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The degree of metastatic outspread in malignant disease is one of the leading factors in determining the appropriate course treatment. Circulating tumor cells (CTCs) represent the population of cells that have acquired the means to gain access to the circulatory system, and the cell population ultimately responsible for the development of metastases at distant sites in the body. While promising as a biomarker for metastatic disease, the widespread study of CTCs has been limited by their rarity, as CTCs are reported to occur as infrequently as 1/mL of whole blood. In this text, we will discuss current and emerging technologies for the size-based enrichment of CTCs from whole blood, and compare some of the advantages and disadvantages of using a size-based approach to CTC enrichment versus affinity-based CTC enrichment platforms.

on mRNA levels, suggesting that androgen-mediated increase in EGFR and ERBB2 mRNAs requires protein neosynthesis. DHT also showed little effects on protein degradation of EGFR and ERBB2 induced by cycloheximide. Furthermore, DHT additionally up-regulated the levels of phosphorylation of EGFR (pEGFR) and its downstream AKT (pAKT) and ERK1/2 (pERK), induced by EGF treatment, in UMUC3 and 5637-AR cells. Immunohistochemical studies on 24 radical cystectomy specimens showed strong associations between expressions of AR and EGFR (p 0.0272), pEGFR (p 0.0064), ERBB2 (p 0.0538), or pERK (p 0.0335), but not pAKT (p 1.0000). KaplanMeier and log-rank tests further revealed that positivity of AR (p 0.0005), EGFR (p 0.2425), pEGFR (p 0.1579), ERBB2 (p 0.2997), or pERK (p 0.1270) and negativity of pAKT (p 0.0483) were associated with tumor progression. CONCLUSIONS: Our results suggest that androgen up-regulates the levels of EGFR and ERBB2 expression, as well as the activity of EGFR signaling, via the AR pathway in bladder cancer cells. Thus, AR signals may play an important role in the regulation of the EGFR/ ERBB2 pathways, leading to the progression of bladder cancer.

Purpose: Sensitive detection and characterization of circulating tumor cells (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. Fifty-nine model system samples were tested to determine 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 five are seeded in 7.5 mL of blood. CTCs were identified in 51 of 57 patients using the microdevice, compared with only 26 patients with the CellSearch method. When CTCs were detected by both methods, greater numbers were recovered by the microfilter device in all but five 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 the clinical setting for the effective management of cancer patients. Clin Cancer Res; 16(20); 5011–8. ©2010 AACR.

Purpose This study sought to determine if alterations in molecular pathways could supplement TNM staging to more accurately predict clinical outcome in patients with urothelial carcinoma (UC). Patients and Methods Expressions of 69 genes involved in known cancer pathways were quantified on bladder specimens from 58 patients with UC (stages Ta-T4) and five normal urothelium controls. All tumor transcript values beyond two standard deviations from the normal mean expression were designated as over- or underexpressed. Univariate and multivariable analyses were conducted to obtain a predictive expression signature. A published external data set was used to confirm the potential of the prognostic gene panels. Results In univariate analysis, six genes were significantly associated with time to recurrence, and 10 with overall survival. Recursive partitioning identified three genes as significant determinants for recurrence, and three for overall survival. Of all genes identified by either univariate or partitioning analysis, four were found to significantly predict both recurrence and survival (JUN, MAP2K6, STAT3, and ICAM1); overexpression was associated with worse outcome. Comparing the favorable (low or normal) expression of ≥ three of four versus ≤ two of four of these oncogenes showed 5-year recurrence probability of 41% versus 88%, respectively (P < .001), and 5-year overall survival probability of 61% versus 5%, respectively (P < .001). The prognostic potential of this four-gene panel was confirmed in a large independent external cohort (disease-specific survival, P = .039). Conclusion We have documented the generation of a concise, biologically relevant four-gene panel that significantly predicts recurrence and survival and may also identify potential therapeutic targets for UC.

Background Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified. Objective To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach. Design, setting, and participants Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence ( n =16), recurrence without progression ( n =16), and progression to carcinoma in situ or invasive disease ( n =16). Measurements Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation. Results and limitations CCND3 ( p =0.003) and HRAS ( p =0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS ( p E2F1 ( p =0.017), BIRC5/Survivin ( p =0.038), and VEGFR2 ( p =0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS , VEGFR2 , and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed. Conclusions Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.

BACKGROUND The concurrent determination of methylation status of E-cadherin gene and E-cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E-cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen. METHODS The methylation of E-cadherin gene was determined by methylation specific-PCR and the protein expression by immunohistochemical method in the consecutive sections of each prostate tissue biopsy specimen. RESULTS The unmethylated E-cadherin gene and homogeneous E-cadherin protein expression was significantly associated with BPH as compared to the primary prostate carcinoma (Fisher's Exact P

We demonstrate two-dimensional multiplexed real-time, label-free antibody-antigen binding assays by optically detecting nanoscale motions of two-dimensional arrays of microcantilever beams. Prostate specific antigen (PSA) was assayed using antibodies covalently bound to one surface of the cantilevers by two different surface chemistries, while the nonreaction surfaces were passivated by poly(ethylene glycol)-silane. PSA as low as 1 ng/mL was detected while 2 mg/microl of bovine serum albumin induced only negligible deflection on the cantilevers.

