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1.  A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma 
Investigational new drugs  2013;32(1):178-187.
Background
DNA aptamers represent a novel strategy in anti-cancer medicine. These compounds are short sequences of DNA that have protein binding effects via shape specific recognition of a target protein in an interaction which is analogous to antibody-antigen binding. AS1411, a DNA aptamer that targets nucleolin (a protein which is overexpressed in many tumor types), was evaluated in patients with metastatic, predominantly clear-cell, renal cell carcinoma (RCC) who had failed treatment with ≥1 previous tyrosine kinase inhibitor. We present the first manuscript reporting the use of this novel anti-cancer agent in humans.
Methods
In this phase II, single-arm study, AS1411 was administered at 40 mg/kg/day by continuous intravenous infusion on days 1–4 of a 28-day cycle, for two cycles. Primary endpoint was overall response rate; progression-free survival (PFS) and safety were secondary endpoints.
Results
35 patients were enrolled and treated; 33 completed two treatment cycles. Median number of prior therapies was 2 (range 1–7). One patient (2.9%) had a response to treatment. The response was dramatic (84% reduction in the sum of longest diameters of selected target tumor lesions) and durable (the patient remains free of progression 2 years after completing therapy). No responses were seen in the other patients. Median PFS was 4 months. Only 34% of patients had an AS1411-related adverse event, all of which were mild or moderate.
Conclusions
AS1411 appears to have limited activity in unselected patients with metastatic RCC. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with nucleolin targeted compounds may benefit from efforts to discover predictive biomarkers of response. Currently, promising pre-clinical studies are ongoing using AS1411 conjugated to traditional cytotoxic agents to selectively deliver these treatments to tumor cells. DNA aptamers represent a novel way to target cancer cells at a molecular level and continue to be developed with a view to improving treatment and imaging in cancer medicine.
doi:10.1007/s10637-013-0045-6
PMCID: PMC4560460  PMID: 24242861
renal cell carcinoma; novel therapeutics; DNA aptamer; nucleotide aptamer; Bcl2
2.  Somatic ERCC2 mutations correlate with cisplatin sensitivity in muscle-invasive urothelial carcinoma 
Cancer discovery  2014;4(10):1140-1153.
Cisplatin-based chemotherapy is the standard of care for patients with muscle invasive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant cisplatin-based chemotherapy is associated with improved survival, although molecular determinants of cisplatin response are incompletely understood. We performed whole exome sequencing on pre-treatment tumor and germline DNA from 50 patients with muscle invasive urothelial carcinoma who received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis “responders”, 25 pT2+ “non-responders”) to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with non-responders (q < 0.01). Expression of representative ERCC2 mutations in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared to wild-type ERCC2. Lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle invasive urothelial carcinoma.
doi:10.1158/2159-8290.CD-14-0623
PMCID: PMC4238969  PMID: 25096233
ERCC2; cisplatin sensitivity; urothelial carcinoma; exceptional responders; nucleotide excision repair defect
3.  Genomic Predictors of Survival in Patients with High-Grade Urothelial Carcinoma of the Bladder 
European urology  2014;67(2):198-201.
Urothelial carcinoma of the bladder (UCB) is genomically heterogeneous, with frequent alterations in genes regulating chromatin state, cell cycle control, and receptor kinase signaling. To identify prognostic genomic markers in high-grade UCB, we utilized capture-based massively-parallel sequencing to analyze 109 tumors.
Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases. The presence of a recurrent PIK3CA mutation was associated with improved recurrence-free survival (RFS; HR=0.35, p=0.014) and cancer-specific survival (CSS; HR=0.35, p=0.040) in patients treated with radical cystectomy. In multivariable analyses controlling for pT and pN stages, PIK3CA mutation remained associated with RFS (HR=0.39, p=0.032). The most frequent alteration, TP53 mutation (57%), was more common in extravesical (69% vs. 32%, p=0.005) and lymph node-positive (77% vs. 56%, p=0.025) disease. Patients with CDKN2A altered tumors experienced worse RFS (HR=5.76, p<0.001) and CSS (HR=2.94, p=0.029) in multivariable analyses. Mutations in chromatin modifying genes were highly prevalent but not associated with outcomes.
In UCB patients treated with radical cystectomy, PIK3CA mutations are associated with favorable outcomes whereas TP53 and CDKN2A alterations are associated with poor outcomes. Genomic profiling may aid in the identification of UCB patients at highest risk following radical cystectomy.
