bluuebunny/crossref_metadata_2025 huggingface dataset:
A kinase-directed RNAi screen identifies GSK-3b/a as a target to overcome drug resistance of p53-null colon cancer cells
Authors: Grassilli Emanuela , Narloch Robert , Federzoni Elena , Leone Biagio Eugenio , Helin Kristian , Lavitrano Marialuisa
Published: May 2015

Evasion from chemotherapy-induced apoptosis strongly contributes to drug resistance and tumor relapse. In particular, p53 loss renders colon carcinoma cells resistant to 5-fluorouracil-induced apoptosis. We have used the 5-fluorouracil-resistant HCT116p53KO colon carcinoma cell line and a kinase-directed retroviral shRNA library to identify kinases whose inactivation reverts resistance to drug-induced apoptosis. In this way we have identified 29 kinases whose downregulation abolished colony growth after 5-fluorouracil treatment. Silencing of these kinases in other drug-resistant p53-null colon cell lines also abolished cellular growth, suggesting that the effect is independent of the genetic background. We focused on GSK-3 since by tissue microarray we found that GSK-3 is overexpressed/activated in tumoral biopsies of infiltrating and invasive colon carcinomas. We demonstrated that GSK-3 silencing in different colon carcinoma cell lines reverts resistance not only to 5-fluorouracil, but also to oxaliplatin and the 2 drugs used in combination. Interestingly, we found that 5-fluorouracil-treated p53-null GSK-3-depleted colon carcinoma cells undergo caspase-independent but PARP-dependent and tBid- and AIF-mediated cell death. Therefore, we propose that GSK-3 is an excellent potential target for the development of anti-cancer therapy. Our results suggest that inhibitors of GSK-3 can be used in combination with chemotherapy to kill cancer cells unable to trigger caspase-dependent apoptosis.
Development of a panel of biomarkers for the diagnosis of prostate cancer
Authors: Fallon Rachel A. , Wheeler Colin , Koopmann Jens , Workman Nick , McAndrew Mike B.
Published: May 2015

The issues surrounding the use of PSA in the diagnosis of prostate cancer (PC) are well documented. In community practice the PSA cut-off point of 4 ng/mL is considered sensitive but relatively non-specific (sensitivity of 86% and specificity of 33%)1 and there is a need for a diagnostic test with greater discriminatory power. The development of autoantibodies associated with prostate cancer has been described2. In general, the appearance of such antibodies can precede disease symptoms by many years, making them attractive as potential biomarkers for early diagnosis.

We have developed a unique “functional protein” array platform which utilises 925 correctly folded proteins chosen for their roles in disease and used this to detect autoantibodies in PC serum samples. We have taken advantage of the multiplex nature of array assays and applied complex data analysis strategies to identify panels of biomarkers which may have clinical utility in the diagnosis of prostate cancer.

Serum samples (case; n = 73 and control; n = 60) were analysed using protein arrays. Data analysis was performed using a multi-scaling and outlier removal step for quadruplicate signals from all 133 protein arrays. Data were arbitrarily split into test and training sets and the data from the training set was then used with both support vector machines and genetic programming to identify classifiers which would successfully distinguish case from control samples. Classifiers were assessed for performance by referring to the combined sensitivity and specificity (S+S score) using the test set. Data were repeatedly split into test and training sets and analysis cycles repeated until a stable set of classifiers was identified.

A set of biomarkers was identified which can distinguish PC from control samples with both sensitivity and specificity above 90%. Further development and validation of this panel is on-going with a larger cohort. 1. Hoffman et al. Prostate-specific antigen testing accuracy in community practice. BMC Family Practice (2002) 3:19. 2. Wang et al. Autoantibody signatures in prostate cancer. N Engl J Med. (2005) 353:1224.
Population-based engineered tumor models as a platform for biomarker discovery in cancer
Authors: Jiang Feng , Lin Jie , Sun Xiaojian , Feng Bin , Nicoletti Richard , Li Guangmu , Chiu M. Isabel , Esteves Brooke , Al-Adhami Mohammed , Bhargava Pankaj , Robinson Murray O.
Published: May 2015

