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1.  Neutrophil gelatinase-associated lipocalin (NGAL) levels in response to unilateral renal ischaemia in a novel pilot two-kidney porcine model 
BJU international  2013;112(4):517-525.
OBJECTIVES
To test a novel porcine two-kidney model for evaluating the effect of controlled acute kidney injury (AKI) related to induced unilateral ischaemia on both renal units (RUs)
To use neutrophil gelatinase-associated lipocalin (NGAL) and physiological serum and urinary markers to assess AKI and renal function.
METHODS
Twelve female Yorkshire pigs had bilateral cutaneous ureterostomies placed laparoscopically with identical duration of pneumoperitoneum for all cases.
An experimental group (n =9) underwent induced unilateral renal ischaemia with left hilar clamping of timed duration (15, 30, 60 min) and a control group (n =3) had no induced renal ischaemia.
Urine was collected and analysed from each RU to assess creatinine and NGAL concentration preoperatively and at multiple postoperative time points. Serum was collected and analysed daily for creatinine and NGAL levels.
Statistical comparisons were made using the rank-sum and sign-rank tests.
RESULTS
Three pigs were excluded because of intra-operative and postoperative complications.
In the RUs that experienced renal ischaemia (n=7),the median urine volume was lower (P=0.04) at 6, 12, 24 and 48 h and the median NGAL concentration was higher (P=0.04) at 12 and 48 h compared with the RUs of control pigs that experienced no renal ischaemia (n=2).
When comparing the ischaemic (left) RU of the pigs in the experimental group with their contralateral non-ischaemic (right) RU, ischaemic RUs had a lower median cumulative urine volume at 6, 12, 24 and 48 h (P = 0.05) and a higher median NGAL concentration at 12, 24 and 48 h (P<0.05).
At 48 h, no significant increase was found in serum NGAL in pigs in the experimental group compared with controls (P=0.2).
Creatinine clearance (CC) was lower in ischaemic RUs compared with non-ischaemic RUs 1 day after surgery (P=0.04) with decreasing CC as the duration of ischaemia increased.
CONCLUSIONS
We have developed a promising novel small-scale pilot surgical model that allowed the evaluation of bilateral RU function separately during and after unilateral renal ischaemia.
The induction of unilateral renal ischaemia corresponds with physiological changes in both the ischaemic and contralateral RU.
AKI as measured by increases in NGAL and decreased renal function as measured by decreases in CC, are specific to the RU exposed to ischaemia.
doi:10.1111/bju.12066
PMCID: PMC4235973  PMID: 23510358
human NGAL protein; ischaemia; acute kidney injury; models; animal; biomarkers
2.  Phase I Study of ARN-509, a Novel Antiandrogen, in the Treatment of Castration-Resistant Prostate Cancer 
Journal of Clinical Oncology  2013;31(28):3525-3530.
Purpose
ARN-509 is a novel androgen receptor (AR) antagonist for the treatment of castration-resistant prostate cancer (CRPC). ARN-509 inhibits AR nuclear translocation and AR binding to androgen response elements and, unlike bicalutamide, does not exhibit agonist properties in the context of AR overexpression. This first-in-human phase I study assessed safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of ARN-509 in men with metastatic CRPC.
Patients and Methods
Thirty patients with progressive CRPC received continuous daily oral ARN-509 at doses between 30 and 480 mg, preceded by administration of a single dose followed by a 1-week observation period with pharmacokinetic sampling. Positron emission tomography/computed tomography imaging was conducted to monitor [18F]fluoro-α-dihydrotestosterone (FDHT) binding to AR in tumors before and during treatment. Primary objective was to determine pharmacokinetics, safety, and recommended phase II dose.
Results
Pharmacokinetics were linear and dose proportional. Prostate-specific antigen declines at 12 weeks (≥ 50% reduction from baseline) were observed in 46.7% of patients. Reduction in FDHT uptake was observed at all doses, with a plateau in response at ≥ 120-mg dose, consistent with saturation of AR binding. The most frequently reported adverse event was grade 1/2 fatigue (47%). One dose-limiting toxicity event (grade 3 abdominal pain) occurred at the 300-mg dose. Dose escalation to 480 mg did not identify a maximum-tolerated dose.
Conclusion
ARN-509 was safe and well tolerated, displayed dose-proportional pharmacokinetics, and demonstrated pharmacodynamic and antitumor activity across all dose levels tested. A maximum efficacious dose of 240 mg daily was selected for phase II exploration based on integration of preclinical and clinical data.
doi:10.1200/JCO.2013.50.1684
PMCID: PMC3782148  PMID: 24002508
3.  Image guided thermal ablation of tumors increases the plasma level of IL-6 and IL-10 
PURPOSE
To identify changes in plasma cytokine levels following image-guided thermal ablation of human tumors and to identify the factors that independently predict changes in plasma cytokine levels.
MATERIALS AND METHODS
Whole blood samples were collected from 36 patients at 3 time points: pre-ablation, post-ablation (within 48 hours), and in follow-up (1–5 weeks after ablation). Plasma levels of IL-1a, IL-2, IL-6, IL-10 and TNFa were measured using a multiplex immunoassay. Univariate and multivariate analyses were performed using cytokine level as the dependent variable and sample collection, time, age, sex, primary diagnosis, metastatic status, ablation site, and ablation type as the independent variables.