The receptor tyrosine kinase ErbB-2 plays an important role in the regulation of growth factor–induced signal transduction cascades in the epithelium, and ErbB-2 is frequently overexpressed in epithelial tumors. Our previous studies on clinical prostate cancer specimens indicated that ErbB-2 expression was increased in patients undergoing hormone ablation therapy. We had also shown that the critical cell cycle regulatory gene cyclin D1 and its promoter were targets of proliferative signaling in prostate cancer cell lines, and that cyclin D1 was required for ErbB-2–induced mammary tumorigenesis. In the current studies, we found that increased ErbB-2 membrane expression correlated with increased nuclear cyclin D1 staining in clinical prostate cancer specimens, and that expression of ErbB-2 was capable of inducing cell cycle progression in human prostate cancer cell lines. We further showed that ErbB-2 induced the cyclin D1 promoter in DU145 cells, and that small interfering RNA knockdown of cyclin D1 protein levels blocked a significant proportion of the heregulin-induced cell cycle progression in LNCaP cells. Probasin promoter–targeted expression of an activated ErbB-2 isoform induced cyclin D1 expression in the mouse prostate, commensurate with prostate intraepithelial neoplasia. Together, these in vitro and in vivo studies identify cyclin D1 as a critical downstream target of ErbB-2 in the prostate epithelium, both of which are possible therapeutic targets for cancer intervention. Furthermore, our novel mouse model provides a useful platform for ongoing in vivo investigations of ErbB-2 signaling in the prostate epithelium. [Cancer Res 2007;67(9):4364–72]

Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.

Background: Induction of molecular chaperone Grp78 (78-kDa glucose-regulated protein) occurs in stress conditions that often characterize tumor microenvironments. We investigated the role of Grp78 in prostate cancer progression and the development of castration resistance, where cancer cells continue to survive despite the stress of an androgen-starved environment. Experimental Design: Immunohistochemistry was done to examine Grp78 expression in 219 prostate cancers from patients with pathologic stage T3N0M0 disease [androgen ablation naive (untreated) and androgen ablation exposed (treated)] and castration-resistant prostate cancer. Classification of tumors was based on intensity of Grp78 cytoplasmic immunoreactivity and percentage of immunoreactive tumor cells. The associations of Grp78 expression with prostate cancer recurrence (clinical and/or serum prostate-specific antigen) and survival were examined in the untreated stage T3N0M0 group. Grp78 expression was also analyzed in the androgen-dependent LNCaP and castration-resistant C42B cell lines. Results: The percentage of tumor cells expressing Grp78 was strongly associated with castration-resistant status (P = 0.005). Increased Grp78 expression was consistently associated with greater risk of prostate cancer recurrence and worse overall survival in patients who had not undergone prior hormonal manipulation. Grp78 expression was also increased in the castration-resistant LNCaP-derived cell line C42B and in LNCaP cells grown in androgen-deprived conditions compared with LNCaP cells grown in androgen-rich media. Conclusion: Our findings show that up-regulation of Grp78 is associated with the development of castration resistance, possibly in part by augmenting cell survival as previously suggested, and may serve as an important prognostic indicator of recurrence in a subset of patients with T3N0M0 disease.

Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44+CD24−/low phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC.Experimental Design: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK+ from early breast cancer patients. CK+ cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging.Results: We detected the putative stem cell–like phenotype in all CK+ specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in Conclusions: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

The role of various molecular determinants involved in the genesis, progression, and outcome of bladder cancer has been the focus of investigations for the past 2 decades. Increasingly, the analysis of the interplay between these molecular factors is taking center stage. We review herein the studies examining the effects of deregulation of the various molecules implicated in the cell cycle, apoptosis, and angiogenesis pathways and analyze the central role of p53 in regulating these pathways. Technological advancements enable detection and quantification of gene transcripts and protein products, helping us move toward achieving the goal of establishing diagnostic, prognostic, and therapeutic marker panels. Recent studies have therefore focused on multiple-marker analyses to generate informative panels that can have greater clinical value for bladder cancer management. The use of molecular marker panels can provide a more objective alternative to clinical parameters for diagnosis and treatment decisions. Clinical trials aimed at treating urothelial carcinoma based on a patient's molecular profile can be predicted to empower clinicians to personalize patient management through increased therapeutic efficacy.

