 1 to 7 and 15 to 21 of every 28-day cycle, in combination with TMZ, was 500 mg daily. Dose-limiting toxicities (DLT) included grade 3 anorexia, grade 3 ALT, and grade 5 hemorrhage in the setting of grade 4 thrombocytopenia. In part 2, the MTD of vorinostat on days 1 to 7 and 15 to 21 of every 28-day cycle, combined with TMZ, was 400 mg daily. No DLTs were encountered, but vorinostat dosing could not be escalated further due to thrombocytopenia. The most common serious adverse events were fatigue, lymphopenia, thrombocytopenia, and thromboembolic events. There were no apparent pharmacokinetic interactions between vorinostat and TMZ. Vorinostat treatment resulted in hyperacetylation of histones H3 and H4 in peripheral mononuclear cells., Conclusion: Vorinostat in combination with temozolomide is well tolerated in patients with HGG. A phase I/II trial of vorinostat with radiotherapy and concomitant TMZ in newly diagnosed glioblastoma is underway., (©2012 AACR.)

Objectives: Human papillomavirus (HPV) infection is a major risk factor for cervical cancer. Imiquimod is a topical medication that enhances the immune response to HPV-induced genital warts. This study evaluated cervical application of imiquimod as an adjunct to standard treatment for cervical dysplasia., Study Design: Fifty-six patients were randomized to standard excisional/ablative treatment vs applications of imiquimod followed by standard treatment. The primary endpoint was dysplasia recurrence within 2 years., Results: There were no differences in dysplasia recurrence between the 2 groups. Treatment was well tolerated, with side effects being mild but significantly worse in women receiving imiquimod for, chills, fatigue, fever, headache, myalgias, and vaginal discharge., Conclusion: This trial does not support the hypothesis that imiquimod, as used in this trial, has an impact on recurrence of cervical dysplasia, but the adequacy of findings are limited by sample size. The trial does support the feasibility and acceptability of the use of imiquimod on the cervix., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Purpose: To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma., Experimental Design: Patients received 7 days of fenretinide: 2,475 mg/m(2)/d divided TID (<18 years) or 1,800 mg/m(2)/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only)., Results: Sixty-two eligible patients, median age 5 years (range 0.6-19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17-506) for stratum 1 and 48 days (range 17-892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40-56%) at day 7 course 1. Toxicities were mild and reversible., Conclusions: Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies., (©2011 AACR)

Purpose: Vorinostat (V) at levels >2.5 µM enhances chemotherapy in vitro. Yet the approved oral dose of 400 mg inconsistently achieves this level in patients. We developed an intermittent oral pulse-dose schedule of V to increase serum levels. We combined V with the cyclin dependent kinase inhibitor flavopiridol (F) which increases V-induced apoptosis., Experimental Design: One week before combination treatment, V alone was given daily for 3d (cycle -1). Then V was given on d1-3 and d8-10, and F on d2 and d9, every 21-d. Due to neutropenia, this was modified to V on d1-3 and d15-17, and F on d2 and d16, every 28-d. Bolus and split-dose F schedules were studied., Results: 34 patients were treated. On the 21-d schedule, the maximum tolerated dose (MTD) was V 600 mg/d and F 60 mg/m(2) bolus. On the 28-d schedule, the MTD was V 800 mg/d and F 30 mg/m(2) over 30 min and 30 mg/m(2) over 4 h. V C(max) at the 800 mg dose was 4.8 µM (± 2.8). V C(max) ≥ 2.5 µM was achieved in 86% of patients at the MTD. F increased the C(max) of V by 27% (95% CI 11%-43%). F C(max) of ≥ 2 µM was achieved in 90% of patients. 8 patients had stable disease for on average 5.5 m (range 1.6-13.2 m)., Conclusions: Intermittent high dose oral V in combination with F is feasible and achieves target serum levels >2.5 µM. V concentrations higher than previously reported with oral dosing were achieved.

