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1.  A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma 
Biomedical chromatography : BMC  2011;26(5):650-654.
2-deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with the glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate/boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45min. The analytes were separated on a YMC ODS C18 reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425nm. The 2-DG calibration curves were linear over the range of 0.63 to 300μg/mL with the limit of detection of 0.5μg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8% and the accuracy ranged from 86.8% to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors.
PMCID: PMC4522920  PMID: 21932382
2-Deoxyglucose; HPLC; Fluorescence derivatization; Pharmacokinetics
2.  Comparative effects of two different forms of selenium on oxidative stress biomarkers in healthy men: a randomized clinical trial 
Epidemiological and laboratory studies indicate that dietary selenium protects against prostate cancer. Results from clinical trials suggest that selenium-enriched yeast (SY) but not selenomethionine (SeMet) may be effective at reducing prostate cancer risk. Our objectives were to directly compare for the first time the effects of SeMet and SY on prostate cancer relevant biomarkers in men. We performed a randomized double blind, placebo-controlled trial of SY (200 or 285 µg/day) and SeMet (200 µg/day) administered for 9 months in 69 healthy men. Primary endpoints included blood levels of selenium-containing compounds and oxidative stress biomarkers (urine 8-hydroxy-2’-deoxyguanosine [8-OHdG] and 8-iso-prostaglandin-F2α [8-iso-PGF2α] and blood glutathione [GSH]). Secondary endpoints included plasma glucose and PSA levels. Compliance was high in all groups (>95%). Plasma selenium levels were increased 93%, 54%, and 86% after 9 months in SeMet and low and high dose SY groups, respectively, and returned to baseline levels after a 3 month washout (P<0.05). Levels of 8-OHdG and 8-iso-PGF2α, were decreased 34% and 28%, respectively, after 9 months in the high dose SY group (P<0.05). These decreases were greatest in individuals with low baseline plasma levels of selenium (<127 ng/ml). No changes in serum PSA or blood glucose and GSH were observed. Overall, we showed for the first time, reductions in biomarkers of oxidative stress following supplementation with SY but not SeMet in healthy men. These findings suggest that selenium-containing compounds other than SeMet may account for the decrease in oxidative stress.
PMCID: PMC4125492  PMID: 24938534
selenium-enriched yeast; selenomethionine; prostate cancer; oxidative stress
3.  Interferon Alpha Plus 13-Cis-Retinoic Acid Modulation of BCL-2 Plus Paclitaxel for Recurrent Small Cell Lung Cancer (SCLC) 
Patients with recurrent small cell lung cancer (SCLC) have dismal outcomes. The failure of salvage therapy is due to the possible development of resistance mechanisms, such as the upregulation of the anti-apoptosis protein, Bcl-2. We conducted a phase II study to evaluate if modulation of Bcl-2 with 13-cis-retinoic acid (13-CRA) and interferon alpha could improve response rates when combined with paclitaxel in patients with recurrent SCLC.
Patients with recurrent SCLC and measurable disease were treated with interferon alpha at 6 million units/m2 subcutaneously and 13-CRA 1 mg/kg orally on days 1 and 2 and paclitaxel 75 mg/m2 intravenously on day 2 of each week for 6 weeks of an 8 week treatment cycle. Treatment was continued until disease progression, development of unacceptable toxicity, or withdrawal of consent. The primary endpoint was response rate with secondary endpoints of progression free survival (PFS) and overall survival (OS). Bcl-2 levels were assessed in peripheral blood mononuclear cells (PBMCs).
37 patients were enrolled; 34 were included in the intention-to-treat analysis as 3 patients were ineligible for the study. There were 3 partial responses (9%) and 5 patients had stable disease (15%) as best response. The median PFS was 2 months and median OS was 6.2 months. Although mean Bcl-2 protein levels decreased with therapy in PBMCs, there was no association between Bcl-2 levels and response rate or survival.
Despite sound pre-clinical evidence, the addition of 13-CRA and interferon alpha to paclitaxel did not improve outcomes for recurrent SCLC.
PMCID: PMC4296897  PMID: 24858462
small cell lung cancer; Bcl-2; 13-cis-retinoic acid; interferon alpha
5.  Next Generation Sequencing As an Aid to Diagnosis and Treatment of an Unusual Pediatric Brain Cancer 
Journal of Personalized Medicine  2014;4(3):402-411.