RESULTS
There was a significant increase in the plasma level of IL-6 post-ablation when compared to pre-ablation (9.6+/−31 fold, p<0.002). IL-10 also showed a significant increase postablation (1.9 +/−2.8 fold, p<0.02). Plasma levels of IL-1a, IL-2, and TNFa were not significantly changed after ablation. Cryoablation resulted in the largest change in IL-6 level (>54 fold), while radiofrequency and microwave ablation showed 3.6 and 3.4-fold changes, respectively. Ablation of melanomas showed the largest change in IL-6 48 hours after ablation (92×), followed by ablation of kidney (26×), liver (8×), and lung (6×) cancers. Multivariate analysis revealed that ablation type (p<0.0003), and primary diagnosis (p<0.03) were independent predictors of changes to IL-6 following ablation. Age was the only independent predictor of IL-10 levels following ablation (p<0.019).
CONCLUSION
Image guided thermal ablation of tumors increases the plasma level of IL-6 and IL-10, without increasing the plasma level of IL-1a, IL-2, or TNFa.
doi:10.1016/j.jvir.2013.02.015
PMCID: PMC4167629  PMID: 23582441
thermal ablation; radiofrequency; microwave; cryoablation; cytokine; inflammation; growth factor
4.  Urine Neutrophil Gelatinase-Associated Lipocalin (uNGAL) as a Marker for Acute Kidney Injury in Kidney Surgery Patients 
The Journal of urology  2013;190(1):159-164.
Purpose
Evaluate uNGAL as a marker for AKI in patients undergoing PN to identify the preoperative clinical features and surgical factors during PN that are associated with renal injury as measured by increased uNGAL levels compared to controls.
Methods
Using RN and thoracic surgery patients as control groups, we prospectively collected and analyzed urine and serum of PN, RN, and thoracic surgery patients between April 2010 and April 2012. Urine was collected preoperatively and at multiple time points postoperatively. Differences in uNGAL levels between the 3 surgical groups were analyzed using a GEE model. The PN group was subdivided based on preoperative eGFR <60 or ≥60 ml/min/1.73m2.
Results
Of 162 patients included in the final analysis, >65% had CVD, and median eGFR was >60 ml/min/1.73m2 for all groups (RN=61, PN=78, thoracic surgery=84.5 ml/min/1.73m2). Preoperatively, a 10-unit increase in eGFR was associated with a 4- unit decrease in uNGAL in the PN group. Postoperatively, uNGAL levels in the PN group were not higher than thoracic surgery or RN control groups, and did not correlate with duration of ischemia. PN patients with preoperative eGFR <60 developed higher uNGAL levels postoperatively compared to those with a higher preoperative eGFR.
Conclusion
uNGAL does not appear to be a useful marker for detection of renal injury in healthy PN patients. However, patients with poorer preoperative renal function have higher baseline uNGAL levels and appear more susceptible to AKI as detected by uNGAL levels and AKIN criteria compared to those with normal eGFR.
doi:10.1016/j.juro.2013.01.101
PMCID: PMC4167359  PMID: 23391468
NGAL protein; human; acute kidney injury; biological markers; nephrectomy; surgery
5.  Analytical and Clinical Validation of a Prostate Cancer-Enhanced messenger RNA Detection Assay in Whole Blood as a Prognostic Biomarker for Survival 
European urology  2013;65(6):1191-1197.
Background
Biomarkers based on detecting prostate cancer-specific transcripts are associated with inferior outcomes, but their validation in a clinical context is lacking.
Objective
To determine whether detecting prostate cancer enhanced transcripts in whole blood using an analytically valid assay has prognostic significance relative to circulating tumor cell (CTC) enumeration.
Design, Setting, and Participants
The predictive value for overall survival of the detection in whole blood by reverse transcription real-time polymerase chain reaction (RT-PCR) of KLK3, KLK2, HOXB13, GRHL2, and FOXA1 was studied in 97 men with metastatic castration-resistant prostate cancer (mCRPC).
Intervention
2.5ml of blood was collected in PAXgene tubes for total RNA extraction and 7.5 ml for CTC enumeration from patients with progressive mCRPC.
Outcome Measurements and Statistical Analysis
Prostate cancer enriched genes were detected using a sensitive RT-PCR assay in whole blood from patients with mCRPC. Analytical validity of the assay was established in a clinical laboratory environment. The frequency of detecting transcripts was compared to CTC enumeration using CellSearch® in an independent data set and survival associations were explored by concordance probability estimate (CPE).
Results and Limitations
Two or more genes were detected by PCR in 53% (51 of 97, 95% CI 43–63%) of patients, and unfavorable CTC counts (≥5cells) were seen in 46% (45 of 97, 95% CI 36–56%). Importantly, transcripts were detectable in 11 of 52 patients with favorable CTC counts (21%, 95% CI 8–35%). Transcript detection predicted overall survival in a proportional hazards model. Significantly, the predictive accuracy of RT-PCR detection in combination with CTC enumeration had a CPE of 0.752 (SE=0.038), although limited by the number of patients.