Irinotecan has radiosensitizing effects and shows synergism with nitrosoureas. We performed a Phase II study of RT and irinotecan, followed by BCNU plus irinotecan in newly-diagnosed GBM. The MTD for patients receiving enzyme-inducing anticonvulsants (EIAC) was as follows: irinotecan 400 mg/m(2)/week on Days 1, 8, 22 and 29 during RT, followed by BCNU 100 mg/m(2) Day 1, and irinotecan, 400 mg/m(2) on Days 1, 8, 22 and 29, every 6 weeks. The MTD for non-EIAC patients was as follows: irinotecan 125 mg/m(2)/week on Days 1, 8, 22 and 29 during RT, followed by BCNU 100 mg/m(2) Day 1 and irinotecan 75 mg/m(2) Days 1, 8, 22 and 29, every 6 weeks. Median OS was 10.8 mos. (95% CI: 7.7-14.9); OS at 12 months was 44.6% (95% CI: 33.3-59.8) and PFS 6 was 28.6% (95% CI: 18.9-43.2). Patients went off treatment due to adverse events (7%), refusal (11%), progressive disease (48%), death (9%), and other (9%); 16% completed protocol treatment. Survival was similar in patients with variant (6/7 or 7/7) and wild-type (6/6) UGT1A1*28 genotypic alleles. Grade 3-4 toxicity was more common in non-EIAC patients with variant alleles. SN-38 C(max) and AUC in EIAC patients receiving 400 mg/m(2) irinotecan were 20.9 ng/ml and 212 ng/ml h, and in non-EIAC patients receiving 125 mg/m(2), 15.5 ng/ml and 207 ng/ml h. SN-38 AUC varied by UGT1A1*28 status in non-EIAC patients. This regimen was not significantly active and radiosensitization was not observed. Non-EIAC patients with UGT1A1*28 variant alleles appear particularly sensitive to toxicity from irinotecan.

The cyclooxygenase (COX)-2 enzyme appears to be an important target for cancer chemoprevention. Given the recent emergence of potentially serious cardiovascular toxicity associated with selective COX-2 inhibitors, nonsteroidal antiinflammatory drugs (NSAIDs), which inhibit both COX-1 and COX-2, have received renewed attention as candidate chemoprevention agents. Sulindac has shown consistent chemopreventive potential in preclinical studies as well as in a limited number of clinical trials reported to date. For the current pharmacokinetic study, sulindac capsules were prepared to facilitate ample agent supplies for future intervention studies. Encapsulation of the parent compound (sulindac sulfoxide) can be readily accomplished, but the effects of alternate formulations on bioavailability have not been rigorously examined. In the present single-dose, two-period crossover trial, we conducted pharmacokinetic analyses of sulindac in capsule (test) versus tablet (reference) formulations. Overall, bioavailability appeared to be higher for the capsule compared with the tablet formulation based on test-to-reference pharmacokinetic variable ratios for the parent compound alone. However, additional analyses based on the sulfide and sulfone metabolites of sulindac with the same pharmacokinetic variables indicated similar chemopreventive exposures between the capsule and tablet formulations. These data support the use of sulindac capsules, which can be readily prepared with matching placebos, in future blinded chemoprevention trials.

Objective: To investigate the prevalence of human papillomavirus (HPV) in patients with vulvar vestibulitis syndrome by using a recently developed polymerase chain reaction (PCR) primer set that detects known papillomavirus types., Study Design: We retrospectively identified 38 patients with vulvar vestibulitis who underwent therapeutic surgical excision of the vestibule. Eleven controls without vestibulitis who underwent vestibular excision for conditions unrelated to HPV infection were identified prospectively. Surgical specimens were examined for the presence of HPV DNA by PCR amplification. DNA sequencing was used to determine HPV type., Results: The prevalence of HPV among patients with vestibulitis was 21% vs. 36% among controls. Group B HPV types accounted for 4 of the 10 (40%) HPV types found in patients with vestibulitis. Overall, in both patient and control samples, a spectrum of HPV types was identified, encompassing many branches of the HPV phylogenetic tree. No etiologic association was apparent., Conclusion: This study did not support an association of HPV with vulvar vestibulitis. The low rate of observed infection in women with and without vestibulitis and the diversity of HPV types identified suggest incidental virus carriage rather than direct cause and effect. The underlying cause of this debilitating condition remains unknown.