doi:10.1016/j.eururo.2014.06.050
PMCID: PMC4312739  PMID: 25092538
bladder cancer; genomics; clinical outcomes; PIK3CA; mutation
4.  Update in Urothelial Carcinoma: Novel Agents and Targeted Therapy 
Urothelial carcinoma (UC) is a chemosensitive disease with high response rates to platinum-based combination chemotherapy in locally advanced or advanced disease. However, de novo or emergence of cisplatin-resistance limits the duration of response, patients are frequently ineligible for cisplatin, and therapies tested thus far have minimal activity as second-line therapy. The first wave of clinical trials of novel agents and targeted therapy have modestly advanced the field and laid the foundations for future studies. These trials include the deployment of monoclonal antibodies and tyrosine kinase inhibitors that target mediators of angiogenesis and growth receptors. Novel cytotoxic agents have also been tested as single-agents in the second-line setting and together with the first-line combination of gemcitabine with cisplatin. To date, these novel agents have yet to demonstrate the ability to substantially improve the overall survival of patients with bladder cancer. Comparative trials of chemotherapy with or without a novel agent are ongoing and have the potential to improve upon current standard therapy. Moreover, state-of-the-art technologies have been developed that will likely identify the molecular alterations which drive both UC and platinum-resistance and in turn provide opportunities for drug development. The latter includes an interrogation of microRNAs and the integrated study of genetic mutations in extreme phenotypes of the disease. In essence, this ongoing work paired with physician and patient commitments to clinical trial participation will ultimately lead to advances in the care of patients with urothelial cancer.
PMCID: PMC4587660  PMID: 26430392
5.  Phase II and Biomarker Study of the Dual MET/VEGFR2 Inhibitor Foretinib in Patients With Papillary Renal Cell Carcinoma 
Journal of Clinical Oncology  2012;31(2):181-186.
Purpose
Foretinib is an oral multikinase inhibitor targeting MET, VEGF, RON, AXL, and TIE-2 receptors. Activating mutations or amplifications in MET have been described in patients with papillary renal cell carcinoma (PRCC). We aimed to evaluate the efficacy and safety of foretinib in patients with PRCC.
Patients and Methods
Patients were enrolled onto the study in two cohorts with different dosing schedules of foretinib: cohort A, 240 mg once per day on days 1 through 5 every 14 days (intermittent arm); cohort B, 80 mg daily (daily dosing arm). Patients were stratified on the basis of MET pathway activation (germline or somatic MET mutation, MET [7q31] amplification, or gain of chromosome 7). The primary end point was overall response rate (ORR).
Results
Overall, 74 patients were enrolled, with 37 in each dosing cohort. ORR by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 was 13.5%, median progression-free survival was 9.3 months, and median overall survival was not reached. The presence of a germline MET mutation was highly predictive of a response (five of 10 v five of 57 patients with and without germline MET mutations, respectively). The most frequent adverse events of any grade associated with foretinib were fatigue, hypertension, gastrointestinal toxicities, and nonfatal pulmonary emboli.
Conclusion
Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations.
doi:10.1200/JCO.2012.43.3383
PMCID: PMC3532390  PMID: 23213094
6.  The role of aberrant VHL/HIF pathway elements in predicting clinical outcome to pazopanib therapy in patients with metastatic clear-cell renal cell carcinoma 
Purpose
Inactivation of von Hippel-Lindau (VHL) gene in clear-cell renal cell carcinoma (RCC) leads to increased levels of hypoxia-inducible factors (HIFs) and overexpression of HIF target genes, such as vascular endothelial growth factor (VEGF) and others. VEGF-targeted agents are standard in advanced clear-cell RCC but biomarkers of activity are lacking.
Patients and Methods
We analyzed tumor tissue samples from metastatic clear-cell RCC patients who received pazopanib as part of clinical trial VEG102616. We evaluated several components of the VHL/HIF pathway: VHL gene inactivation (mutation and/or methylation), HIF1α and HIF2α immunohistochemistry staining, and HIF1α transcriptional signature. We evaluated the association of these biomarkers with best overall response rate and progression-free survival to pazopanib, a standard first-line VEGF-targeted agent.
Results
The VEG102616 trial enrolled 225 patients, from whom 78 samples were available for tumor DNA extraction. Of these, 70 patients had VHL mutation or methylation. VHL gene status did not correlate with overall response rate or progression-free survival. Similarly, HIF1α (65 samples) and HIF2α (66 samples) protein levels (high vs. low) did not correlate with overall response rate or progression-free survival to pazopanib. The HIF1α transcriptional signature (46 samples) was enriched in tumors expressing high HIF1α levels. However, the HIF1α gene expression signature was not associated with clinical outcome to pazopanib.
Conclusion
In patients with advanced clear-cell RCC, several potential biomarkers along the VHL/HIF1α/HIF2α axis were not found to be predictive for pazopanib activity. Additional efforts must continue to identify biomarkers associated with clinical outcome to VEGF-targeted agents in metastatic RCC.
doi:10.1158/1078-0432.CCR-13-0491
PMCID: PMC4522695  PMID: 23881929
renal cell carcinoma; VEGF; HIF; VHL; biomarkers; pazopanib
7.  Double-Blind, Randomized Trial of Docetaxel Plus Vandetanib Versus Docetaxel Plus Placebo in Platinum-Pretreated Metastatic Urothelial Cancer 
Journal of Clinical Oncology  2011;30(5):507-512.