Classification of pediatric brain tumors with unusual histologic and clinical features may be a diagnostic challenge to the pathologist. We present a case of a 12-year-old girl with a primary intracranial tumor. The tumor classification was not certain initially, and the site of origin and clinical behavior were unusual. Genomic characterization of the tumor using a Clinical Laboratory Improvement Amendment (CLIA)-certified next-generation sequencing assay assisted in the diagnosis and translated into patient benefit, albeit transient. Our case argues that next generation sequencing may play a role in the pathological classification of pediatric brain cancers and guiding targeted therapy, supporting additional studies of genetically targeted therapeutics.
PMCID: PMC4263965  PMID: 25563358
glioblastoma; pediatric glioma; metastatic glioma; gene mutation; next generation sequencing; BRAF V600E; vemurafenib; CKDN2A
6.  TargetingTumor MetabolismWith 2-Deoxyglucose in Patients With Castrate-Resistant Prostate Cancer and Advanced Malignancies 
The Prostate  2010;70(13):1388-1394.
A profound difference between cancer and normal tissues is the preferential utilization of glycolysis by cancer cells. To translate this paradigm in the clinic, we completed a phase I study of 2-deoxyglucose (2DG), and assessed 2DG uptake with fluorodeoxyglucose (FDG) positron emission tomography (PET) and the autophagy substrate p62 as a marker of 2DG resistance.
Patients received 2DG orally on days 1–14 of a 21-day cycle in cohorts of three in a dose-escalating manner. Correlative assessments included PET scans at baseline and day 2 and p62 protein in peripheral blood mononuclear cells as a potential marker of 2DG resistance.
The dose of 45 mg/kg was defined as the recommended phase II dose, secondary to dose-limiting toxicity of grade 3 asymptomatic QTc prolongation at a dose of 60 mg/kg. PK evaluation of 2DG revealed linear pharmacokinetics with Cmax 45 μg/ml (277 μM), 73.7 μg/ml (449 μM), and 122 μg/ml (744 μM) in dose levels 30, 45, and 60 mg/kg, respectively. Five of eight patients assessed with FDG-PET scanning demonstrated decreased FDG uptake by day 2 of therapy, suggesting competition of 2DG with FDG. Five of six patients assessed for p62 had a decrease in p62 at 24 hr.
These data support the safety of 2DG, defined 2DG PK, demonstrated the effect of 2DG on FDG-PET imaging, and demonstrated the feasibility of assessment of p62 as an autophagic resistance marker. These data support future studies of 2DG alone or in combination with approaches to abrogate autophagy.
PMCID: PMC4142700  PMID: 20687211
deoxyglucose; metabolism; prostate cancer; autophagy; p62
7.  An Active Learning Approach for Rapid Characterization of Endothelial Cells in Human Tumors 
PLoS ONE  2014;9(3):e90495.
Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.
PMCID: PMC3946171  PMID: 24603893
8.  Enrichment of human prostate cancer cells with tumor initiating properties in mouse and zebrafish xenografts by differential adhesion 
The Prostate  2013;74(2):187-200.
Prostate tumor-initiating cells (TICs) have intrinsic resistance to current therapies. TICs are commonly isolated by cell sorting or dye exclusion, however, isolating TICs from limited primary prostate cancer (PCa) tissues is inherently inefficient. We adapted the collagen adherence feature to develop a combined immunophenotypic and time-of-adherence assay to identify human prostate TICs.
PCa cells from multiple cell lines and primary tissues were allowed to adhere to several matrix molecules, and fractions of adherent cells were examined for their TIC properties.
Collagen-I rapidly-adherent PCa cells have significantly higher clonogenic, migration, and invasion abilities, and initiated more tumor xenografts in mice when compared to slowly-adherent and no-adherent cells. To determine the relative frequency of TICs among PCa cell lines and primary PCa cells, we utilized zebrafish xenografts to define the tumor initiation potential of serial dilutions of rapidly-adherent α2β1hi/CD44hi cells compared to non-adherent cells with α2β1low/CD44low phenotype. Tumor initiation from rapidly-adherent α2β1hi/CD44hi TICs harboring the TMPRSS2:ERG fusion generated xenografts comprising of PCa cells expressing Erg, AMACR, and PSA. Moreover, PCa-cell dissemination was consistently observed in the immune-permissive zebrafish microenvironment from as-few-as 3 rapidly-adherent α2β1hi/CD44hi cells. In zebrafish xenografts, self-renewing prostate TICs comprise 0.02–0.9% of PC3 cells, 0.3–1.3% of DU145 cells, and 0.22–14.3% of primary prostate adenocarcinomas.