Purpose: Pemetrexed, a multi-targeted antifolate that disrupts synthesis of both purines and pyrimidines, is approved for use in malignant pleural mesothelioma and non-small cell lung cancer. Pemetrexed is currently being evaluated for anti-tumor activity in a variety of solid and central nervous system tumors. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of pemetrexed in a non-human primate model that is highly predictive of human CSF penetration., Methods: Pemetrexed, 20 mg/kg (400 mg/m2), was administered intravenously to four non-human primates. Serial blood samples were obtained from all animals and serial CSF samples were obtained from three animals. Plasma and CSF concentrations of pemetrexed were measured using LC/MS/MS and the resulting concentration versus time data were evaluated using model independent and dependent methods., Results: Pemetrexed disappearance from plasma was best described by a two compartment model with a mean distribution half-life of 13.8 +/- 3.2 min and an elimination half-life of 70.0 +/- 16.0 min. The volume of distribution of and the clearance from the central compartment were 0.066 +/- 0.017 l/kg and 3.6 +/- 0.6 ml/min/kg, respectively. Peak CSF concentrations occurred 40-71 min after the start of the infusion with an average of 0.26 +/- 0.15 microM., Conclusion: The CSF penetration of pemetrexed was less than 2% (range 0.33-1.58%), suggesting that it should be used in conjunction with other CNS preventive strategies when used in the treatment of malignancies with a predilection for CNS or leptomeningeal metastases.

The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G-G interstrand cross-links via reactive N(10)-C(11)/N(10')-C(ll') imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557-m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8-1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22-720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 microg/kg doses for five consecutive days.

Human papillomaviruses (HPVs) have been identified in 99% of cervical tumors. Considerable speculation exists about the role of HPV in cancer at other sites, such as skin, esophagus, and lung. One barrier to the study of HPV in extragenital tumors may be the inability to detect novel HPV types. Our objective was to design broad-range primers for detecting papillomaviruses from any group in the HPV Sequence Database. Complete L1 protein sequences were aligned and 11 blocks of conserved protein sequences were identified. Primers were designed in the corresponding nucleotide regions of five blocks. One pair of primers, ME and MH, derived from blocks E and H, appeared promising when compared with established consensus primers. To improve the sensitivity for detection of multiple papillomavirus types, ME/MH primers were split into several primers with the 5' regions matched to individual papillomavirus supergroups. Compared with the commonly used MY09/11 primer set, our primer sets were broader in range and more sensitive against most papillomavirus types tested. Application of these primers to esophageal and tonsillar carcinomas identified no novel HPV types but confirmed a high level of sensitivity for detecting HPV from these clinical samples. These primers should facilitate the search for novel papillomaviruses.