Purpose
Vandetanib is an oral once-daily tyrosine kinase inhibitor with activity against vascular endothelial growth factor receptor 2 and epidermal growth factor receptor. Vandetanib in combination with docetaxel was assessed in patients with advanced urothelial cancer (UC) who progressed on prior platinum-based chemotherapy.
Patients and Methods
The primary objective was to determine whether vandetanib 100 mg plus docetaxel 75 mg/m2 intravenously every 21 days prolonged progression-free survival (PFS) versus placebo plus docetaxel. The study was designed to detect a 60% improvement in median PFS with 80% power and one-sided α at 5%. Patients receiving docetaxel plus placebo had the option to cross over to single-agent vandetanib at progression. Overall survival (OS), overall response rate (ORR), and safety were secondary objectives.
Results
In all, 142 patients were randomly assigned and received at least one dose of therapy. Median PFS was 2.56 months for the docetaxel plus vandetanib arm versus 1.58 months for the docetaxel plus placebo arm, and the hazard ratio for PFS was 1.02 (95% CI, 0.69 to 1.49; P = .9). ORR and OS were not different between both arms. Grade 3 or higher toxicities were more commonly seen in the docetaxel plus vandetanib arm and included rash/photosensitivity (11% v 0%) and diarrhea (7% v 0%). Among 37 patients who crossed over to single-agent vandetanib, ORR was 3% and OS was 5.2 months.
Conclusion
In this platinum-pretreated population of advanced UC, the addition of vandetanib to docetaxel did not result in a significant improvement in PFS, ORR, or OS. The toxicity of vandetanib plus docetaxel was greater than that for vendetanib plus placebo. Single-agent vandetanib activity was minimal.
doi:10.1200/JCO.2011.37.7002
PMCID: PMC4104290  PMID: 22184381
8.  DNA copy number analysis of metastatic urothelial carcinoma with comparison to primary tumors 
BMC Cancer  2015;15:242.
Background
To date, there have been no reports characterizing the genome-wide somatic DNA chromosomal copy-number alteration landscape in metastatic urothelial carcinoma. We sought to characterize the DNA copy-number profile in a cohort of metastatic samples and compare them to a cohort of primary urothelial carcinoma samples in order to identify changes that are associated with progression from primary to metastatic disease.
Methods
Using molecular inversion probe array analysis we compared genome-wide chromosomal copy-number alterations between 30 metastatic and 29 primary UC samples. Whole transcriptome RNA-Seq analysis was also performed in primary and matched metastatic samples which was available for 9 patients.
Results
Based on a focused analysis of 32 genes in which alterations may be clinically actionable, there were significantly more amplifications/deletions in metastases (8.6% vs 4.5%, p < 0.001). In particular, there was a higher frequency of E2F3 amplification in metastases (30% vs 7%, p = 0.046). Paired primary and metastatic tissue was available for 11 patients and 3 of these had amplifications of potential clinical relevance in metastases that were not in the primary tumor including ERBB2, CDK4, CCND1, E2F3, and AKT1. The transcriptional activity of these amplifications was supported by RNA expression data.
Conclusions
The discordance in alterations between primary and metastatic tissue may be of clinical relevance in the era of genomically directed precision cancer medicine.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1192-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1192-2
PMCID: PMC4392457  PMID: 25886454
9.  Six-Month Progression-Free Survival as the Primary Endpoint to Evaluate the Activity of New Agents as Second-line Therapy for Advanced Urothelial Carcinoma 
Clinical genitourinary cancer  2013;12(2):130-137.
This study examined the association of progression-free survival at 6 months with overall survival in the context of second-line therapy of advanced urothelial carcinoma in pooled patient-level data from 10 phase II trials and then externally validated in a large phase III trial. Progression-free survival at 6 months was significantly correlated with overall survival and is an innovative primary endpoint to evaluate new agents in this setting.
Objective
Second-line systemic therapy for advanced urothelial carcinoma (UC) has substantial unmet needs, and current agents show dismal activity. Second-line trials of metastatic UC have used response rate (RR) and median progression-free survival (PFS) as primary endpoints, which may not reflect durable benefits. A more robust endpoint to identify signals of durable benefits when investigating new agents in second-line trials may expedite drug development. PFS at 6 months (PFS6) is a candidate endpoint, which may correlate with overall survival (OS) at 12 months (OS12) and may be applicable across cytostatic and cytotoxic agents.
Methods
Ten second-line phase II trials with individual patient outcomes data evaluating chemotherapy or biologics were combined for discovery, followed by external validation in a phase III trial. The relationship between PFS6/RR and OS12 was assessed at the trial level using Pearson correlation and weighted linear regression, and at the individual level using Pearson chi-square test with Yates continuity correction.