Conclusions
This validated RT-PCR assay detecting prostate-specific RNA in whole blood is prognostic for survival, and may assess patient risk complimentary with CellSearch CTC enumeration. Its clinical utility is being prospectively explored.
doi:10.1016/j.eururo.2013.07.006
PMCID: PMC4113474  PMID: 23954088
biomarker; circulating tumor cells; prostate cancer; prostate-specific markers
6.  Rare cell capture technology for the diagnosis of leptomeningeal metastasis in solid tumors 
Neurology  2013;80(17):1598-1605.
Objective:
To evaluate the utility of rare cell capture technology (RCCT) in the diagnosis of leptomeningeal metastasis (LM) from solid tumors through identification of circulating tumor cells (CTCs) in the CSF.
Methods:
In this pilot study, CSF samples from 60 patients were analyzed. The main patient cohort consisted of 51 patients with solid tumors undergoing lumbar puncture for clinical suspicion of LM. Those patients underwent initial MRI evaluation and had CSF analyzed through conventional cytology and for the presence of CTCs using RCCT, based on immunomagnetic platform enrichment utilizing anti–epithelial cell adhesion molecule antibody-covered magnetic nanoparticles. An additional 9 patients with CSF pleocytosis but without solid tumors were separately analyzed to ensure accurate differentiation between CTCs and leukocytes.
Results:
Among the 51 patients with solid tumors, 15 patients fulfilled criteria for LM. CSF CTCs were found in 16 patients (median 20.7 CTCs/mL, range 0.13 to >150), achieving a sensitivity of 100% as compared with 66.7% for conventional cytology and 73.3% for MRI. One patient had a false-positive CSF CTC result (specificity = 97.2%); however, that patient eventually met LM criteria 6 months after the tap. CSF CTCs were not found in any of the additional 9 patients with CSF pleocytosis.
Conclusion:
RCCT is an accurate, novel method for the detection of LM in solid tumors, potentially providing earlier diagnostic confirmation and sparing patients from repeat lumbar punctures.
doi:10.1212/WNL.0b013e31828f183f
PMCID: PMC3662321  PMID: 23553479
7.  Circulating Tumors Cells as Biomarkers 
Cancer journal (Sudbury, Mass.)  2011;17(6):438-450.
Personalized cancer medicine requires the development of tumor-specific biomarkers to optimize selection of targeted therapies and to better assess response to therapy. Current efforts in several tumor types have shown that patients in whom circulating tumor cells (CTCs) are detected have an inferior prognosis relative to those in whom CTCs are not detected and that the elimination or decrease of CTCs following treatment is associated with improved clinical outcomes. Technological advances in the detection, isolation, capture, and characterization of CTCs from phlebotomy samples obtained in a routine clinical practice setting have enabled the evaluation of different CTC biomarkers. Unmet needs in cancer diagnosis and treatment where CTC biomarkers have been studied include determining prognosis, assessing the effects of treatment, and as a source of tumor for the biologic identification and characterization of determinants to predict sensitivity to one form of treatment versus another and to understand mechanisms of treatment resistance.
At present, there is no single definition of a CTC and no single CTC “biomarker.” Rather, multiple assays (tests) are in development for CTC biomarkers. However, before the role of any biomarker in medical decision making can be determined, it is essential that the assays used to measure the biomarker are analytically validated in a sequence of trials to generate the evidence to support the biomarker’s use in the given context of use. It is against this background that this review focuses on the process of developing CTC biomarker assays, with the objective of outlining the necessary steps to qualify specific CTC tests for medical decision making in clinical practice or drug development. The potential for point-of-care tests is clear.
doi:10.1097/PPO.0b013e31823e69ac
PMCID: PMC3985131  PMID: 22157288
Circulating tumor cells; biomarker; regulatory qualification; personalized medicine
8.  Identification of a recurrent germline PAX5 mutation and susceptibility to pre-B cell acute lymphoblastic leukemia 
Nature genetics  2013;45(10):1226-1231.
doi:10.1038/ng.2754
PMCID: PMC3919799  PMID: 24013638
9.  Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors 
Cancer research  2012;72(23):6142-6152.
Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPCs), due to several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCas) and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by qRT-PCR in PCa cell lines and circulating tumor cells (CTCs) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, while expression of SRD5A2, CYP3A4, CYP3A5 and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts were detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis targeting approach via simultaneous, frontline enzymatic blockade and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent anti-androgens, would represent a powerful future strategy for PCa management.
doi:10.1158/0008-5472.CAN-12-1335
PMCID: PMC3685485  PMID: 22971343
Prostate cancer; androgen synthesis; testosterone; dihydrotestosterone; CYP17; AKR1C3; abiraterone; MDV3100 (enzalutamide)
10.  Circulating tumor cells as biomarkers in prostate cancer 
Unmet needs in prostate cancer drug development and patient management are the ability to monitor treatment benefit and to identify the target of interest in a tumor at the time treatment is being considered. This review focuses on establishing analytical valid biomarkers for specific contexts of use in patients with castration-resistant prostate cancer (CRPC), emphasizing a biomarker currently in clinical use, circulating tumor cells (CTC). The Oncology Biomarker Qualification Initiative provides a road map for these investigations, which, if followed, will facilitate the incorporation of these types of assays into clinical decision-making.