Objectives: Microarray expression analysis of cervical tumors has revealed differential expression of genes that may be useful as markers or targets for treatment. We question the application of array findings across the major categories of cervical cancer. We sought to identify differences between normal squamous epithelium (NSQ) and glandular epithelium (NGL) of the uterine cervix and their malignant variants: squamous cell cancer (SCC) and adenocarcinoma (ACA)., Methods: Eight genes were selected: 12-lipoxygenase (12-LOX), keratin 4, trypsinogen 2 (TRY2), Rh glycoprotein C (RhGC), collagen type V alpha 2, integrin alpha 5, integrin alpha 6, and c-myc. Ten cases each of SCC and ACA of the cervix were selected from our tumor bank. NSQ and NGL epithelia were obtained from consecutive patients undergoing surgery for benign disease. RNA extraction, cDNA synthesis, and DNA amplification of all samples were performed according to an established protocol. Electrophoresis of the multiplexed polymerase chain reaction (PCR) products was performed under standard conditions, followed by digital image capture. A ratio of target to control gene (beta-actin) was obtained for each sample. Analysis of variance was applied to the mean ratios for each tissue to establish significant differences. Individual pairwise comparisons were made by Student t tests and verified with the Tukey-Kramer test., Results: Clinically valid comparisons are NSQ to NGL, NSQ to SCC, NGL to ACA, and SCC to ACA. Various expression patterns were observed between the epithelia and their malignant phenotypes. Significant differences in gene expression were observed between benign squamous and glandular epithelium in four of the eight genes and between malignant squamous and glandular epithelium in three of the eight genes. Significant differences in gene expression between benign and malignant tissues were demonstrated in four of the eight genes., Conclusions: We have defined significant differential expression changes between the two principal cervical tumor types. Differences in genes are demonstrated and must be considered if array technology is applied to the study of the biologic behavior of these tumors as well as their screening and management. The observed differential expression should be a compelling argument to perform type-specific expression analysis for other tumors with histological variants.

Objective: The likelihood of developing cervical cancer has been shown to be increased in persons with certain HLA alleles. We evaluated immune response genes in the HLA region of chromosome 6 to see if individual or interactive associations with cervical cancer risk could be identified., Methods: Tissue was obtained from 127 women undergoing surgical treatment for cervical cancer. Blood samples were obtained from 175 control subjects. A combination of polymerase chain reaction (PCR), sequence-specific PCR, and DNA sequencing was used to evaluate polymorphic alleles, including HLA class I B7, TNF alpha, HLA class II DR2, TAP1, and TAP2 genes. Fisher's exact test and logistic regression modeling were used for statistical analysis., Results: A significantly greater proportion of the patients with cervical cancer were found to have the HLA class II DR2 1501 allele (P = 0.023) and the TAP2 A/B heterozygous pattern of alleles (P = 0.0006) than were women without cervical cancer. A proportion of patients with cervical cancer significantly smaller than that of the control women had a polymorphism at the -238 position of the TNF promoter and the TAP1 C/C homozygous pattern of alleles. With logistic modeling, the markers that showed consistent association with the occurrence of cervical cancer were TAP2 A/B, HLA-DR2 1501, and TAP1 C/C., Conclusions: We demonstrated a significant association between immune response genes and the risk of cervical cancer. Our data create a compelling argument for a gene or a cluster of genes in the HLA region of chromosome 6 that regulates host immune responses to human papillomavirus infection in a manner that results in inherited susceptibility or resistance to the transforming properties of oncogenic papillomaviruses.

Background: The natural history of cervical cancer comprises a latency period that probably involves long-term immunologic tolerance of human papillomavirus infection. Identifying host determinants of viral persistence may help to better understand the mechanisms of tolerance and may lead to the development of tests that can allow more focused follow-up of high-risk individuals., Methods: Genotypic frequencies of 12 polymorphic loci in four candidate genes from 127 cervical cancer patients were compared with a control group of 108 female blood donors. Genotypes were determined by PCR amplification and direct sequencing of isolated genomic DNA., Results: The tumor necrosis factor-alpha (TNFalpha) -238 polymorphism was significantly underrepresented in cervical cancer patients [heterozygotes (HETs), odds ratio (OR) = 0.33; 95% confidence interval (CI), 0.11-0.96], as was the TNFalpha -376 polymorphism (P = 0.02; 0% for any variant genotype in cases vs 4.7% in controls). The NRAMP1 3' untranslated region STP+86 polymorphism also appeared to be inversely associated with cervical cancer, but this result did not reach statistical significance (HET, OR = 0.57; 95% CI, 0.32-1.02). The p53 codon 72 arginine allele showed a suggestive negative association with cervical cancer (HET, OR = 0.49; 95% CI, 0.14-1.63; homozygotes, OR = 0.35; 95% CI, 0.11-1.17). The remaining alleles tested showed no association with cervical cancer., Conclusions: We identified host genetic polymorphisms that may be associated with cervical cancer risk, some of which have been linked to potential functional effects on cellular immune responses or antigen processing. We failed to confirm earlier reports of increased cervical cancer susceptibility in women who harbor the p53 P72R allele. Although our findings support the general hypothesis that host immunogenetic determinants other than class II MHC may be important in the development of cervical cancer, further analysis of the HLA gene cluster comprising the implicated TNFalpha single-nucleotide polymorphisms will be required to determine whether their association is linkage independent.