Results
In the discovery dataset, a significant correlation was observed between PFS6 and OS12 at the trial (R2 = 0.55, Pearson correlation = 0.66) and individual levels (82%, Қ = 0.45). Response correlated with OS12 at the individual level less robustly (78%, Қ = 0.36), and the trial level association was not statistically significant (R2 = 0.16, Pearson correlation = 0.37). The correlation of PFS6 (81%, Қ = 0.44) appeared
doi:10.1016/j.clgc.2013.09.002
PMCID: PMC4142680  PMID: 24220220
Advanced urothelial carcinoma; Intermediate endpoint; Overall survival; Progression-free survival at 6 months; Second-line treatment
10.  Synthetic lethality in ATM-deficient RAD50-mutant tumors underlie outlier response to cancer therapy 
Cancer discovery  2014;4(9):1014-1021.
Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small cell cancer to combined checkpoint kinase 1 (Chk1) inhibition and DNA damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of Chk1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy whereby checkpoint inhibition in combination with DNA damaging chemotherapy is synthetically lethal in tumor but not normal cells with somatic mutations that impair Mre11 complex function.
doi:10.1158/2159-8290.CD-14-0380
PMCID: PMC4155059  PMID: 24934408
DNA damage and repair; cancer genomics; exceptional responders; targeted and systemic therapy; RAD50
11.  Phase I Study of Ixabepilone, Mitoxantrone, and Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer Previously Treated With Docetaxel-Based Therapy: A Study of the Department of Defense Prostate Cancer Clinical Trials Consortium 
Journal of Clinical Oncology  2009;27(17):2772-2778.
Purpose
Mitoxantrone plus prednisone and ixabepilone each have modest activity as second-line chemotherapy in docetaxel-refractory castration-resistant prostate cancer (CRPC) patients. Clinical noncrossresistance was previously observed.
Patients and Methods
Metastatic CRPC patients progressing during or after taxane-based chemotherapy enrolled in a phase I multicenter study of ixabepilone and mitoxantrone administered every 21 days along with prednisone. Ixabepilone and mitoxantrone doses were alternately escalated in a standard 3 + 3 design. Patients were evaluated for toxicity and disease response. Dose-limiting toxicities (DLTs) were defined as treatment related, occurring during cycle 1, and included grade 4 prolonged or febrile neutropenia, thrombocytopenia (grade 4 or grade 3 with bleeding), or ≥ grade 3 nonhematologic toxicity.
Results
Thirty-six patients were treated; 59% of patients experienced grade 3/4 neutropenia. DLTs included grade 3 diarrhea (n = 1), prolonged grade 4 neutropenia (n = 4), and grade 5 neutropenic infection (n = 1). Due to prolonged neutropenia, the highest dose levels were repeated with pegfilgrastim on day 2 of each cycle. The maximum tolerated dose in combination with pegfilgrastim was not exceeded. The recommended phase II dose is mitoxantrone 12 mg/m2 and ixabepilone 35 mg/m2 every 21 days, pegfilgrastim 6 mg subcutaneously day 2, and continuous prednisone 5 mg twice per day. Thirty-one percent of patients have experienced ≥ 50% prostate-specific antigen (PSA) declines, and two experienced objective responses. Of 21 patients treated with mitoxantrone 12 mg/m2 plus ixabepilone ≥ 30 mg/m2, nine (43%) experienced ≥ 50% PSA declines (95% CI, 22% to 66%).
Conclusion
These results suggest that the combination of ixabepilone and mitoxantrone is feasible and active in CRPC and requires dosing with pegfilgrastim.
doi:10.1200/JCO.2008.19.8002
PMCID: PMC2698016  PMID: 19349545
12.  Activating mTOR mutations in a patient with an extraordinary response on a phase I trial of everolimus and pazopanib 
Cancer discovery  2014;4(5):546-553.
Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole exome sequencing of a patient with a 14-month complete response on this trial revealed two simultaneous mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biological mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or “personalized”) medicine approaches to screen cancer patients for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR.
doi:10.1158/2159-8290.CD-13-0353
PMCID: PMC4122326  PMID: 24625776
13.  Integrative analysis of 1q23.3 copy number gain in metastatic urothelial carcinoma 
Purpose
Metastatic urothelial carcinoma (UC) of the bladder is associated with multiple somatic copy number alterations (SCNAs). We evaluated SCNAs to identify predictors of poor survival in patients with metastatic UC treated with platinum-based chemotherapy.
Experimental Design
We obtained overall survival (OS) and array DNA copy number data from metastatic UC patients in two cohorts. Associations between recurrent SCNAs and OS were determined by a Cox proportional hazard model adjusting for performance status and visceral disease. mRNA expression was evaluated for potential candidate genes by Nanostring nCounter to identify transcripts from the region that are associated with copy number gain. In addition, expression data from an independent cohort was used to identify candidate genes.
Results
Multiple areas of recurrent significant gains and losses were identified. Gain of 1q23.3 was independently associated with a shortened OS in the both cohorts (adjusted HR 2.96; 95% CI, 1.35 to 6.48; P = 0.01 and adjusted HR 5.03; 95% CI 1.43-17.73; P < 0.001). The F11R, PFDN2, PPOX, USP21 and DEDD genes, all located on 1q23.3, were closely associated with poor outcome.