Zebrafish PCa xenografts were used to determine that the frequency of prostate TICs varies among PCa cell lines and primary PCa tissues. These data support a paradigm of utilizing zebrafish xenografts to evaluate novel therapies targeting tumor initiating cells in prostate cancer.
PMCID: PMC3939797  PMID: 24154958
Prostate cancer stem cells; tumor-initiating cells; zebrafish
9.  Eastern Cooperative Oncology Group Phase II Trial of Lapatinib in Men with Biochemically Relapsed, Androgen Dependent Prostate Cancer 
Urologic oncology  2011;31(2):211-218.
Activation of the epidermal growth factor pathway is important in prostate cancer development and the transcription of androgen receptor regulated genes. This study evaluated the potential activity of lapatinib in men with biochemically-relapsed androgen-dependent (stage D0) prostate cancer.
Experimental Design
Patients with a rising PSA after primary therapy for prostate cancer were enrolled. A PSA doubling time (PSADT) <12 months was required. Lapatinib was administered at 1,500 mg orally daily. Outcome measures were changes in PSA kinetics. Primary tumor blocks were obtained and assessed for EGFR expression, EGFR Q787Q polymorphism, and Kras 38 mutational status.
49 patients were enrolled (14 ineligible), resulting in 35 pts for analysis. No PSA response was observed; best response was stable disease (n=28, 80.0%). Pre-treatment average slope was 0.19 log (PSA)/month (PSADT=3.70 months), in contrast to on-treatment average slope of 0.13 log (PSA)/month (PSADT=5.44 months) using linear mixed effects models (p=0.006). Median progression-free survival (PFS) was 17.4 months for the high EGFR group and 6.0 months for the low EGFR group (p=0.50). Patients with Kras 38 mutation had shorter PFS than those without Kras 38 mutation (p=0.09).
Although no PSA responses (primary endpoint) was observed, lapatinib may have biologic activity in men with stage D0 prostate cancer as evidenced by a decrease in PSA slope in this non-randomized study. Additional trials assessing the role of EGFR overexpression and Kras wild type status in prostate cancer should be investigated.
PMCID: PMC3223557  PMID: 21784672
Epidermal growth factor receptor; tyrosine kinase inhibitors; clinical trial
10.  Does Primary Androgen-Deprivation Therapy Delay the Receipt of Secondary Cancer Therapy for Localized Prostate Cancer? 
European urology  2012;62(6):966-972.
Despite evidence that shows no survival advantage, many older patients receive primary androgen-deprivation therapy (PADT) shortly after the diagnosis of localized prostate cancer (PCa).
This study evaluates whether the early use of PADT affects the subsequent receipt of additional palliative cancer treatments such as chemotherapy, palliative radiation therapy, or intervention for spinal cord compression or bladder outlet obstruction.
Design, setting, and participants
This longitudinal population-based cohort study consists of Medicare patients aged ≥66 yr diagnosed with localized PCa from 1992 to 2006 in areas covered by the Surveillance Epidemiology and End Results (SEER) program. SEER-Medicare linked data through 2009 were used to identify the use of PADT and palliative cancer therapy.
Outcome measurements and statistical analysis
Instrumental variable analysis methods were used to minimize confounding effects. Confidence intervals were derived from the bootstrap estimates.
Results and limitations
This study includes 29 775 men who did not receive local therapy for T1–T2 PCa within the first year of cancer diagnosis. Among low-risk patients (Gleason score 2–7 in 1992–2002 and Gleason score 2–6 in 2003–2006) with a median age of 78 yr and a median follow-up of 10.3 yr, PADT was associated with a 25% higher use of chemotherapy (hazard ratio [HR]: 1.25; 95% confidence interval [CI], 1.08–1.44) and a borderline higher use of any palliative cancer surgery (HR: 1.07; 95% CI, 0.97– 1.19) within 10 yr of diagnosis in regions with high PADT use compared with regions with low PADT use. Because this study was limited to men >65 yr, the results may not be applicable to younger patients.