Background: Human papillomavirus (HPV) is an independent risk factor for select head and neck carcinomas and most uterine cervix carcinomas. We report two patients with synchronous diagnoses of cervical cancer and HPV-related head and neck cancer., Case: One patient was a 53-year-old woman with regionally metastatic tonsillar carcinoma treated surgically and with adjuvant radiation. Abnormal vaginal bleeding developed. Gynecologic examination showed advanced cervical carcinoma. The other patient was a 78-year-old woman surgically treated for carcinoma of the left anterior nose. Five months later, symptoms of recurrent nasal carcinoma and concurrent vaginal bleeding developed. Gynecologic examination showed advanced cervical carcinoma., Conclusions: These cases of coincident tumors demonstrate possible systemic susceptibility to the carcinogenic effects of HPV. The common association of HPV with both uterine cervix cancers and select head and neck cancers should prompt early evaluation of gynecologic or upper aerodigestive tract symptoms for patients with known HPV-related cancers., ((c) 2002 Elsevier Science (USA).)

"High-risk" human papillomaviruses (HPVs) are associated with intraepithelial neoplasia and cancer of the uterine cervix. HPV has also been found in nonmelanoma skin cancer (NMSC), especially in squamous cell carcinomas (SCCs) of immunosuppressed patients. Recently, lesions of psoriasis have been shown to harbor HPV, and patients with psoriasis often have a history of extensive therapy with ultraviolet radiation (UVR). UVR is the major known risk factor in the occurrence of NMSC, in which HPV may be a cofactor for SCC. We report an otherwise healthy, nonimmunosuppressed patient with psoriasis who had a history of extensive exposure to UVR and experienced multiple SCCs on UV-exposed body sites. By the polymerase chain reaction method, we detected HPV in 5 of 9 SCCs. Automated sequencing showed HPV types 12 and 17. Only 1 of 3 normal skin specimens was HPV positive (HPV type 17). This positive specimen was from UV-exposed skin; one of the two HPV-negative, normal skin specimens was located on a body site not exposed to sun. In addition, HPV type 62 was found in a brush specimen of the uterine cervix. This case report suggests an association between psoriasis, HPV infection, and UVR exposure, in onset of SCC.