CTC enumeration at baseline and post-treatment is prognostic of survival, with no threshold effect, and the shedding of cells into the circulation represents an intrinsic property of the tumor, distinct from extent of disease. The clinical utility of monitoring CTC changes with treatment as an efficacy-response surrogate biomarker of survival is currently being tested in large phase III trials with the novel antiandrogen therapies abiraterone acetate and MDV3100.
Molecular biomarkers can be characterized in CTC as potential predictive biomarkers of tumor sensitivity to a therapeutic modality. Additionally, we discuss novel technologies to enrich and characterize CTC from more patients, and the potential clinical uses of CTC's in determining prognosis and monitoring treatment effects, and as a source of tissue to identify predictive markers of drug sensitivity to guide treatment selection. Prospective studies, designed around the biomarker itself and the specific clinical context for which it is applied, are needed to further assess the role of these and novel markers in clinical practice.
doi:10.1158/1078-0432.CCR-10-2650
PMCID: PMC3743247  PMID: 21680546
11.  Tissue and serum mesothelin are potential markers of neoplastic progression in Barrett’s–associated esophageal adenocarcinoma 
Background
Mesothelin is overexpressed in several malignancies and is purportedly a specific marker of malignant transformation. In this pilot study, we investigated whether tissue and serum mesothelin are potential markers of neoplastic progression in Barrett’s esophagus (BE) and in esophageal adenocarcinoma (EAC).
Methods
Mesothelin expression was retrospectively evaluated in normal, BE, and EAC tissue from surgically resected esophageal specimens (n = 125). In addition, soluble mesothelin-related peptide (SMRP) levels were measured in serum.
Results
Normal esophageal mucosa did not express mesothelin. BE tissue with high-grade dysplasia specifically expressed mesothelin, whereas BE tissue with low-grade or without dysplasia did not. Fifty-seven (46%) EAC tumors were positive for mesothelin. EAC tumors with BE expressed mesothelin more often than those without BE (58% vs 35%, P = 0.01). SMRP levels were elevated in 70% of EAC patients (mean, 0.89 nM; range, 0.03-3.77 nM), but not in patients with acid reflux and/or BE.
Conclusions
Mesothelin is commonly expressed in BE-associated esophageal adenocarcinoma. Based on this pilot study, a prospective study is under way to evaluate tissue and serum mesothelin are potential markers of neoplastic progression in BE and in EAC (NCT01393483).
Impact
Current surveillance methods in Barrett’s esophagus are invasive and neither cost-effective nor sensitive. This pilot study suggests that serum mesothelin is a marker of neoplastic transformation in BE and may provide a noninvasive method to improve identification of malignant transformation.
doi:10.1158/1055-9965.EPI-11-0993
PMCID: PMC3297720  PMID: 22237988
Mesothelin; SMRP; Barrett’s esophagus; esophageal cancer; screening
12.  TMPRSS2-ERG Status in Circulating Tumor Cells as a Predictive Biomarker of Sensitivity in Castration-Resistant Prostate Cancer Patients Treated With Abiraterone Acetate 
European urology  2011;60(5):897-904.
Background
Abiraterone acetate (AA) is an androgen biosynthesis inhibitor shown to prolong life in patients with castration-resistant prostate cancer (CRPC) already treated with chemotherapy. AA treatment results in dramatic declines in prostate-specific antigen (PSA) in some patients and no declines in others, suggesting the presence of molecular determinants of sensitivity in tumors.
Objective
To study the role of transmembrane protease, serine 2 (TMPRSS2)–v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion, an androgen-dependent growth factor, in circulating tumor cells (CTCs) as a biomarker of sensitivity to AA.
Design, setting, and participants
The predictive value of TMPRSS2-ERG status was studied in 41 of 48 men with postchemotherapy-treated CRPC enrolled in sequential phase 2 AA trials.
Intervention
Patients received AA 1000 mg daily and continuously.
Measurements
TMPRSS2-ERG status was characterized by a sensitive, analytically valid reverse transcription polymerase chain reaction assay in CTCs enriched from ethylene-diaminetetraacetic acid anticoagulated blood obtained prior to AA treatment. Outcomes were measured by PSA Working Group 1 criteria.
Results and limitations
Standard procedures for specimen acquisition, processing, and testing using the validated TMPRSS2-ERG assay on a multiplex platform gave intra-assay and interassay coefficients of variation <7%. TMPRSS2-ERG fusion was present in 15 of 41 patients (37%), who had a median baseline CTC count of 17 (interquartile range: 7–103 cells per 7.5 ml). A PSA decline ≥50% was observed in 7 of 15 patients (47%) with the fusion and in 10 of 26 patients (38%) without the fusion. Although limited by the low number of patients, a posttherapy CTC count of less than five per 7.5 ml was prognostic for longer survival relative to a CTC count five or more. TMPRSS2-ERG status did not predict a decline in PSA or other clinical outcomes.