Early treatment of low-risk, localized PCa with PADT does not delay the receipt of subsequent palliative therapies and is associated with an increased use of chemotherapy.
PMCID: PMC3472155  PMID: 22608160
Prostatic neoplasm; Medicare; SEER program; Antineoplastic agents–hormonal
11.  Inhibition of IL-6 expression in LNCaP prostate cancer cells by a combination of atorvastatin and celecoxib 
Oncology Reports  2013;31(2):835-841.
In the present study, we investigated the effect of a combination of atorvastatin and celecoxib on the formation of interleukin (IL)-6, a cytokine that is increased during the progression of LNCaP tumors from androgen dependence to androgen independence. Culturing LNCaP cells in androgen-depleted (AD) medium increased the levels of IL-6 and survivin, and treatment of the cells in AD medium with a combination of atorvastatin and celecoxib strongly inhibited the increase in IL-6 and survivin which is one of the downstream targets of the IL-6 signaling pathway. Addition of recombinant IL-6 partially abrogated the combined effect of atorvastatin and celecoxib on apoptosis in LNCaP cells cultured in AD medium. In SCID mice, we found that the levels of IL-6 and survivin expression were increased when LNCaP tumors became androgen-independent. Treatment of the mice with atorvastatin or celecoxib alone caused decrease in the levels of IL-6 and survivin as LNCaP tumors became androgen-independent, but treatment of the mice with a combination of celecoxib and atorvastatin resulted in a much stronger inhibition in the increase in IL-6 and survivin expression. Our results indicate that decreases in IL-6 and survivin levels by atorvastatin and celecoxib administration are associated with increased apoptosis in LNCaP cells treated with this drug combination. Our in vivo studies indicate that the inhibitory effect of a combination of atorvastatin and celecoxib on the progression of androgen-dependent LNCaP xenograft tumors to androgen independence is associated with inhibition of the increase in IL-6 and survivin that occurs when androgen-dependent LNCaP prostate tumors become androgen-independent.
PMCID: PMC3981114  PMID: 24296978
prostate cancer; IL-6; atorvastatin; celecoxib; xenograft tumor
The Prostate  2012;72(12):10.1002/pros.22487.
Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737.
Experimental Design
Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter.
Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity.
We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.
PMCID: PMC3840901  PMID: 22241682
Prostate Cancer; Autophagy; Metabolism; Bcl-2; BH3; ABT-737; ABT-263
13.  Obesity and prostate cancer detection: insights from three national surveys 
The American journal of medicine  2010;123(9):10.1016/j.amjmed.2010.05.011.
Previous studies suggest that obesity is associated with higher prostate cancer progression and mortality despite an association with lower prostate cancer incidence. This study aims to better understand these apparently inconsistent relationships among obese men, by combining evidence from three nationally representative cross-sectional surveys.
We evaluated relationships between obesity and (1) testosterone concentrations in the Third National Health and Nutrition Examination Survey (NHANES III; n=845), (2) prostate-specific antigen (PSA) in NHANES 2001–2004 (n=2,458) and (3) prostate biopsy rates in the National Health Interview Survey (NHIS 2000; n=4,789) population. Mean testosterone, PSA concentrations and biopsy rates were computed for body mass index (BMI) categories.
Testosterone concentrations were inversely associated with obesity (p-trend<0.0001) in NHANES III. In NHANES 2001–2004 obese (BMI >35) versus lean (BMI <25) men were less likely to have PSA concentrations that reached the biopsy threshold of >4 ng/ml (3% versus 8%; p<0.0001). Among NHIS participants all BMI groups had similar rates of PSA testing (p=0.24). However, among men who had PSA tests, 11% of men with BMI >30 versus 16% with BMI <25, achieved a PSA threshold of 4 ng/ml; p=0.01. Furthermore, biopsy rates were lower among men with BMI >30 versus BMI <25 in NHIS participants (4.6% vs. 5.8%; p=0.05).
Obesity was associated with lower PSA-driven biopsy rates. These data support further studies to test the hypothesis that obesity affects prostate cancer detection independent of prostate cancer risk by decreasing the PSA-driven biopsy rates.