A retrospective evaluation of the Steffee metacarpophalangeal (MCP) thumb joint prostheses was performed to determine the long-term outcome and survivorship of the prosthesis. Fifty-four primary thumb arthroplasties (49 patients) were performed for pain, weakness, or instability involving the thumb MCP joint secondary to arthritis. Underlying etiology included rheumatoid (49 thumbs), psoriatic (1 thumb), scleroderma (2 thumbs), and degenerative (2 thumbs) arthritis. Thirty-one thumbs had concomitant interphalangeal joint instability and underwent interphalangeal joint fusions. At an average follow-up period of 57 months, the average motion of the MCP joint was 21 degrees (range, 0 degrees to 40 degrees ), with a significant improvement in position and stability. Thumb axis length was maintained or increased in 98%. Although there was not a consistent long-term improvement in grip or pinch strength, 87% of the patients reported subjective improvement in strength and function as a result of surgery. Pain was relieved in all thumbs with preoperative pain. Complications included a periprosthetic fracture, 2 late infections, and 1 gross loosening of the implant. The survivorship of the implant was 93% survivorship at 5 years and 89% survivorship at 10 years, with only 4 failures in 54 thumbs. The Steffee thumb MCP arthroplasty resulted in excellent long-term survivorship, patient satisfaction, and functional outcome.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Objective: The E6 regions of the oncogenic human papillomaviruses (HPVs) are important in carcinogenesis and immune recognition. We examined the E6 DNA sequence from HPV-16-associated cervical cancers to determine the frequency and degree of variation from the consensus sequence in selected populations., Methods: Samples positive for HPV-16 were analyzed using polymerase chain reaction followed by automated DNA sequencing: 62 from U.S. women, 20 each from Italian and Indian women, and 21 from Thai women., Results: Of 151 codons, 18 contained 24 base substitutions, reflecting the overall conserved nature of this region. The HPV-16 E6 region from U. S. women showed considerably more sequence variation than that from European and Asian women. Five patterns common to U.S. and European and Asian samples accounted for 78% of all tumor-associated viruses. The E6 regions known to be involved in p53 binding and degradation are involved with a surprising degree of sequence variation, whereas the carboxy end of the molecule is highly conserved., Conclusions: The area of greatest sequence variation includes a proposed human leukocyte antigen interaction site. A novel large deletion in one sample results in loss of all functional regions of E6. These findings were analyzed for possible significance with regard to immune selection and functional importance of the carboxy end of the E6 protein., (Copyright 2000 Academic Press.)

Warts can be difficult to diagnose and to treat in the setting of human immunodeficiency virus (HIV) infection. A 37-year-old woman with a background of HIV presented with a large verrucous plaque involving her right foot. Human papillomavirus (HPV)-66 was identified in the lesional skin biopsy sample and in scrapings obtained from her cervix. The wart rapidly responded to topical cidofovir therapy. HPV-66 is a novel HPV type to be associated with verruca vulgaris. Topical cidofovir should be further investigated as an alternative treatment modality for verruca vulgaris.

Objective: The purpose of this study was to evaluate cervical cancers in older women to determine whether they differed from tumors in younger women with respect to human papillomavirus types, frequencies of p53 mutations, and presence of a proposed high-risk HLA-DR2 haplotype., Study Design: Cervical tissue was obtained from women undergoing surgical treatment of in situ or invasive carcinoma of the cervix. Viral and genomic deoxyribonucleic acid was extracted. The presence of human papillomavirus deoxyribonucleic acid was detected by polymerase chain reaction amplification. Viral subtypes were assigned by means of a combination of type-specific amplification and automated sequencing of the L1 region. The presence of p53 mutations was evaluated by direct sequencing of exons 5 through 9. The HLA-DR locus was screened for the presence of the high-risk DRB1*1501 allele by means of selective polymerase chain reaction amplification followed by agarose gel electrophoresis of HLA-DR2 types., Results: Tumors from 39 women 62 to 85 years old were analyzed. Tumors from 104 younger women formed a reference group. Human papillomavirus 16 was found in 41% and 54% and human papillomavirus 18 was found in 10% and 12% of the tissue samples from older and younger women, respectively. The overall distributions of human papillomavirus types did not differ statistically between the groups. One of the 25 older patients tested had a p53 mutation. This tumor also had a positive test result for human papillomavirus 18. The DR*1501 allele was present in 33% of the older patients and 28% of the younger patients. The expected frequency of this allele in white Americans is 19.8%. The increased frequency of this allele among both older and younger women with cervical cancer was statistically significant (P < .05)., Conclusions: We hypothesized that cervical cancer in older women might differ from that in younger women with respect to human papillomavirus types, natural host immunity, or the frequency of nonviral origins of the cancer. The findings show, however, that tumors from older women are extremely similar to those from younger women with respect to the human papillomavirus types present and the infrequent occurrence of p53 mutations. In addition we found that an HLA-DR allele that is associated with a risk of cervical cancer in younger women is also associated with risk in older women. These findings are most consistent with a model similar to that in younger women but with an unusually long latency for the transforming effect of the virus in some hosts.