Conclusions
Molecular profiles of CTCs with an analytically valid assay identified the presence of the prostate cancer–specific TMPRSS2-ERG fusion but did not predict for response to AA treatment. This finding demonstrates the role of CTCs as surrogate tissue that can be obtained in a routine practice setting.
doi:10.1016/j.eururo.2011.07.011
PMCID: PMC3185163  PMID: 21802835
Abiraterone; Biomarker; Circulating tumor cells; Prostate cancer; Prostate-specific antigen; TMPRSS2-ERG fusion
13.  Serum YKL-40 is a marker of prognosis and disease status in high-grade gliomas† 
Neuro-Oncology  2011;13(11):1244-1251.
The objective of this study was to evaluate whether longitudinal levels of serum YKL-40 correlate with disease status or survival in adults with gliomas. Patients with histologically confirmed gliomas were eligible for this longitudinal study. Serum samples were collected prospectively and concurrently with MRI scans at multiple time points during the course of the disease. YKL-40 levels determined by ELISA were correlated with radiographic disease status and survival. We performed a multivariate survival analysis including well-known prognostic factors such as age, performance status, and extent of surgical resection. Three hundred and forty-three patients with gliomas (41 low-grade, 105 anaplastic, and 197 glioblastoma) were accrued. Two-year survival from registration was 29% for glioblastomas, 62% for anaplastic gliomas, and 83% for low-grade gliomas. A total of 1740 serum samples were collected, and 95.6% of samples had matching MRI scans. Serum YKL-40 level was significantly lower in patients with no radiographic disease compared with patients with radiographic disease in both the anaplastic glioma (P= .0008) and the glioblastoma (P= .0006) cohorts. Serum levels of YKL-40 in patients with low-grade gliomas were not associated with radiographic disease status. Increases in YKL-40 were independently associated with worse survival in anaplastic gliomas (hazard ratio [HR] = 1.4, P= .01) and glioblastomas (HR = 1.4, P< .0001). Longitudinal increases in serum YKL-40 are associated with increased risk of death in patients with glioblastomas and anaplastic gliomas. YKL-40 is also a putative indicator of disease status in these patients.
doi:10.1093/neuonc/nor117
PMCID: PMC3199155  PMID: 21831900
glioblastoma; glioma; serum marker; YKL-40
14.  An In Vivo Platform for Tumor Biomarker Assessment 
PLoS ONE  2011;6(10):e26722.
Tumor biomarkers provide a quantitative tool for following tumor progression and response to therapy. However, investigations of clinically useful tumor biomarkers are time-consuming, costly, and limited by patient and tumor heterogeneity. In addition, assessment of biomarkers as indicators of therapy response is confounded by the concomitant use of multiple therapeutic interventions. Herein we report our use of a clinically relevant orthotopic animal model of malignant pleural mesothelioma for investigating tumor biomarkers. Utilizing multi-modality imaging with correlative histopathology, we demonstrate the utility and accuracy of the mouse model in investigating tumor biomarkers – serum soluble mesothelin-related peptide (SMRP) and osteopontin (OPN). This model revealed percentage change in SMRP level to be an accurate biomarker of tumor progression and therapeutic response – a finding consistent with recent clinical studies. This in vivo platform demonstrates the advantages of a validated mouse model for the timely and cost-effective acceleration of human biomarker translational research.
doi:10.1371/journal.pone.0026722
PMCID: PMC3202552  PMID: 22046338
15.  Portable Filter-Based Microdevice for Detection and Characterization of Circulating Tumor Cells 
Purpose
Sensitive detection and characterization of circulating tumor cell (CTC) could revolutionize the approach to patients with early stage and metastatic cancer. The current methodologies have significant limitations including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood.
Experimental Design
We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. 59 model system samples were tested for determining the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch® platform enumeration for direct comparison.
Results
Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when 5 are seeded in 7.5 ml of blood. CTCs were identified in 51 out of 57 patients using the microdevice, compared to only 26 patients with the CellSearch® method. When CTC were detected by both methods, greater numbers were recovered by the microfilter device in all but 5 patients.
Conclusions
This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in clinical setting for effective management of cancer patients.
doi:10.1158/1078-0432.CCR-10-1105
PMCID: PMC2955786  PMID: 20876796
16.  Phase II Multicenter Study of Abiraterone Acetate Plus Prednisone Therapy in Patients With Docetaxel-Treated Castration-Resistant Prostate Cancer 
Journal of Clinical Oncology  2010;28(9):1496-1501.
Purpose
Persistence of ligand-mediated androgen receptor signaling has been documented in castration-resistant prostate cancers (CRPCs). Abiraterone acetate (AA) is a potent and selective inhibitor of CYP17, which is required for androgen biosynthesis in the testes, adrenal glands, and prostate tissue. This trial evaluated the efficacy and safety of AA in combination with prednisone to reduce the symptoms of secondary hyperaldosteronism that can occur with AA monotherapy.
Patients and Methods
Fifty-eight men with progressive metastatic CRPC who experienced treatment failure with docetaxel-based chemotherapy received AA (1,000 mg daily) with prednisone (5 mg twice daily). Twenty-seven (47%) patients had received prior ketoconazole. The primary outcome was ≥ 50% prostate-specific antigen (PSA) decline, with objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and changes in Eastern Cooperative Oncology Group (ECOG) performance status (PS) and circulating tumor cell (CTC) numbers. Safety was also evaluated.