Nodal staging has been identified as an independent indicator of prognosis. Quantitative RT-PCR data was taken for 70 genes associated with bladder cancer and genetic programming was used to develop classification rules associated with nodal stages of bladder cancer. This study suggests involvement of several key genes for discriminating between samples with and without nodal metastasis.

Background Previous studies on bladder cancer have shown nodal involvement to be an independent indicator of prognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis from molecular profiles of primary urothelial carcinoma tissues. Methods The study included primary bladder tumor tissues from 60 patients across different stages and 5 control tissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positive and node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardized competitive RT-PCR and the expression values of the training set samples were run through an iterative machine learning process called genetic programming that employed an N-fold cross validation technique to generate classifier rules of limited complexity. These were then used in a voting algorithm to classify the validation set samples into those associated with or without nodal metastasis. Results The generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when compared to the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in gene expression motifs that cumulatively suggested a pattern ICAM1>MAP2K6>KDR for node positive cases. Additionally, the motifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors. Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producing an accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis. Conclusion Our study demonstrates the use of standardized quantitative gene expression values from primary bladder tumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status. Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematical combinations in the progression towards nodal positivity. Further studies are needed to identify more class-specific signatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

In the true spirit of interdisciplinary expansion of scientific knowledge, results from the applicability of nanotechnology in the fields of physical sciences and chemistry have now matured enough to extend into biology and medicine. The advances in nanotechnology in the past decade have offered novel opportunities for sensing clinically relevant markers, molecular dsease imaging and tools for therapeutic intervention, which have a potential to transform the field of medicine. We describe the currently available components of the nano-toolbox, which are likely to be incorporated in clinical practice in the forseeable future. These include nanowires, cantilevers and nanopores for sensing, naoparticles and quantum dots for molecular imaging and nanoparticles for therapy. We also discuss the issue of toxicity of these nanomaterials and other limitations in the application of nanotechnology to medicine.

The basis of cancer treatment has come a long way since the days of the the classical TNM staging. Identification of novel biomarkers for various cancers and their specific correlations with prognosis have increased our understanding of carcinogenesis and tumour progression on the molecular level. Recent advances in technologies for simultaneous detection of multiple biomarkers have opened up new avenues for multimarker profiling on a more individual scale. We summarize the key molecular determinants and their prognostic role in various cancers, and examine the available techniques for analysing biomarker panels that can have a major impact on cancer diagnostics and therapeutics.

Alterations in global levels and regional patterns of deoxyribonucleic acid methylation are among the earliest and most common events known to occur in human cancer. The mutational and epigenetic effects of this covalent deoxyribonucleic acid modification to the development of bladder cancer are well recognized. The contribution of aberrant methylation to mutational hot spots located within genes, transcriptional silencing, and chromosomal instability is reviewed in the context of its relevance to bladder carcinogenesis. Understanding how such processes evolve during the progression of bladder cancer is essential for using these molecular changes in the clinical setting. The recent development of sensitive and specific techniques for quantifying methylation changes in urine specimens and bodily fluids underscores the potential use of this molecular marker for early detection and surveillance of bladder cancer. Further refinement of these molecular biological techniques holds much promise for the use of methylation markers for bladder cancer diagnosis, risk stratification, and disease prognostication.

We report complementary detection of prostate-specific antigen (PSA) using n-type In 2 O 3 nanowires and p-type carbon nanotubes. Our innovation involves developing an approach to covalently attach antibodies to In 2 O 3 NW surfaces via the onsite surface synthesis of phosphoric acid-succinylimide ester. Electronic measurements under dry conditions revealed complementary response for In 2 O 3 NW and SWNT devices after the binding of PSA. Real time detection in solution has also been demonstrated for PSA down to 5 ng/mL, a benchmark concentration significant for clinical diagnosis of prostate cancer, which is the most frequently diagnosed cancer.

We report complementary detection of prostate-specific antigen (PSA) using n-type In2O3 nanowires and p-type carbon nanotubes. Our innovation involves developing an approach to covalently attach antibodies to In2O3 NW surfaces via the onsite surface synthesis of phosphonic acid−succinylimide ester. Electronic measurements under dry conditions revealed complementary response for In2O3 NW and SWNT devices after the binding of PSA. Real-time detection in solution has also been demonstrated for PSA down to 5 ng/mL, a benchmark concentration significant for clinical diagnosis of prostate cancer, which is the most frequently diagnosed cancer.

Attempts are being made in many laboratories to find new biomarkers for and new methods of molecular staging of bladder cancer. At the forefront of this are the authors from Los Angeles who have contributed the first of four mini-reviews in this section. Other mini-reviews examine the role of hand-assisted laparoscopy in urology, a contribution to the sometimes heated argument about whether laparoscopy should be ‘pure’ or hand-assisted: in addition, two mini-reviews describe the role of photodynamic diagnosis in managing superficial bladder cancer, and the contribution of non-surgical factors to the success of hypospadias repair.