PMCID: PMC3826172  PMID: 20800152
obesity; prostate cancer; prostate-specific antigen; biopsy
14.  Abiraterone in Prostate Cancer: a new angle to an old problem 
Abiraterone acetate is an orally administered potent inhibitor of cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1 or CYPc17), which is essential for synthesis of testosterone from cholesterol. While decreasing serum testosterone through inhibition of testicular function is the first line of treatment for men with metastatic prostate cancer, residual androgens may still be detected in patients treated with LHRH agonists. Treatment with abiraterone results in rapid, and complete, inhibition of androgen synthesis in the adrenal glands and within the tumor itself. An overall survival benefit of maximal androgen suppression was recently demonstrated in a randomized placebo controlled phase III clinical trial of abiraterone with prednisone versus prednisone in men with metastatic castrate resistant prostate cancer previously treated with docetaxel chemotherapy. Abiraterone’s efficacy demonstrates the importance of androgen signaling in patients with castrate resistant metastastic disease, and the importance of studies of other novel agents such as MDV3100, an androgen receptor inhibitor, that additionally targets androgen receptor translocation. These promising results now pose a new angle to an old problem regarding hormonal therapy and raise new questions about how resistance develops, how to best sequence therapy, and how to optimize combinations with other emerging novel targeted agents.
PMCID: PMC3761800  PMID: 22451619
Abiraterone; Prostate Cancer; CYP17A1; CYP17; CYPc17
15.  A validated HPLC assay for the determination of R-(-)-gossypol in human plasma and its application in clinical pharmacokinetic studies 
R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiff's base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C18 at 30°C. The mobile phase consists of 10 mmol/L KH2PO4 (pH=3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R2=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.
PMCID: PMC3358459  PMID: 22483642
R-(-)-gossypol; HPLC-UV; pharmacokinetics
16.  Inference of synergy/antagonism between anticancer drugs from the pooled analysis of clinical trials 
Drug interactions can have a significant impact on the response to combinatorial therapy for anticancer treatment. In some instances these interactions can be anticipated based on pre-clinical models. However, the anticipation of drug interactions in the clinical context is in general a challenging task.
Here we propose the pooled analysis of clinical trials as a mean to investigate drug interactions in anticancer therapy. To this end we collected 1,163 Phase II clinical trials with response data on over 53,745 subjects.
We provide statistical definitions of drugs resulting in clinical synergy and antagonism and identify drug combinations in each group. We also quantify the possibility of inferring interactions between three or more drugs from parameters characterizing the action of single and two-drugs combinations.
Our analysis provides a statistical methodology to track the performance of drug combinations in anticancer therapy and to quantify drug interactions in the clinical context.
PMCID: PMC3694030  PMID: 23758906
Cancer therapy; Clinical trial; Overall response rate; Combinatorial therapies; Clinical synergy; Systems biology
17.  Phase II Trial of Weekly Ixabepilone in Men With Metastatic Castrate-Resistant Prostate Cancer (E3803): A Trial of the Eastern Cooperative Oncology Group 
Clinical genitourinary cancer  2012;10(2):99-105.
Ixabepilone is an epothilone B analogue with activity in a variety of solid malignancies, including prostate cancer. The main dose-limiting toxicity of ixabepilone is myelosuppression when administered by using an every 3-week schedule. Here we evaluate the activity of a weekly ixabepilone in men with metastatic castrate-resistant prostate cancer to minimize hematologic toxicity.
BMS-247550 (ixabepilone) is an epothilone B analogue with activity in taxane-resistant cancer cell lines. Here we report the activity and toxicity of ixabepilone, administered by using a weekly schedule, in men with metastatic castrate-resistant prostate cancer (CRPC).
Experimental Design
Patients with metastatic CRPC received ixabepilone at 20 mg/m2 intravenous weekly × 3, in 4-week cycles. This noncomparative study stratified patients to either a chemotherapy naive (CN), prior taxane (Tax) only, or 2 prior cytotoxic (TCx) chemotherapy arm. The primary endpoint was prostate-specific antigen response by using PCWG (Prostate Cancer Working Group) 1 criteria. Secondary endpoints included radiographic response when using RECIST (Response Evaluation Criteria In Solid Tumors).