Results
A ≥ 50% decline in PSA was confirmed in 22 (36%) patients, including 14 (45%) of 31 ketoconazole-naïve and seven (26%) of 27 ketoconazole-pretreated patients. Partial responses were seen in four (18%) of 22 patients with RECIST-evaluable target lesions. Improved ECOG PS was seen in 28% of patients. Median time to PSA progression was 169 days (95% CI, 82 to 200 days). CTC conversions with treatment from ≥ 5 to < 5 were noted in 10 (34%) of 29 patients. The majority of AA-related adverse events were grade 1 to 2, and no AA-related grade 4 events were seen.
Conclusion
AA plus prednisone was well tolerated, with encouraging antitumor activity in heavily pretreated CRPC patients. The incidence of mineralocorticoid-related toxicities (hypertension or hypokalemia) was reduced by adding low-dose prednisone. The combination of AA plus prednisone is recommended for phase III investigations.
doi:10.1200/JCO.2009.25.9259
PMCID: PMC3040042  PMID: 20159814
17.  A Phase II Trial of Docetaxel with Rapid Androgen Cycling as a Treatment for Patients with Prostate Cancer 
Purpose
We evaluated rapid androgen cycling in combination with docetaxel for men with progressive non-castrate prostate cancers.
Methods
Non-castrate patients with ≤ 6 months of hormones were eligible. Cohort 1 (63 patients ) received 6 28-day cycles of docetaxel (75 mg/m2), leuprolide and 7 days of topical testosterone. Cohort 2 (39 patients) received 9 21-day cycles of docetaxel (70 mg/m2), leuprolide and 3 days of testosterone. The primary endpoint was the proportion of patients at 18 months who achieved non -castrate testosterone levels (>150 ng/dl) and an undetectable PSA (≤ 0.05, ≤0.5, or ≤2.0 ng/ml with prior prostatectomy, radiotherapy, or no definitive therapy, respectively). Cytochrome P450 3A4 (CYP3A4) activity and docetaxel pharmacokinetics were evaluated.
Results
A higher proportion of patients achieved the undetectable PSA outcome at 18 months in cohort 2 relative to cohort 1 (13% vs. 0%). The 16% incidence of febrile neutropenia was higher than observed in patients was castration-resistant disease, which may have been related to a 50% reduction in overall docetaxel clearance in the non-castrate group. There was no alteration in CYP3A4 activity (P=0.87) or docetaxel clearance (P=0.88) between cycles.
Conclusions
The undetectable PSA endpoint allows for a rapid screening of interventions for further study. Increasing the number of docetaxel cycles following a shorter period of testosterone repletion, and a longer duration of testosterone depletion, increased the proportion of men who achieved an undetectable PSA. The higher-than-expected incidence of febrile neutropenia may have been related to the reduced overall docetaxel clearance in patients with non-castrate vs castrate testosterone levels.
doi:10.1200/JCO.2007.15.1928
PMCID: PMC3051836  PMID: 18565882
18.  Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study 
Lancet  2010;375(9724):1437-1446.
Background
MDV3100 is a rationally-designed androgen receptor antagonist that blocks androgen receptor (AR) binding, nuclear translocation, and co-activator recruitment more effectively than the androgen receptor antagonists currently in use. MDV3100 is also unique in that it prevents DNA binding, induces apoptosis, and has no agonist activity when AR is overexpressed. Because growth of castration-resistant prostate cancer (CRPC) appears to depend upon continued androgen receptor signaling, we hypothesized that MDV3100 could be effective therapy for men with CRPC. Antitumor activity and safety were assessed in a phase 1-2 trial.
Methods
Eligible patients with progressive metastatic CRPC were enrolled in cohorts of 3-6 patients. Once the safety of a dose was established, cohorts were expanded to include at least 12 chemotherapy-naïve and 12 post-chemotherapy treated patients.
Findings
140 patients were treated with doses ranging from 30 to 600 mg daily. Positron emission tomography (PET) imaging to assess androgen receptor blockade showed decreased 18-fluorodihydrotestosterone binding at dosages of 60 mg/day and above. Antitumor effects were observed at all dosages including declines in serum PSA of 50% or more in 56% of patients, responses in soft tissue, stabilized bone disease, and conversion from unfavourable to favourable circulating tumour cell counts. The median time to progression was 47 weeks for radiological progression. The maximal tolerated dose for sustained treatment (>28 days) was 240 mg and the most common adverse event was dose-dependent fatigue, which generally resolved following dose reduction.
Interpretation
Encouraging antitumor activity on all outcomes assessed was observed for MDV3100 in both chemotherapy-naïve and post-chemotherapy patients with CRPC, establishing that patients with CRPC are not uniformly hormone-refractory. A phase 3 trial in patients with progressive disease after docetaxel treatment is underway.
doi:10.1016/S0140-6736(10)60172-9
PMCID: PMC2948179  PMID: 20398925
19.  Phase II-I-II Study of Two Different Doses and Schedules of Pralatrexate, a High-Affinity Substrate for the Reduced Folate Carrier, in Patients With Relapsed or Refractory Lymphoma Reveals Marked Activity in T-Cell Malignancies 
Journal of Clinical Oncology  2009;27(26):4357-4364.
Purpose
To determine the maximum-tolerated dose (MTD) and efficacy of pralatrexate in patients with lymphoma.