Comparison of acquired mutations in the p53 tumor suppressor gene can illuminate factors contributing to carcinogenesis among cancer cohorts. Japan has an ethnically homogeneous population with a low incidence of breast cancer. Previously we reported an unusual frequency, allelic status, and clustering of mutations in breast cancers from the northern part of the main Japanese island. To extend these findings, exons 2-11 and adjacent intronic sequences were analysed in tumors of women from northern (Hokkaido) and southern (Tokushima) Japan. The frequency of breast cancers with p53 gene mutations in the Hokkaido group is the highest reported (81%) while that in Tokushima (28%) is similar to most other populations. Thirteen of the 19 mutations (68.4%) in the Hokkaido cohort were heterozygous, an unusually high frequency for p53 mutations in any tumor type. There were three missense mutations at codon 175, a known hotspot for alterations in the p53 gene, and three missense mutations at codon 179, a rare site for p53 changes. In addition, the patterns of p53 gene mutation differed between the two Japanese cohorts (P=0.04). The multiple differences in acquired p53 mutations suggest unsuspected biological differences among breast cancers in northern and southern Japan. In addition, the high frequency of p53 mutations in breast cancers from Hokkaido predicted a poorer prognosis for this population which was confirmed on examination of mortality data.

Alterations of the p53 tumor suppressor gene are the most frequent genetic abnormalities in human malignancies, but the role of p53 in the etiology of malignant melanomas is unclear. Fifty unselected malignant melanomas were analyzed for p53 overexpression by immunohistochemistry using 3 monoclonal antibodies (MAbs). Fifteen tumors (29.4%) showed positive staining with at least 2 different antibodies. In the first 20 consecutive tumors exons 5-9 and adjacent splice sites of the p53 gene were analyzed by genomic sequencing. There were 4 mutations in 20 metastatic melanomas. Three of 4 mutations were C:G-->T:A transitions. A search of our database of p53 mutations revealed that out of 8 p53 mutations reported by others, 4 are C:G-->T:A transitions at dipyrimidine sites, and one is a tandem CC-->TT mutation. This mutational pattern is comparable with the pattern of p53 mutations in squamous cell and basal cell carcinomas of the skin and is related to exposure to ultraviolet B (UV-B) wavelength radiation. Taken together with a predominance of UV-induced mutations in the CDKN2/ p16 gene demonstrated in melanoma cell lines, our data support a role of sunlight exposure in the etiology of malignant melanoma. The low frequency of p53 mutants in melanomas compared with other types of skin cancers suggests that although mutations in this gene are likely to be involved in the development of some malignant melanomas, they do not play as large a role as in squamous and basal cell carcinomas of the skin.

Most studies of mutations in the p53 tumor suppressor gene in tumors have examined only exons 5-8. Our laboratory previously found 64 mutations in exons 5-8 of the p53 gene in 194 primary breast cancers. Herein, we report 18 additional mutations found outside of exons 5-8. Mutations are present in exons 4, 9 and 10, and flanking splice junctions, but not in the promotor region or in exons 1, 2, 3 and 11. No missense mutations are found outside of exons 5-8. Instead, there is a predominance of frameshift mutations with lesser numbers of nonsense and splice site mutations. In contrast, the majority of mutations in exons 5-8 in this sample are missense changes and all of these are at amino acids that are identical in the 11 known p53 sequences that represent about 1.6 billion years of evolutionary divergence. The difference in mutational pattern between these two regions of the p53 gene is due to a lack of missense mutations and inframe microdeletions outside of exons 5-8. A review of our database of p53 mutations (De Vries et al., in preparation) shows that the patterns of mutation inside and outside of exons 5-8 differ in other types of cancers as well. The paucity of missense mutations in exons 2-4 and 9-11 in breast and other cancers (even at amino acids identical throughout p53 gene evolution) suggest that at least some missense mutations result in a phenotype other than malignant transformation. These data also illustrate the importance of examining identical exons when comparing the pattern of p53 gene mutations in different populations.