In total, 124 patients were enrolled, of whom, 109 were eligible (35 CN, 42 Tax, and 32 TCx) for the primary response determination in this study. Prostate-specific antigen responses were seen in 12 (34.3%) of 35, 12 (28.6%) of 42, and 7 (21.9%) of 32 patients with the partial objective response in 5 (22.7%) of 22, 2 (8.0%) of 25, and 0 (0.0%) of 24 patients for the CN, Tax, and TCx arms, respectively. Significant (grade 3/4) neutropenia was seen in 6 (15.4%), 7 (14.6%), and 9 (25.0%); and grade 3/4 sensory neuropathy was seen in 8 (20.5%), 12 (25.0%), and 12 (33.3%) for CN, Tax, and TCx, respectively. Grade 3/4 thrombocytopenia was infrequent and seen in only one patient on the CN and the TCx arm.
Ixabepilone was found to have an acceptable toxicity profile when administered by using a weekly schedule with less myelosuppression compared with prior studies when using the every 3-week schedule. Single-agent activity was observed and met prespecified activity levels for the Tax treated arm.
PMCID: PMC3535436  PMID: 22386239
BMS-247550; Chemotherapy; Epothilone; Microtubule-inhibitor
18.  Risk Profiles and Treatment Patterns among Men diagnosed with Prostate Cancer and a Prostate Specific Antigen Level Below 4.0 ng/ml 
Archives of internal medicine  2010;170(14):1256-1261.
Despite controversy over the benefit of prostate specific antigen (PSA) screening, little is known about risk profiles and treatment patterns in men diagnosed with prostate cancer who have a PSA value less than or equal to 4 ng/mL.
We utilized data from the Surveillance, Epidemiology, End Results system to describe patient characteristics and treatment patterns of 123,934 men with newly diagnosed prostate cancer in 2004–2006. Age-standardized treatment rates were calculated in five-year age strata. Logistic regression was used to quantify the odds ratios (OR) of men with low– and high–risk disease and the use of radical prostatectomy (RP) or radiation therapy (RT).
Men with a PSA of 4.0 ng/ml or less represent 14% of incident prostate cancer cases. Fifty-four percent of men diagnosed with prostate cancer and PSA ≤ 4.0 ng/mL harbor low-risk disease (stage ≤ T2a, PSA level ≤ 10 ng/mL, and Gleason score ≤ 6), but over 75% of them received RP or RT. Men with screen-detected prostate cancer and PSA values ≤ 4 ng/mL were 1.49 (CI 1.38–1.62) and 1.39 (CI 1.30–1.49) times more likely to receive RP and RT, respectively, and were less likely to have high-grade disease than men who had non-screen detected prostate cancer (OR=0.67; 95% CI:0.60–0.76).
Most men diagnosed with prostate cancer with a PSA threshold ≤ 4.0 ng/mL had low-risk disease but underwent aggressive local therapy. Lowering biopsy threshold, while lacking the ability to distinguish indolent cancers from aggressive cancers, may increase overdiagnosis and overtreatment.
PMCID: PMC3651841  PMID: 20660846
19.  Cilengitide (EMD 121974, NSC 707544) in asymptomatic metastatic castration resistant prostate cancer patients: A randomized phase II trial by the Prostate Cancer Clinical Trials Consortium 
Investigational new drugs  2010;29(6):1432-1440.
Integrins are involved in prostate cancer metastasis by regulating cell adhesion, migration, invasion, motility, angiogenesis and bone metabolism. We evaluated the efficacy of two dose levels of cilengitide in patients (pts) with castrate resistant prostate cancer (CRPC).
Chemotherapy-naïve, asymptomatic metastatic CRPC pts were randomized to cilengitide 500mg or 2000mg IV twice weekly using parallel 2-stage design. The primary endpoint was rate of objective clinical progression at six-months. Secondary endpoints included clinical and PSA response rates, safety and effects of cilengitide treatment on circulating tumor cells (CTCs) and bone remodeling markers.
Forty-four pts were accrued to first stage (22/arm). Median number of cycles was three in both arms (500mg arm: 1–8; 2000 mg arm: 1–15). At six months, two pts (9%) on the 500mg arm and five pts (23%) on the 2000mg arm had not progressed. Best objective response was stable disease (SD) in seven pts for 9.9[8.1,20.9] months. There were three grade 3 and no grade 4 toxicities. At 12 weeks, analysis of bone markers did not reveal significant trends. At progression, bone specific alkaline phosphatase and N-telopeptide increased in all pts, less so in pts on the 2000mg arm and in pts on both arms who obtained SD at 6 months. CTCs increased over time in both arms.