Patients and Methods
Pralatrexate, initially given at a dose of 135 mg/m2 on an every-other-week basis, was associated with stomatitis. A redesigned, weekly phase I/II study established an MTD of 30 mg/m2 weekly for six weeks every 7 weeks. Patients were required to have relapsed/refractory disease, an absolute neutrophil greater than 1,000/μL, and a platelet count greater than 50,000/μL for the first dose of any cycle.
Results
The every-other-week, phase II experience was associated with an increased risk of stomatitis and hematologic toxicity. On a weekly schedule, the MTD was 30 mg/m2 weekly for 6 weeks every 7 weeks. This schedule modification resulted in a 50% reduction in the major hematologic toxicities and abrogation of the grades 3 to 4 stomatitis. Stomatitis was associated with elevated homocysteine and methylmalonic acid, which were reduced by folate and vitamin B12 supplementation. Of 48 assessable patients, the overall response rate was 31% (26% by intention to treat), including 17% who experienced complete remission (CR). When analyzed by lineage, the overall response rates were 10% and 54% in patients with B- and T-cell lymphomas, respectively. All eight patients who experienced CR had T-cell lymphoma, and four of the six patients with a partial remission were positron emission tomography negative. The duration of responses ranged from 3 to 26 months.
Conclusion
Pralatrexate has significant single-agent activity in patients with relapsed/refractory T-cell lymphoma.
doi:10.1200/JCO.2008.20.8470
PMCID: PMC3651599  PMID: 19652067
20.  Fluorescence in situ Hybridization Analysis of Circulating Tumor Cells in Metastatic Prostate Cancer 
Purpose
To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration resistant metastatic prostate cancer (CRPC).
Experimental Design
We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with metastatic CRPC.
Results
High-level chromosomal amplification of AR was detected in 37.5% of samples analyzed, and relative gain of MYC in 55.8%. No such abnormalities were detected in samples with CTC counts of less than 10, reflecting ascertainment difficulty in these lower count samples.
Conclusion
The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors, and thus demonstrate that analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. Furthermore, we demonstrate that as many as 50% of these patients have substantial amplification of the AR locus, indicating that androgen signaling continues to play an important role in late-stage prostate cancer.
doi:10.1158/1078-0432.CCR-08-2036
PMCID: PMC2875199  PMID: 19276271
circulating tumor cells; prostate cancer; tumor markers; FISH; androgen receptor
21.  Circulating Tumor Cell Number as a Prognostic Marker in Progressive Castration-Resistant Prostate Cancer: Use in Clinical Practice and Clinical Trials 
The lancet oncology  2009;10(3):233-239.
SUMMARY
Background
To assess the use of circulating tumor cell (CTC) number as a continuous variable as a prognostic factor for survival, and for the clinical management of patients with progressive metastatic castration-resistant prostate cancer receiving first-line chemotherapy.
Methods
The study included 164 men with progressive metastatic castration-resistant prostate cancer. CTCs were isolated by immunomagnetic capture from blood samples drawn at baseline and after the initiation of first-line chemotherapy. Baseline variables including CTC number, prostate-specific antigen (PSA), and lactate dehydrogenase (LDH), and posttreatment variables (fold change in CTCs and PSA) were tested for association with survival using the Cox proportional hazards models. The concordance probability estimate was used to gauge the discriminatory strength of the informative factors in separating low- and high-risk patients.
Findings
At baseline, variables associated with increased risk of death were a high LDH (hazard ratio [HR] 6.44), CTC number (HR 1.58), and PSA (HR 1.26), low albumin (HR 0.10), and low hemoglobin (HR 0.72) (all p<0.001). At 4, 8, and 12 weeks posttreatment, changes in CTC number were strongly associated with risk (all p≤0.001), while changes in PSA were modestly associated (p=0.04 to 0.8). The combination of factors most predictive of survival were LDH and CTC number (concordance probability estimate 0.72–0.75). Time to CTC progression was modestly associated with time to death.
Interpretation
CTC number, analyzed as a continuous variable, was more predictive of survival than PSA at baseline and during patient follow-up, and can be used to monitor disease status. A model including baseline and posttreatment CTC, independent of discrete cutoff values, and baseline LDH was most predictive. The prospective evaluation of CTC number as an intermediate endpoint of survival in randomized prospective clinical trials is warranted.
Funding
The Prostate Cancer Foundation.