Dideoxy fingerprinting (ddF) is an efficient method for detecting single base and other sequence changes in PCR-amplified DNA segments. This screening method is a hybrid between single-strand conformation polymorphism analysis (SSCP) and Sanger dideoxy sequencing. It involves a Sanger sequencing reaction with one dideoxynucleotide followed by non-denaturing gel electrophoresis. We are using ddF to screen for mutations in the p53 tumor suppressor gene in primary breast cancers. ddF detected more than 100 mutations in different regions of the gene, including all types of single-base mutations and microdeletions/microinsertions of various sizes. Furthermore, ddF reliably detected heterozygous mutations, if the region of interest was screened in both directions. In a blinded, prospective study, ddF detected all 25 mutations within exons 4-10 and adjacent flanking intronic regions previously found by direct sequencing. ddF was also useful in scoring two common polymorphisms within the p53 gene. Guidelines for preventing false-positive and false-negative results are summarized.

Since mutagens produce an extraordinary diversity of mutational patterns, differential mutational exposures among populations are expected to produce different patterns of mutation. Classical epidemiological methods have been successful in implicating specific mutagens in cancers such as those of lung and skin in which one mutagen predominates. In breast cancer, however, no mutagens have been implicated in an unequivocal manner. In an attempt to facilitate epidemiological studies, we have been studying the pattern of p53 gene mutations in breast cancers from multiple populations with high and low breast cancer incidences. We previously reported that breast cancers from Midwest United States, predominantly rural Caucasian women, have a different pattern of p53 gene mutation from populations of Western European women. Herein, we analyze patterns of p53 mutations from Graz, Austria, another population with a high incidence of breast cancer. Among the 60 Austrian breast cancers analyzed, 14 (23%) have a p53 gene mutation in exons 5-9 or in adjacent splice junctions. Analysis of the patterns of mutation shows differences between the "Western European" profile and the Austrian and Midwest United States groups (P = 0.027 and 0.024, respectively). The Austrian pattern is characterized by a high frequency of A:T-->T:A transversions (P = 0.006). The presence of distinct patterns of mutation among the limited number of analyzed populations of Western European origin supports the idea that differential mutagenic exposure and/or genetic differences contribute to breast cancer mutagenesis among geographically distinct Caucasians of Western European origin.

We determined the pattern of mutations in exons 2-11 and adjacent intronic regions in breast cancers from Midwestern US white women. Twenty-one mutations were detected in 53 tumors (39.6%). Comparisons of the pattern of mutations within exons 5-9 showed that the frequency of missense mutations (44%) was lower in breast cancers of US Midwestern women than in most tumor types including breast cancers in other populations. Compared to breast cancers reported in a Scottish population, US women had a high frequency of G:C-->T:A transversions (P = 0.046). These findings suggest that environmental or endogenous factors contribute to p53 mutagenesis in mammary tissue to different extents among different populations. With a median follow-up of 19 months, the presence of a mutation was associated with shorter time to disease recurrence (P = 0.05) and shorter survival (P = 0.003). Putative dominant negative missense-type mutations (missense and in-frame microdeletions; P = 0.001) and null mutations (hemizygous nonsense and frameshift mutations; P = 0.007) were equally ominous. Thus, tumors with missense p53 mutations resulting in over-expression of a dysfunctional but otherwise intact protein have a clinical outcome similar to tumors with null mutations resulting in a truncated or garbled protein.

The pattern of acquired mutations in the p53 gene can be used to study differences in factors contributing to carcinogenesis. We investigated mutations in exons 5-9 and adjacent intronic regions in 47 breast cancers of black women from Michigan, a population with the highest breast-cancer mortality in the US. The 16 mutations detected differed from those of other populations. In particular, the black women had an excess of A:T-->G:C transitions compared with rural white US midwest women. While the causes of the different pattern of acquired mutation remain to be determined, this molecular epidemiological approach detects the consequences of mutagenic processes in specific populations. Mutation patterns will constrain hypotheses to mechanisms consistent with the observed biochemical alterations.