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.

A label-free technique capable of rapidly screening human blood samples simultaneously for multiple serum tumor markers would enable accurate and cost-effective diagnosis of cancer before physiological symptoms appear. Recently, microfabricated, bimaterial cantilever sensors have been demonstrated to detect DNA hybridization and antigen-antibody binding at clinically relevant concentrations. Cantilever sensors deflect measurably under the surface stress resulting when biomolecules immobilized on one surface of the sensor interact with their binding partners [1]. We present an array of cantilever sensors (silicon nitride with a gold coated surface) capable of simultaneously interrogating 100 different biomolecular interactions.Copyright © 2004 by ASME

Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.

As the determination of gene sequences and their function gains speed at the dawn of the third millennium, biomedical research efforts are oriented towards definition of the genetic and molecular expression patterns that may drive different disease. A major part of these efforts is addressed to the definition of inter-individual variations that are expected to become integral for treatment planning, in terms of efficacy and adverse effects of drugs. It is this thrust on genome-based 'rational therapeutics' that is hoped to progressively lead to the era of 'personalized medicine'. This approach uses the technological expertise from genomics and functional genomics to define, predict and monitor the nature of the response of an individual to drugs, and to rationally design newer drugs. In the present review we will conduct our readers through an understanding of the fundamentals of pharmacogenomics and of the technologies currently available that are advancing this relatively new science. Conversely, there are issues raised that concern how medical practice is preparing itself to implement new alternatives for therapeutical interventions and finally, how to respect patient confidentiality and rights.

We designed a new separable bilayer (SB) microfiltration device for the viable enrichment of circulating tumor cells (CTCs) independent of antigen expression. Bilayer pore structures at the micro scale decrease cell damage to preserve viability, while maintaining throughput to allow rapid enrichment. The large pore size (40 um) of the upper layer facilitates the CTCs proliferation on chip. The separability enables the efficient release of the CTCs after successful on chip culture of captured CTCs. We characterized the device performances including capture efficiency, enrichment against leukocytes, viability and proliferability. The SB device can enrich tumor cells with 80% capture efficiency, higher than 103 enrichment, and better than 70% viability from 1 mL whole blood samples on a 1 cm2 device. Multiple cell lines were shown to be able to proliferate after captured on SB device. Furthermore, using CTCs derived from an in vivo model system, cell cultures were established by releasing the captured CTCs from the chip. The cultured CTCs and their corresponding parental cell lines were injected into mice. Tumors were established with no discernable differences in volume and rate of growth, demonstrating similar tumorigenicity of viable CTCs recovered by the SB device. In a feasibility study, we successfully detected CTCs from 1 mL of clinical whole blood sample enriched via SB device. Citation Format: Ming-Da Zhou, Sijie Hao, Anthony J. Williams, Bo Lv, Jiyue Zhu, Richard J. Cote, Ram H. Datar, Yu-Chong Tai, Siyang Zheng. Separable bilayer microfiltration device for viable label-free enrichment of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2014-LB-193

Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of stan- dard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this chal- lenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM's ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R 2 ¼ 0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported

This paper presents a comprehensive experimental study and characterization of material and bonding of PDMS based structures to various substrates. A previously published method [1] of bonding is further improved with the inclusion of more substrate material and additional characteristics. Uncured PDMS is used as an adhesive to bond PDMS devices reversibly to various substrates including a number of commonly used substrate materials that are not supported by the widely used plasma treatment method. We have optimized parameters such as PDMS base to curing agent ratio, curing temperature, and PDMS device age to obtain better bond strengths and quality. Bond strengths are presented for semiconductor substrates (silicon, zinc oxide, and silicon dioxide), metals (gold, aluminum), photoresists (SU-8, AZxx) and glass. Silicon based substrates experienced minor amounts of surface residue, but the method is fully reversible for other tested substrates. Bond strengths were measured as maximum endurable pressure between PDMS and substrates. Maximum average bond strengths of more than 0.4 MPa were achieved for substrates with Si-O groups. Other substrates exhibited maximum average bond strengths in the range 0.2–0.3 MPa. Also presented is a method that avoids alignment step for PDMS microfluidic device bonding, named the non-aligned method. This method provides bond strengths of more than 0.1 MPa. Presented methods do not need special equipment or processes such as plasma generators or temperature increases. Biocompatibility tests are performed for materials used in fabrications to ensure applicability in bio-sensing related devices.

e22018 Background: CTCs can be detected in the blood of men with prostate cancer and have been used as a biomarker. The only FDA-approved technology (CellSearch) utilizes epithelial cell adhesion m...