Conclusions
1q23.3 copy number gain displayed association with poor survival in two cohorts of metastatic UC. The identification of the target of this copy number gain is ongoing, and exploration of this finding in other disease states may be useful for the early identification of poor risk UC patients. Prospective validation of the survival association is necessary to demonstrate clinical relevance.
doi:10.1158/1078-0432.CCR-13-0759
PMCID: PMC3975677  PMID: 24486590
14.  Time from Prior Chemotherapy Enhances Prognostic Risk Grouping in the Second-line Setting of Advanced Urothelial Carcinoma: A Retrospective Analysis of Pooled, Prospective Phase 2 Trials 
European urology  2012;63(4):717-723.
Background
Outcomes for patients in the second-line setting of advanced urothelial carcinoma (UC) are dismal. The recognized prognostic factors in this context are Eastern Cooperative Oncology Group (ECOG) performance status (PS) >0, hemoglobin level (Hb) <10 g/dl, and liver metastasis (LM).
Objectives
The purpose of this retrospective study of prospective trials was to investigate the prognostic value of time from prior chemotherapy (TFPC) independent of known prognostic factors. Design, setting, and participants: Data from patients from seven prospective trials with available baseline TFPC, Hb, PS, and LM values were used for retrospective analysis (n = 570). External validation was conducted in a second-line phase 3 trial comparing best supportive care (BSC) versus vinflunine plus BSC (n = 352).
Outcome measurements and statistical analysis
Cox proportional hazards regression was used to evaluate the association of factors, with overall survival (OS) and progression-free survival (PFS) being the respective primary and secondary outcome measures.
Results and limitations
ECOG-PS >0, LM, Hb <10 g/dl, and shorter TFPC were significant prognostic factors for OS and PFS on multivariable analysis. Patients with zero, one, two, and three to four factors demonstrated median OS of 12.2, 6.7, 5.1, and 3.0 mo, respectively (concordance statistic = 0.638). Setting of prior chemotherapy (metastatic disease vs perioperative) and prior platinum agent (cisplatin or carboplatin) were not prognostic factors. External validation demonstrated a significant association of TFPC with PFS on univariable and most multivariable analyses, and with OS on univariable analyses. Limitations of retrospective analyses are applicable.
Conclusions
Shorter TFPC enhances prognostic classification independent of ECOG-PS>0, Hb<10 g/ dl, and LM in the setting of second-line therapy for advanced UC. These data may facilitate drug development and interpretation of trials.
doi:10.1016/j.eururo.2012.11.042
PMCID: PMC4127896  PMID: 23206856
Urothelial carcinoma; Second line; Prognosis; Time from prior chemotherapy; Hemoglobin; Liver metastasis; Performance status
15.  Identification of ALK Gene Alterations in Urothelial Carcinoma 
PLoS ONE  2014;9(8):e103325.
Background
Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome.
Methods
Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case.
Results
70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5′ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease.
Conclusions
ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.
doi:10.1371/journal.pone.0103325
PMCID: PMC4118868  PMID: 25083769
16.  FGFR3 expression in primary and metastatic urothelial carcinoma of the bladder 
Cancer Medicine  2014;3(4):835-844.
While fibroblast growth factor receptor 3 (FGFR3) is frequently mutated or overexpressed in nonmuscle-invasive urothelial carcinoma (UC), the prevalence of FGFR3 protein expression and mutation remains unknown in muscle-invasive disease. FGFR3 protein and mRNA expression, mutational status, and copy number variation were retrospectively analyzed in 231 patients with formalin-fixed paraffin-embedded primary UCs, 33 metastases, and 14 paired primary and metastatic tumors using the following methods: immunohistochemistry, NanoString nCounterTM, OncoMap or Affymetrix OncoScanTM array, and Gain and Loss of Analysis of DNA and Genomic Identification of Significant Targets in Cancer software. FGFR3 immunohistochemistry staining was present in 29% of primary UCs and 49% of metastases and did not impact overall survival (P = 0.89, primary tumors; P = 0.78, metastases). FGFR3 mutations were observed in 2% of primary tumors and 9% of metastases. Mutant tumors expressed higher levels of FGFR3 mRNA than wild-type tumors (P < 0.001). FGFR3 copy number gain and loss were rare events in primary and metastatic tumors (0.8% each; 3.0% and 12.3%, respectively). FGFR3 immunohistochemistry staining is present in one third of primary muscle-invasive UCs and half of metastases, while FGFR3 mutations and copy number changes are relatively uncommon.