Cilengitide was well tolerated with modest clinical effect in favor of the higher dose. The unique trial design including a shift from response rate to objective progression as the endpoint, and not acting on PSA increases was feasible.
PMCID: PMC2917503  PMID: 20336348
prostate cancer; metastatic disease; integrins; angiogenesis; cilengitide; bone biomarkers
20.  Phase II study of Cilengitide (EMD 121974, NSC 707544) in patients with non-metastatic castration resistant prostate cancer, NCI-6735. A study by the DOD/PCF Prostate Cancer Clinical Trials Consortium 
Investigational New Drugs  2010;30(2):749-757.
Integrins mediate invasion and angiogenesis in prostate cancer bone metastases. We conducted a phase II study of Cilengitide, a selective antagonist of αvβ3 and αvβ5 integrins, in non-metastatic castration resistant prostate cancer with rising PSA.
Patients were observed for 4 weeks with PSA monitoring, and then treated with 2,000 mg IV of cilengitide twice weekly until toxicity/progression. PSA, circulating tumor cells (CTCs) and circulating endothelial cells (CECs) were monitored each cycle with imaging performed every 3 cycles. Primary end point was PSA decline by ≥ 50%. Secondary endpoints were safety, PSA slope, time to progression (TTP), overall survival (OS), CTCs, CECs and gene expression.
16 pts were enrolled; 13 were eligible with median age 65.5 years, baseline PSA 8.4 ng/mL and median Gleason sum 7. Median of 3 cycles was administered. Treatment was well tolerated with 2 grade 3 toxicities and no grade 4 toxicities. There were no PSA responses; 11 patients progressed by PSA after 3 cycles. Median TTP was 1.8 months and median OS has not been reached. Median pre- and on-treatment PSA slopes were 1.1 and 1.8 ng/mL/month. Baseline CTCs were detected in 1/9 patients. CTC increased (0 to 1; 2 pts), remained at 0 (2 pts) or decreased (23 to 0; 1 patient) at progression. Baseline median CEC was 26 (0–61) and at progression, 47 (15–148). Low cell counts precluded gene expression studies.
Cilengitide was well tolerated but had no detectable clinical activity. CTCs are of questionable utility in non-metastatic prostate cancer.
PMCID: PMC3175265  PMID: 21049281
EMD 121974; cilengitide; non-metastatic prostate cancer
21.  Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition 
PLoS ONE  2012;7(7):e41831.
mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.
PMCID: PMC3406086  PMID: 22848625
22.  A phase I trial of MK-0731, a Kinesin Spindle Protein (KSP) inhibitor, in patients with solid tumors 
Investigational New Drugs  2011;30(3):1088-1095.
The kinesin spindle protein (KSP) is essential for separation of spindle poles during mitosis. Its inhibition results in mitotic arrest. This phase I trial examined safety, tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetic parameters, and anti-tumor activity of MK-0731, a potent inhibitor of KSP.
Experimental design
In part 1, patients with advanced solid tumors received MK-0731 intravenously over 24 h every 21 days starting at 6 mg/m2, escalating until MTD was reached. In part 2, patients with taxane-resistant tumors received the MTD. Plasma samples were collected to analyze the pharmacokinetics of MK-0731. Tumor response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) v1.0.
In part 1, 21 patients (median age 63 years) were treated with MK-0731 at doses ranging from 6 to 48 mg/m2/24 h for median four cycles. The dose-limiting toxicity was neutropenia and the MTD was 17 mg/m2/24 h. At the MTD, AUC (±SD) was 10.5 (±7.3) μM × hour, clearance (±SD) was 153 mL/min (±84), and t1/2 was 5.9 h. In part 2, 22 patients received the MTD and there were no DLTs. Although there were no objective tumor responses, four patients (with cervical, non-small cell lung, and ovarian cancers) had prolonged stable disease.
MK-0731 at the MTD of 17 mg/m2/day every 21 days in patients with solid tumors had few grade 3 and 4 toxicities with the major DLTs at higher doses being myelosuppression. Anti-tumor efficacy was suggested by the length of stable disease in selected patients with taxane-resistant tumors.