481 Background: Detection of circulating tumor cells (CTCs) in renal cell carcinoma (RCC) is limited by inconsistent expression of epithelial cell adhesion molecule. We have previously detected CTCs using microfilter devices that exploit size differences between larger epithelial tumor and smaller non-tumor blood cells. Methods: Treatment naive pts with clear cell metastatic RCC (mRCC) were enrolled on a prospective phase II trial of intermittent sunitinib. Peripheral blood samples were collected at baseline, day 28 of cycles 1, 2, 4, and after the 8-week off-treatment period. Peripheral blood was diluted 1:1 with PBS and 1% formalin. Cells were captured with microfilters, stained with pan cytokeratin (CK) and CD45, conjugated with Alexa Fluor (AF) 594 and AF 488. Microfilters were cover-slipped with a DAPI containing medium for nuclear visualization. CTCs were identified as large CK+/CD45- events. Results: Pt characteristics (n=17) included 13 male/4 female; median age 65 years; median performance status KPS 90%; 100% prior nephrectomy. By Heng criteria, 4 pts were favorable, 12 intermediate and 1 poor risk. Site of metastases included lungs (59%), bone (41%) and lymph nodes (35%). Baseline CTCs (35 total samples collected) were detectable in at least one sample in 9 patients (53%), with a median CTC count of 6 (range, 1-31/ml); overall median CTC count was 1 CTC/ml including undetectable patients at baseline. Intrapatient variability was observed with the maximum intrapatient range of 4 to 58 CTCs/ml. After cycle 1 of sunitinib, median CTCs increased to 23 cells/ml (range, 0-94). Median CTCs after cycle 2 was 2 cells/ml (range, 0-322) and cycle 4 was 4.5 cells/ml (range, 0-500). After the 8 week off-treatment period, a median of 3.5 CTCs/ml (range, 0-242) were detected. In 6 patients with undetectable CTCs at baseline, CTCs were detected during the first 2 cycles of treatment, and in 4 of these patients, they declined or became undetectable by the last sampling period. Conclusions: A high rate of CTC detection is demonstrated using micro-filters in mRCC. CTC levels tend to increase during the first few weeks of treatment with sunitinib, and then decline with continued sunitinib and after sunitinib is held.

BACKGROUND: Traditional single-marker and multimarker molecular profiling approaches in bladder cancer do not account for major risk factors and their influence on clinical outcome. This study examined the prognostic value of molecular alterations across all disease stages after accounting for clinicopathological factors and smoking, the most common risk factor for bladder cancer in the developed world, in a population-based cohort. METHODS: Primary bladder tumors from 212 cancer registry patients (median follow-up, 13.2 years) were immunohistochemically profiled for Bax, caspase-3, apoptotic protease-activating factor 1 (Apaf-1), Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. “Smoking intensity” quantified the impact of duration and daily frequency of smoking. RESULTS: Age, pathological stage, surgical modality, and adjuvant therapy administration were significantly associated with survival. Increasing smoking intensity was independently associated with worse outcome (P < .001). Apaf-1, E-cadherin, and p53 were prognostic for outcome (P = .005, .014, and .032, respectively); E-cadherin remained prognostic following multivariable analysis (P = .040). Combined alterations in all 9 biomarkers were prognostic by univariable (P < .001) and multivariable (P = .006) analysis. A multivariable model that included all 9 biomarkers and smoking intensity had greater accuracy in predicting prognosis than models composed of standard clinicopathological covariates without or with smoking intensity (P < .001 and P = .018, respectively). CONCLUSIONS: Apaf-1, E-cadherin, and p53 alterations individually predicted survival in bladder cancer patients. Increasing number of biomarker alterations was significantly associated with worsening survival, although markers comprising the panel were not necessarily prognostic individually. Predictive value of the 9-biomarker panel with smoking intensity was significantly higher than that of routine clinicopathological parameters alone. Cancer 2013. © 2013 American Cancer Society.