doi:10.1002/cam4.262
PMCID: PMC4303151  PMID: 24846059
Biomarker; bladder cancer; FGFR3; metastatic urothelial carcinoma; muscle-invasive urothelial carcinoma; targeted therapy
17.  Prognostic Model for Predicting Survival of Patients With Metastatic Urothelial Cancer Treated With Cisplatin-Based Chemotherapy 
A prognostic model that predicts overall survival (OS) for metastatic urothelial cancer (MetUC) patients treated with cisplatin-based chemotherapy was developed, validated, and compared with a commonly used Memorial Sloan-Kettering Cancer Center (MSKCC) risk-score model. Data from 7 protocols that enrolled 308 patients with MetUC were pooled. An external multi-institutional dataset was used to validate the model. The primary measurement of predictive discrimination was Harrell’s c-index, computed with 95% confidence interval (CI). The final model included four pretreatment variables to predict OS: visceral metastases, albumin, performance status, and hemoglobin. The Harrell’s c-index was 0.67 for the four-variable model and 0.64 for the MSKCC risk-score model, with a prediction improvement for OS (the U statistic and its standard deviation were used to calculate the two-sided P = .002). In the validation cohort, the c-indices for the four-variable and the MSKCC risk-score models were 0.63 (95% CI = 0.56 to 0.69) and 0.58 (95% CI = 0.52 to 0.65), respectively, with superiority of the four-variable model compared with the MSKCC risk-score model for OS (the U statistic and its standard deviation were used to calculate the two-sided P = .02).
doi:10.1093/jnci/djt015
PMCID: PMC3691944  PMID: 23411591
18.  Prospective Analysis of Genetic Polymorphisms and Risk of Recurrence in Renal Cell Cancer 
The lancet oncology  2012;14(1):81-87.
Summary
Background
Germline genetic polymorphisms may affect the risk of recurrence in patients with localized renal cell carcinoma (RCC). Our aim was to investigate the association of genetic polymorphisms with RCC recurrence.
Patients and Methods
We analyzed germline DNA samples extracted from 554 (discovery cohort of 403 and an independent validation cohort of 151) patients with localized RCC treated at Dana-Farber/Harvard Cancer Center (DF/HCC) and of European-American ancestry (Caucasians). The discovery cohort was selected from a prospective database at Dana-Farber/Harvard Cancer Center and the validation cohort was identified from the Brigham and Women’s Hospital surgery and pathology department records. Single nucleotide polymorphims (SNPs) residing in 70 genes involved in RCC pathogenesis including the VHL/HIF/VEGF, PI3K/AKT/mTOR pathways, and genes involved in immune regulation and metabolism were genotyped for the discovery cohort (total 285 SNPs successfully genotyped and assessable for analysis). The analyses of genotype associations with recurrence free survival (RFS) were assessed using Cox proportional hazards model, Kaplan-Meier method and logrank test. False discovery rate (FDR) q-value was used to adjust for multiple comparisons in selecting potential SNPs with RFS association. The finding from the discovery cohort was validated in an external independent cohort.
Findings
We report the significant association between genotype variants of SNP rs11762213 (c.144G>A; p.Ala48Ala, located in exon two c-MET) and primary analysis endpoint of RFS using both univariate and multivariable analysis. Specifically, patients carrying one or two copies of the minor (risk) allele had an increased risk of recurrence or death (hazard ratio (HR) =1·86, 95% confidence interval (CI), 1·17,2·95; p=0·0084) in the multivariate analysis adjusted for clinical and pathological factors. The median RFS for carriers of the risk allele was 19 months (95%CI: 9,*) compared to 50 months (95%CI: 37,75) for homozygotes of the non-risk allele. The significant association was validated using data from the validation cohort with a HR of 2·45 (95%CI: 1·01,5·95; p=0·048), although of borderline significance. The rs11762213 results in a synonymous aminoacid change in cMET gene. * unable to estimate due to small sample.
Interpretation
Patients with localized RCC and c-MET polymorphism (rs11762213) may have an increased risk of recurrence after nephrectomy. If these results are further validated, it may be incorporated in future prognostic tools, potentially aiding in the design of adjuvant clinical trials with c-MET inhibitors, and clinical management.
Funding
This project is funded by the Conquer Cancer Foundation and ASCO under a Career Development Award (CDA) for Dr. Choueiri, The Trust Family Research for Kidney cancer for Dr. Choueiri and the NIH/NCI Kidney cancer SPORE.
doi:10.1016/S1470-2045(12)70517-X
PMCID: PMC3769687  PMID: 23219378
localized renal cell cancer; nephrectomy; recurrence free interval; genetic polymorphisms; single nucleotide polymorphisms; MET; VEGF
19.  Loss of Sh3gl2/Endophilin A1 Is a Common Event in Urothelial Carcinoma that Promotes Malignant Behavior12 
Neoplasia (New York, N.Y.)  2013;15(7):749-760.
Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis, we identified Sh3gl2 (endophilin A1) as a bladder urothelium-enriched transcript. The gene encoding Sh3gl2 is located on chromosome 9p, a region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to nontumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, which is a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 19 of 20 human UC cell lines but preserved in the low-grade cell line RT4. Stable silencing of Sh3gl2 in RT4 cells by RNA interference 1) enhanced proliferation and colony formation in vitro, 2) inhibited EGF-induced EGFR internalization and increased EGFR activation, 3) stimulated phosphorylation of Src family kinases and STAT3, and 4) promoted growth of RT4 xenografts in subrenal capsule tissue recombination experiments. Conversely, forced re-expression of Sh3gl2 in T24 cells and silenced RT4 clones attenuated oncogenic behaviors, including growth and migration. Together, these findings identify loss of Sh3gl2 as a frequent event in UC development that promotes disease progression.