doi:10.1016/S1470-2045(08)70340-1
PMCID: PMC2774131  PMID: 19213602
circulating tumor cells; prostate cancer; PSA; LDH; prognosis
22.  Bone Disease in Thalassemia: A Frequent and Still Unresolved Problem 
Adults with β thalassemia major frequently have low BMD, fractures, and bone pain. The purpose of this study was to determine the prevalence of low BMD, fractures, and bone pain in all thalassemia syndromes in childhood, adolescence, and adulthood, associations of BMD with fractures and bone pain, and etiology of bone disease in thalassemia. Patients of all thalassemia syndromes in the Thalassemia Clinical Research Network, ≥6 yr of age, with no preexisting medical condition affecting bone mass or requiring steroids, participated. We measured spine and femur BMD and whole body BMC by DXA and assessed vertebral abnormalities by morphometric X-ray absorptiometry (MXA). Medical history by interview and review of medical records, physical examinations, and blood and urine collections were performed. Three hundred sixty-one subjects, 49% male, with a mean age of 23.2 yr (range, 6.1–75 yr), were studied. Spine and femur BMD Z-scores < −2 occurred in 46% and 25% of participants, respectively. Greater age, lower weight, hypogonadism, and increased bone turnover were strong independent predictors of low bone mass regardless of thalassemia syndrome. Peak bone mass was suboptimal. Thirty-six percent of patients had a history of fractures, and 34% reported bone pain. BMD was negatively associated with fractures but not with bone pain. Nine percent of participants had uniformly decreased height of several vertebrae by MXA, which was associated with the use of iron chelator deferoxamine before 6 yr of age. In patients with thalassemia, low BMD and fractures occur frequently and independently of the particular syndrome. Peak bone mass is suboptimal. Low BMD is associated with hypogonadism, increased bone turnover, and an increased risk for fractures.
doi:10.1359/jbmr.080505
PMCID: PMC3276604  PMID: 18505376
DXA; BMD; fractures; vertebral morphometry; thalassemia
23.  Circulating prostate tumor cells detected by RT-PCR in men with localized or castration-refractory prostate cancer: concordance with CellSearch assay and association with bone metastases and with survival 
Clinical chemistry  2009;55(4):765-773.
BACKGROUND
Reverse transcription-PCR (RT-PCR) assays for detecting or analyzing expression profiles of circulating tumor cells (CTCs) are currently of uncertain clinical value. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs by real-time RT-PCR for KLK3 (PSA) and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival.
METHODS
KLK3, KLK2, and prostate stem cell antigen (PSCA) mRNAs were determined by standardized, quantitative real-time RT-PCR assays using blood from 180 localized disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™) approved for clinical use.
RESULTS
All healthy volunteers were negative for KLK mRNAs. KLK3 or KLK2 mRNAs were positive (≥80 mRNAs per mL blood) in 37 (49%) patients with CRPC, but in only 15 (8%) patients with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80-85%) and correlated (Kendall’s tau 0.60-0.68). Among patients with CRPC, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival, but only modestly correlated with serum PSA. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with positive KLK mRNA status.
CONCLUSION
Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases, but rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.
doi:10.1373/clinchem.2008.117952
PMCID: PMC2782624  PMID: 19233911
24.  Evaluation of molecular forms of PSA and human kallikrein 2 in predicting biochemical failure following radical prostatectomy 
Most pretreatment risk-assessment models to predict biochemical recurrence (BCR) after radical prostatectomy (RP) for prostate cancer rely on total prostate-specific antigen (PSA), clinical stage, and biopsy Gleason grade. We investigated whether free PSA (fPSA) and human glandular kallikrein-2 (hK2) would enhance the predictive accuracy of this standard model. Preoperative serum samples and complete clinical data were available for 1,382 patients who underwent RP for localized prostate cancer from 1993 − 2005. A case-control design was utilized, and conditional logistic regression models were used to evaluate the association between preoperative predictors and BCR after RP. We constructed multivariable models with fPSA and hK2 as additional preoperative predictors to the base model. Predictive accuracy was assessed with the area under the ROC curve (AUC). There were 146 BCR cases; the median follow up for patients without BCR was 3.2 years. Overall, 436 controls were matched to 146 BCR cases. The AUC of the base model was 0.786 in the entire cohort; adding free PSA and hK2 to this model enhanced the AUC to 0.798 (p=0.053), an effect largely driven by free PSA. In the subgroup of men with tPSA ≤10 ng/ml (48% of cases), adding free PSA and hK2 enhanced the AUC of the base model to a similar degree (from 0.720 to 0.726, p=0.2). Free PSA is routinely measured during prostate cancer detection. We suggest that the role of fPSA in aiding preoperative prediction should be investigated in further cohorts.
doi:10.1002/ijc.23983
PMCID: PMC2776773  PMID: 19003994
free PSA; PSA; human kallikrein 2; prostate cancer; biochemical recurrence; radical prostatectomy
25.  Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits 
Nature  2008;455(7214):804-807.
Infection with antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a dangerous and costly complication of broad-spectrum antibiotic therapy1,2. How antibiotic-mediated elimination of commensal bacteria promotes infection by antibiotic-resistant bacteria is a fertile area for speculation with few defined mechanisms. Here we demonstrate that antibiotic treatment of mice notably downregulates intestinal expression of RegIIIγ (also known as Reg3g), a secreted C-type lectin that kills Gram-positive bacteria, including VRE. Downregulation of RegIIIγ markedly decreases in vivo killing of VRE in the intestine of antibiotic-treated mice. Stimulation of intestinal Toll-like receptor 4 by oral administration of lipopolysaccharide re-induces RegIIIγ, thereby boosting innate immune resistance of antibiotic-treated mice against VRE. Compromised mucosal innate immune defence, as induced by broad-spectrum antibiotic therapy, can be corrected by selectively stimulating mucosal epithelial Toll-like receptors, providing a potential therapeutic approach to reduce colonization and infection by antibiotic-resistant microbes.
doi:10.1038/nature07250
PMCID: PMC2663337  PMID: 18724361

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