Circulating tumor cells (CTCs) play a critical role in metastasis. Functional characterization of CTCs is limited by a lack of available methods to enrich viable CTCs. We have developed a next-generation (next-gen) platform, modifying our current size-based CTC enrichment technology, for viable CTC capture. Here, we employed our next-gen microfilter device for viable CTC capture in a syngeneic breast cancer mouse model system to show that such cells can be cultured with high efficiency for further characterization. Balb/c mice received subcutaneous injection of 4T1 (highly metastatic) or 4T07 (poorly metastatic) mouse mammary tumor cell lines originally derived from the same spontaneously arising tumor. Primary tumors were excised on day 21, digested and placed in culture, and viable CTCs were isolated using the next-gen microfilter device from 0.5ml whole blood draws for culture directly on-chip. Successfully established cell cultures were characterized by morphology, IHC for markers associated with epithelial-mesenchymal transition (EMT), and anchorage-independent growth to compare abilities to form tumorispheres. Tumors developed in 100% (6/6) of mice injected; cultures were obtained from 100% dissociated primary tumors (n=3 4T1, n=3 4T07), and isolated CTCs yielded cultures from 33% of mice injected with both cell lines (1/3 each). While 4T07-CTC cultures demonstrate homogenous morphology similar to the corresponding primary tumor, 4T1-CTC cultures demonstrate two morphologically distinct subclones: “A," spheroid-like, different from the primary tumor, and “B," spindle-like, similar to the primary tumor. “A” cells demonstrate the ability to produce progeny with spherical as well as spindle-like morphology in vitro. EMT profiling revealed that both 4T1-CTC subclones demonstrate epithelial and mesenchymal expression characteristics, while 4T07-CTCs predominately show mesenchymal character. CTCs had a greater ability to form tumorispheres in anchorage-independent growth conditions than corresponding primary tumors, with 4T1 “A” having greater tumorisphere-forming ability than 4T1 “B”. These studies demonstrate our ability to capture and culture viable CTCs from mice injected with tumor cells of varying metastatic capability. The characterization of CTCs may help identify functionally important subsets of CTCs. Molecular and functional analyses of CTCs are ongoing, and include epigenomic, transcriptomic and proteomic expression profiling, assessment of stem cell-like properties, and their mechanistic role in metastatic disease. Our work could be the basis to establish CTC cultures in human patients, and provide a transformative approach to the functional characterization of CTCs, leading to an exciting new technology for improved cancer research and individualized patient management. Citation Format: Anthony Williams, Brett Schrand, Siddarth Rawal, Zheng Ao, Randall Brenneman, Eli Gilboa, Siyang Zheng, Ram Datar, Richard Cote. Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1447. doi:10.1158/1538-7445.AM2013-1447

4575 Background: Urothelial carcinoma of the bladder (UCB) is a disease of alterations in several cellular pathways. Routine molecular profiling studies do not account for smoking, a well established risk factor for UCB, and its influence on outcome. This study assessed the prognostic potential of a multi-pathway protein panel across all UCB stages in a population-based cohort after accounting for clinicopathologic factors and smoking history. Methods: 212 UCB patients from the LA CSP, part of the NCI/SEER cancer registry, were included. "Smoking intensity" analyzed biologic and molecular impact of smoking by combining smoking status, duration of smoking and number of cigarettes smoked daily into a composite covariate. Tumors were profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX2, VEGF and E-cadherin alterations by IHC. Univariate analyses and multivariable modeling examined associations with outcome. Results: Median follow up was 13.2 years. Age, pathologic stage, adjuvant therapy (all p< 0.001) and surgical modality (p = 0.05) were associated with survival. Increasing smoking intensity was associated with worse outcome (P < 0.001). Apaf-1 (p = 0.005), E-cadherin (p = 0.014) and p53 (p = 0.032) were univariately prognostic; E-cadherin remained prognostic after multivariate analysis (p = 0.04). Combined alterations in all 9 biomarkers were prognostic by univariate (p < 0.001) and multivariate (p = 0.006) analysis. A multivariate model that included all 9 biomarkers and smoking intensity was more accurate in predicting prognosis than models comprising of standard clinicopathologic covariates without (p < 0.001) or with (p = 0.018) smoking intensity. Conclusions: The study confirms detrimental effects of smoking on UCB prognosis. Apaf-1, E-cadherin and p53 individually predicted UCB survival. Increasing number of biomarker alterations was significantly associated with worsening survival, although markers contained in the panel were not necessarily prognostic individually. Predictive value of the nine-biomarker panel with smoking intensity was significantly higher than that of routine clinicopathologic parameters alone.

10503 Background: CTC are promising biomarkers in mCRPC but have not been prospectively validated for docetaxel treatment (Rx). Using CellSearch technology (J&J), we enumerated CTC in this Phase 3 trial & assessed prognostic value for OS. The aim of this correlative study nested within 0421 was to compare CTC via CellSearch technology to newer microfilter technologies (Cote & Goldkorn PIs RO1 CA141077). Comparative data analysis is ongoing. Methods: CTC were drawn at baseline (d1) & pre-cycle 2 (d21) of Rx & shipped overnight to a central site for enumeration (CTC/7.5 ml). Cox regression evaluated the association between OS and (i) baseline CTC counts & (ii) CTC dynamics (d1 to d21) in pts with good (=5) baseline CTC counts. Receiver operator characteristic (ROC) analysis and Characteristics & Regression Trees (CART) were used to explore further prognostic CTC cutpoints for 2-yr survival. Results: Of 263 patients (pts) consented, 238 were evaluable at d1 & 232 at d21. At d1 median CTC was 5 (range 0-5916) & d1 CTC < vs. >= 5 was associated with baseline PSA (mean 99 vs. 320 ng/ml, p=0.004) and worse bone pain (36% vs. 51%, p=0.03). There was a significant difference in OS for d1 CTC < vs. >=5, with a hazard ratio (HR) of 2.92 (95% CI 1.92-4.43, p=5), a >=2-fold decrease in CTC was associated with longer OS, HR 0.45 (95%CI 0.24-0.84, p=0.012); adjusting for risk factors. D1 CTC and 2-year survival had ROC AUC of 0.781. CART analysis identified prognostic subgroups based on CTC of 0, 1-5, 6-53, and >53: (HR 0.36, 0.77,1.3 and 2.8). Conclusions: In this phase 3 trial, d1 CTC was prognostic of OS after risk factor adjustment. CTC dynamics from d1 to d21 were also prognostic of OS. These data are an exploratory subset analysis of the overall study. Yet, they comprise the largest docetaxel-based prospective cohort to date, which validates a 5 CTC prognostic threshold for OS & identifies new potential prognostic subgroups that may extend the clinical utility of CTC enumeration in mCRPC.