PMCID: PMC3394096  PMID: 21424701
Kinesin spindle protein; Oncology; Neutropenia
23.  Transdermal estradiol in castrate and chemotherapy resistant prostate cancer 
Given prior studies demonstrating the marked clinical activity of oral estrogens in prostate cancer, more recent data demonstrating the safety of transdermal estradiol, and the renewed interest in targeting testosterone metabolism and androgen receptor pathways, we report the results of a trial of transdermal estradiol in advanced heavily pre-treated castrate and chemotherapy refractory patients.
Patients with prostate cancer progressing after androgen ablation therapy and chemotherapy were treated with transdermal estradiol patches (0.4 mg per 24 hours total) applied weekly and assessed for tolerability and biochemical activity.
Twenty-two patients were treated on study with all patients evaluable for safety and 20 patients evaluable for response. All patients had aggressive and resistant disease, as demonstrated by a median PSA of 170 ng/mL (range 14 to 5030 ng/mL), with more than 60% having been treated with two or more prior chemotherapy regimens, and 20% with visceral disease. Nine patients had a decrease in PSA, of which two patients had a PSA response defined as a decline in PSA by 50%. Therapy was well tolerated and no thrombotic events were observed.
In heavily pre-treated patients with advanced castrate and chemotherapy refractory metastatic prostate cancer, transdermal estradiol was safe and had biochemical activity. These data support further studies to understand if transdermal estradiol can be useful following multiple standard therapies.
PMCID: PMC3560819  PMID: 22460098
estradiol; abiraterone; testosterone; prostate cancer
24.  Principles and Current Strategies for Targeting Autophagy for Cancer Treatment 
Autophagy is an evolutionarily conserved, intracellular self-defense mechanism where organelles and proteins are sequestered into autophagic vesicles (AVs) that are subsequently degraded through fusion with lysosomes. Cells thereby prevent the toxic accumulation of damaged or unnecessary components, but also recycle these components to sustain metabolic homoeostasis. Heightened autophagy is a mechanism of resistance for cancer cells faced with metabolic and therapeutic stress, revealing opportunities for exploitation as a therapeutic target in cancer. We summarize recent developments in the field of autophagy and cancer, and build upon the results presented at the Cancer Therapeutics and Evaluation Program (CTEP) Early Drug Development meeting in March, 2010. Herein, we describe our current understanding of the core components of the autophagy machinery, the functional relevance of autophagy within the tumor microenvironment and outline how this knowledge has informed preclinical investigations combining the autophagy inhibitor hydroxychloroquine (HCQ) with chemotherapy, targeted therapy and immunotherapy. Finally, we describe ongoing clinical trials involving HCQ as a first generation autophagy inhibitor, as well as strategies for the development of novel, more potent and specific inhibitors of autophagy.
PMCID: PMC3075808  PMID: 21325294
25.  Inhibitory Effect of a γ-Tocopherol-Rich Mixture of Tocopherols on the Formation and Growth of LNCaP Prostate Tumors in Immunodeficient Mice 
Cancers  2011;3(4):3762-3772.
In the present study, we determined the effects of a γ-tocopherol-rich mixture of tocopherols (γ-TmT) on the growth and apoptosis of cultured human prostate cancer LNCaP cells. We also determined the effects of dietary γ-TmT on the formation and growth of LNCaP tumors in immunodeficient mice. In the in vitro study, we found that the activity of γ-TmT was stronger than α-tocopherol for inhibiting the growth and stimulating apoptosis in LNCaP cells. In the animal study, treatment of severe combined immunodeficient (SCID) mice with dietary γ-TmT inhibited the formation and growth of LNCaP xenograft tumors in a dose-dependent manner. Mechanistic studies showed that γ-TmT administration inhibited proliferation as reflected by decreased mitosis and stimulated apoptosis as reflected by increased caspase-3 (active form) expression in LNCaP tumors. In addition, dietary administration of γ-TmT increased the levels of α-, γ- and δ- tocopherol in plasma, and increased levels of γ- and δ- tocopherol were also observed in the prostate and in tumors. The present study demonstrated that γ-TmT had strong anticancer activity both in vitro and in vivo. Additional studies are needed to determine the potential preventive effect of γ-TmT for prostate cancer in humans.
PMCID: PMC3763395  PMID: 24213110
prostate cancer; tocopherol; immunodeficient mice; xenograft tumor

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