PMCID: PMC3689238  PMID: 23814487
20.  Combination of a novel gene expression signature with a clinical nomogram improves the prediction of survival in high-risk bladder cancer 
Purpose
We aimed to validate and improve prognostic signatures for high-risk urothelial carcinoma of the bladder.
Experimental Design
We evaluated microarray data from 93 bladder cancer patients managed by radical cystectomy to determine gene expression patterns associated with clinical and prognostic variables. We compared our results with published bladder cancer microarray datasets comprising 578 additional patients, and with 49 published gene signatures from multiple cancer types. Hierarchical clustering was utilized to identify subtypes associated with differences in survival. We then investigated whether the addition of survival-associated gene expression information to a validated post-cystectomy nomogram utilizing clinical and pathologic variables improves prediction of recurrence.
Results
Multiple markers for muscle invasive disease with highly significant expression differences in multiple datasets were identified, such as FN1, NNMT, POSTN and SMAD6. We identified signatures associated with pathologic stage and the likelihood of developing metastasis and death from bladder cancer, as well as with two distinct clustering subtypes of bladder cancer. Our novel signature correlated with overall survival in multiple independent datasets, significantly improving the prediction concordance of standard staging in all datasets (mean ΔC-statistic: 0.14, 95% CI 0.01–0.27; P < 0.001). Tested in our patient cohort, it significantly enhanced the performance of a postoperative survival nomogram (ΔC-statistic: 0.08, 95% CI −0.04–0.20; P < 0.005).
Conclusions
Prognostic information obtained from gene expression data can aid in post-treatment prediction of bladder cancer recurrence. Our findings require further validation in external cohorts and prospectively in a clinical trial setting.
doi:10.1158/1078-0432.CCR-11-2271
PMCID: PMC3569085  PMID: 22228636
Bladder cancer; gene expression analysis; molecular subtypes; survival analysis; bioinformatics
21.  Advanced Urothelial Carcinoma: Overcoming Treatment Resistance through Novel Treatment Approaches 
The current standard of care for metastatic urothelial carcinoma is cisplatin-based chemotherapy but treatment is generally not curative. Mechanisms of resistance to conventional cytotoxic regimens include tumor cell drug efflux pumps, intracellular anti-oxidants, and enhanced anti-apoptotic signaling. Blockade of signaling pathways with small molecule tyrosine kinase inhibitors has produced dramatic responses in subsets of other cancers. Multiple potential signaling pathway targets are altered in Urothelial carcinoma (UC). Blockade of the PI3K/Akt/mTOR pathway may prove efficacious because 21% have activating PI3K mutations and another 30% have PTEN inactivation (which leads to activation of this pathway). The fibroblast growth factor receptor 3 protein may be overactive in 50–60% and agents which block this pathway are under development. Blockade of multiple other pathways including HER2 and aurora kinase also have potential efficacy. Anti-angiogenic and immunotherapy strategies are also under development in UC and are discussed in this review. Novel therapeutic approaches are needed in UC. We review the various strategies under investigation and discuss how best to evaluate and optimize their efficacy.
doi:10.3389/fphar.2013.00003
PMCID: PMC3565214  PMID: 23390417
urothelial cancer; bladder cancer; oncogenes; chemotherapy; resistance mechanisms
22.  Personalized therapy for urothelial cancer: review of the clinical evidence 
Clinical investigation  2011;1(4):546-555.
Despite a detailed understanding of the molecular aberrations driving the development of urothelial cancers, this knowledge has not translated into advances for the treatment of this disease. Urothelial cancers are chemosensitive, and platinum-based combination chemotherapy remains the standard of care for advanced disease, as well as neoadjuvant and adjuvant therapy for locally advanced disease. However, nearly half of patients who undergo resection of locally advanced urothelial cancer will relapse and eventually develop platinum-resistant disease. Clinical trials of targeted agents against angiogenesis and growth factors, as well as novel chemotheraputics, have generally been unsuccessful in urothelial cancers. Improvements in the theraputic arsenal for urothelial cancer depend upon identification of new targets and strategies to overcome platinum resistance.
doi:10.4155/cli.11.26
PMCID: PMC3384687  PMID: 22754656
23.  Abiraterone acetate: oral androgen biosynthesis inhibitor for treatment of castration-resistant prostate cancer 
Prostate cancer is the second leading cause of cancer death in men in the US and Europe. The treatment of advanced-stage prostate cancer has been androgen deprivation. Medical castration leads to decreased production of testosterone and dihydrotestosterone by the testes, but adrenal glands and even prostate cancer tissue continue to produce androgens, which eventually leads to continued prostate cancer growth despite castrate level of androgens. This stage is known as castrate-resistant prostate cancer (CRPC), which continues to be a challenge to treat. Addition of androgen antagonists to hormonal deprivation has been successful in lowering the prostate-specific antigen levels further, but has not actually translated into life-prolonging options. The results of several contemporary studies have continued to demonstrate activation of the androgen receptor as being the key factor in the continued growth of prostate cancer. Blockade of androgen production by nongonadal sources has led to clinical benefit in this setting. One such agent is abiraterone acetate, which significantly reduces androgen production by blocking the enzyme, cytochrome P450 17 alpha-hydroxylase (CYP17). This has provided physicians with another treatment option for patients with CRPC. The landscape for prostate cancer treatment has changed with the approval of cabazitaxel, sipuleucel-T and abiraterone. Here we provide an overview of abiraterone acetate, its mechanism of action, and its potential place for therapy in CRPC.