The most important determinant of prognosis and management of cancer is the incidence of metastasis. As a result, circulating tumor cell (CTC) detection and enumeration has been demonstrated to be a useful tool in assessing prognosis and monitoring therapeutic response. Current approaches to CTC detection largely depend on the antibody-mediated magnetic capture of the CTC using antibodies against the epithelial cell adhesion molecule (EpCAM), a feature which can be exploited in only a select few malignancies, and even in these cases, CTC capture is directly impacted by the variable expression of EpCAM. Responding to these issues, we have developed a novel parylene-based microfilter device for the capture, enumeration, and molecular characterization of CTC by exploiting size differences between larger epithelial tumor cells and smaller non-tumor blood cells. Our size-based approach, in contrast to immunoaffinity-based platforms, is ‘antigen expression-agnostic’; allowing analysis of diverse CTC populations and CTC from tumor types lacking target capture antigens. In the present study, the prospective collection of 500 blood samples from patients being treated at the Cleveland Clinic-Taussig Cancer Institute in Cleveland, OH is currently underway: 450 with advanced breast (BC), prostate (PC), and colorectal cancer (CRC) (150 from each disease site), and 50 with advanced renal cell carcinoma (RCC) and melanoma (25 from each disease site). To date, we have analyzed 28 patient blood samples by the microfilter device, of which 26 (92.8%) where positive for CTC (total at primary disease site, % samples positive, range of CTC detected) - BC: 1, 100%, 48; PC: 13, 84.6%, 1 to 144; CRC: 7, 100%, 18 to >500; RCC: 7, 100%, 1-128.* Further analysis demonstrates a number of interesting molecular characteristics on CTC, including heterogeneity in intensity of cytokeratin (CK) expression, differential patterns of CK staining on CTC (filamentous vs granular), and the occurrence of CTC as individual events or in clusters. Further, our data in blood samples from RCC demonstrate an ability to analyze CTC from non-EpCAM expressing tumor types, an area of study where many platforms have limited utility. With the ability to perform multiple, repeated sample analyses to detect recurrent disease early and enable drug response surveillance, we believe our technology has the potential to provide a faster, more efficient alternative to currently available methods for CTC analysis. The molecular characteristics on CTC we have identified herein could have critical biological relevance and implications for predicting aggressiveness of CTC and improving therapeutic monitoring. In ongoing studies, we are investigating the correlation of CTC enumeration, morphology, and clustering, with tumor type and treatment efficacy. *Data to be updated prior to presentation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2372. doi:1538-7445.AM2012-2372

The basis for identification of disseminated tumor cells (DTC) and circulating tumor cells (CTC) is evaluation of antigenic differences [for epithelial tumors, generally antigens which identify their epithelial origin, such as cytokeratins (CK)] coupled with the morphology of the CTC (reviewed in

INTRODUCTION: Urothelial carcinoma of the bladder develops through multiple cellular alterations. Traditional single-marker and multimarker molecular profiling approaches in bladder cancer do not account for risk factors and their influence on clinical outcome. Cigarette smoking is the most well established risk factor for bladder cancer in the western world. This study sought to examine the prognostic value of molecular alterations across all disease stages after stratifying for clinicopathologic factors and smoking in a population-based cohort. METHODS: 212 patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. To analyze the biologic and molecular impact of smoking, we introduced a novel “smoking intensity” variable that took into account a patient's smoking status, duration of smoking and number of cigarettes smoked daily to quantify the impact of exposure to cigarette smoke. Primary bladder tumors were immunohistochemically profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. Univariate analyses and multivariable modeling were used to examine associations with outcome. RESULTS: Median follow up was 13.2 years. For smokers (n=184), median age to start smoking was 17 years (range, 12-40 years), and median smoking duration was 35 years (range, 0.5-50 years). Median number of cigarettes smoked daily was 25 (range, 2-100). Increasing pathologic stage and smoking intensity were independently associated with worsening survival (P CONCLUSION: The study confirms detrimental effects of smoking on bladder cancer prognosis. Apaf-1, E-cadherin and p53 can individually predict survival in bladder cancer patients, with Apaf-1 being the most prognostic individual marker. The nine-biomarker panel can significantly predict outcome independent of stage and smoking history. Increasing biomarker alterations was significantly associated with worsening survival, although markers comprising the panel were not necessarily prognostic individually. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2258. doi:10.1158/1538-7445.AM2011-2258

Background: Circulating tumor cell (CTC) quantitation has demonstrated prognostic significance in multiple tumor types. Experience utilizing CTC to study tumor gene expression is limited and unvalidated. We explored our ability to perform RNA extraction from CTC's using a novel membrane microfilter device (MD). Our objectives were to evaluate (a) the feasibility to extract RNA from cells on the MD, (b) the detection of CTC-specific gene expression on the MD above basal levels of normal cells, and (c) the ability to perform quantitative gene expression analysis in clinical blood samples using the MD. Methods: RNA was extracted from MDA-MB-231 breast cancer cells as follows: (1) fresh, unfixed cells as a control, (2) cells fixed in 1% formalin for 10 min at room temperature, (3) cells resuspended into 5ml PBS, fixed in 1% formalin for 10 min at room temperature, processed by the MD, and either (3.1) removed from the microfilter by LCM for RNA recovery, or (3.2) lysed from the entire filtration area for RNA recovery. We compared respective expression levels by qRT-PCR of 44 genes with known relevance in metastasis from each of the 3 sources described above to the expression levels of the cells from the control source. We also evaluated RNA integrity from each source by 260/280 ratio. Additionally, varying dilutions of tumor cells were spiked into blood from normal healthy donors, processed by the MD, and the expression levels of 92 genes by qRT-PCR with known relevance in metastasis were compared to expression levels from a healthy donor blood sample. In clinical samples, 10ml blood was drawn from 4 patients with metastatic bladder cancer, processed by the MD, RNA recovered, and the expression levels of 92 genes by qRT-PCR with known relevance in metastasis were compared to the expression levels from a healthy donor blood sample. All cDNA samples prepared from the microfilters were amplified using custom designed gene pools by PCR prior to qPCR analysis. Results: Our data indicate an ability to extract RNA with good integrity from tumor cells (by 260/280 ratio) captured by the MD despite pre-incubation of samples in a mild fixative, that RNA can be extracted from tumor cells that have been removed from the MD by LCM without any significant degradation, and that low yields of RNA can be reverse transcribed and amplified prior to qPCR analysis. In both spiked and clinical blood samples, we detected differential expression in numerous genes as compared to healthy donor blood. Conclusion: While the enumeration of CTC's has been the primary means to collect clinically useful data, molecular characterization of CTC is yet to be widely accomplished. Our data demonstrate potential to quantitatively characterize relative gene expression levels in CTC, which will enhance our understanding of the metastatic process, and improve prediction of therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5254. doi:10.1158/1538-7445.AM2011-5254

INTRODUCTION: Urothelial carcinoma (UC) of the bladder develops through alterations in several cellular processes. Previous UC multimarker studies do not account for risk factor exposure that can influence clinical outcome. Cigarette smoking is the most well established risk factor for UC in the U.S. This study sought to identify molecular alterations associated with smoking and their prognostic value in a population-based cohort. METHODS: 212 UC patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. Median follow up was 13.2 years. To analyze the biologic and molecular impact of smoking, we introduced novel variables - “smoke exposure” considered smoker status and duration of smoking, and “smoking intensity” considered number of cigarettes smoked daily in addition to the above smoking parameters. For ex-smokers, the “relative cessation measure” considered duration of smoking cessation until diagnosis and duration of smoking. Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX-2, VEGF, and ECAD immunohistochemical expressions were analyzed on archival UC sections. Covariate and clinical outcome (overall survival) associations were examined. RESULTS: Stage was associated with p53 (P CONCLUSION: The study confirms the detrimental effect of smoking on UC prognosis and identifies key molecules that are deregulated by the carcinogenic exposure. Apaf-1, ECAD and p53 were important individual predictors of outcome, with Apaf-1 being prognostic after stratifying for stage and/or smoking. Number of altered markers was the most robust outcome predictor, independent of standard clinicopathologic and epidemiologic criteria, and can be used as a tool to identify patients who are in need of more aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4702.

Glucose responsive polymer brushes were synthesized on gold substrates and microcantilever arrays. The response properties of these brushes were evaluated by exposing them to different glucose concentrations for a range of pH values. This work demonstrates the potential for polymer brush-functionalized micromechanical cantilevers as glucose detectors. Furthermore, the work demonstrates that stimulus-responsive polymer brushes on micromechanical cantilevers have a significantly larger bending response due to glucose binding compared with self-assembled monolayers.

9561 Background: p53 alterations at the protein or gene level are important in the development and progression of bladder transitional cell carcinoma (TCC). We studied the association of p53 phenot...