doi:10.2147/DDDT.S15850
PMCID: PMC3267518  PMID: 22291466
CRPC; abiraterone; CYP17; inhibitors; androgens; castration resistant prostate cancer
24.  Phase III Trial of Bevacizumab Plus Interferon Alfa Versus Interferon Alfa Monotherapy in Patients With Metastatic Renal Cell Carcinoma: Final Results of CALGB 90206 
Journal of Clinical Oncology  2010;28(13):2137-2143.
Purpose
Bevacizumab is an antibody that binds vascular endothelial growth factor and has activity in metastatic renal cell carcinoma (RCC). Interferon alfa (IFN-α) is the historic standard initial treatment for RCC. A prospective, randomized, phase III trial of bevacizumab plus IFN-α versus IFN-α monotherapy was conducted.
Patients and Methods
Patients with previously untreated, metastatic clear cell RCC were randomly assigned to receive either bevacizumab (10 mg/kg intravenously every 2 weeks) plus IFN-α (9 million units subcutaneously three times weekly) or the same dose and schedule of IFN-α monotherapy in a multicenter phase III trial. The primary end point was overall survival (OS). Secondary end points were progression-free survival (PFS), objective response rate, and safety.
Results
Seven hundred thirty-two patients were enrolled. The median OS time was 18.3 months (95% CI, 16.5 to 22.5 months) for bevacizumab plus IFN-α and 17.4 months (95% CI, 14.4 to 20.0 months) for IFN-α monotherapy (unstratified log-rank P = .097). Adjusting on stratification factors, the hazard ratio was 0.86 (95% CI, 0.73 to 1.01; stratified log-rank P = .069) favoring bevacizumab plus IFN-α. There was significantly more grade 3 to 4 hypertension (HTN), anorexia, fatigue, and proteinuria for bevacizumab plus IFN-α. Patients who developed HTN on bevacizumab plus IFN-α had a significantly improved PFS and OS versus patients without HTN.
Conclusion
OS favored the bevacizumab plus IFN-α arm but did not meet the predefined criteria for significance. HTN may be a biomarker of outcome with bevacizumab plus IFN-α.
doi:10.1200/JCO.2009.26.5561
PMCID: PMC2860433  PMID: 20368558
25.  Phase I Clinical Trial of the CYP17 Inhibitor Abiraterone Acetate Demonstrating Clinical Activity in Patients With Castration-Resistant Prostate Cancer Who Received Prior Ketoconazole Therapy 
Journal of Clinical Oncology  2010;28(9):1481-1488.
Purpose
Abiraterone acetate is a prodrug of abiraterone, a selective inhibitor of CYP17, the enzyme catalyst for two essential steps in androgen biosynthesis. In castration-resistant prostate cancers (CRPCs), extragonadal androgen sources may sustain tumor growth despite a castrate environment. This phase I dose-escalation study of abiraterone acetate evaluated safety, pharmacokinetics, and effects on steroidogenesis and prostate-specific antigen (PSA) levels in men with CPRC with or without prior ketoconazole therapy.
Patients and Methods
Thirty-three men with chemotherapy-naïve progressive CRPC were enrolled. Nineteen patients (58%) had previously received ketoconazole for CRPC. Bone metastases were present in 70% of patients, and visceral involvement was present in 18%. Three patients (9%) had locally advanced disease without distant metastases. Fasted or fed cohorts received abiraterone acetate doses of 250, 500, 750, or 1,000 mg daily. Single-dose pharmacokinetic analyses were performed before continuous daily dosing.
Results
Adverse events were predominantly grade 1 or 2. No dose-limiting toxicities were observed. Hypertension (grade 3, 12%) and hypokalemia (grade 3, 6%; grade 4, 3%) were the most frequent serious toxicities and responded to medical management. Confirmed ≥ 50% PSA declines at week 12 were seen in 18 (55%) of 33 patients, including nine (47%) of 19 patients with prior ketoconazole therapy and nine (64%) of 14 patients without prior ketoconazole therapy. Substantial declines in circulating androgens and increases in mineralocorticoids were seen with all doses.
Conclusion
Abiraterone acetate was well tolerated and demonstrated activity in CRPC, including in patients previously treated with ketoconazole. Continued clinical study is warranted.
doi:10.1200/JCO.2009.24.1281
PMCID: PMC2849769  PMID: 20159824

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