This abstract is being presented as a short talk in Plenary Session 1. A full abstract is printed in the Proffered Abstracts section (PR1) of the Conference Proceedings.
Functional analysis of human tumor response to signal transduction inhibitors: Ex vivo analysis of programmed cell death (EVA-PCD) in 1° culture microspheroids
Authors: Nagourney Robert A. , Kenyon Kaylee R. , Frederico Eric , Bernard Paula , Evans Steven S.
Published: May 2015

Advances in cancer therapy increasingly reflect the application of genomics and proteomics to target tumor-specific phenomena. Several insights have accelerated this process including the primacy cell survival signals in carcinogenesis and drug resistance and the growing appreciation of tumor biology as contextual. Ex vivo analysis of programmed cell death (EVA/PCD™) addresses both issues by measuring metabolic and morphologic features of drug induced cell death (apoptotic and non-apoptotic) using native state 1° cultures isolated from surgical specimens and cytologically (+) fluids. The predictive validity of the EVA/PCD™ established for cytotoxics, e.g,. 2CDA in hairy cell leukemia; purine-alkylator synergy in NHL (Nagourney et al, B.J. Cancer, ‘93) and platin-gemcitabine activity/synergy in breast and gynecologic cancers (Nagourney et al, Deoxynucleoside analogs in cancer therapy, Humana Press, 2006) has led to this platforms current focus on signal transduction with a growing emphasis on NSCLC. A recent report in NSCLC provided an ORR of 62%, med TTP of 9.5 mos and med OS of 22.3 mos, including the identification of durable responders to EGFr-TKIs (Nagourney RA Proc ASCO, 2010). An EVA/PCD™ exploration of “vertical” and “horizontal” signal inhibition (Nagourney et al Proc AACR, 2010) favored “horizontal” strategies in NSCLC providing very durable clinical responses with dual EGFr/VEGFr and clinical response for dual EGFr/mTOR salvage following single agent EGFr-TKI failure. By expanding beyond current FDA-approved drugs into the new agents that target PI3K, AKT, TORC1&2, MEK/ERK and c-MET, the EVA/PCD™ can facilitate developmental therapeutics. Analyses are now examining the activity and synergy of EGFr-TKIs with VEGF, mTOR, MEK/ERK, PI3K, AKT and c-MET inhibitors in NSCLC and other diseases as will be reported. Supported in part by a research grant from Biophysica, Ltd, the Vanguard Cancer Foundation and The Nagourney Institute.
Pathways of angiogenic resistance in glioblastoma: Adaptations in growth factor signaling in response to VEGF and HGF attenuation
Authors: Graves Christian A. , Camphausen Kevin
Published: May 2015

Background: Glioblastoma is the most common and lethal central nervous system malignancy with a median survival often less than 18 months. Despite promising initial responses to anti-angiogenic therapies many patients with this disease develop resistance to treatment via poorly understood mechanisms. Here we demonstrate that glioblastoma cell lines grown under growth factor antagonizing conditions exhibit an altered phenotype with changes in growth factor signaling, gene expression as well as in vivo tumorigenesis.

Methods: U87 cells were cultured under constant pressure with bevacizumab (3.0 mg/mL), AMG102 (1.0 µg/mL), or a combination of both drugs long term (seven or more passages) in adherent culture. In vitro expression analysis was carried out by growth factor and receptor tyrosine kinase arrays, cDNA microarray, and ELISA. Intracranial xenograft studies demonstrated resultant differences in growth kinetics and survival.

Results: Hepatocyte growth factor (HGF), glial derived neurotrophic factor (GDNF), and platelet-derived growth factor AA (PDGFAA) were elevated at 5.3, 4.5, and 3.9 fold in combinatorially treated cells versus controls, respectively. These changes correlated with relative increases in corresponding receptor mRNA and protein expression. Furthermore, AMG102 and combinatorially pressured cells were highly tumorigenic as compared to control and bevacizumab treated cells in vivo (Median survival 10 ± 1 vs. 32 ± 2 days, respectively).

Conclusion: Herein we describe concomitant increases of various oncogenic growth factors which might potentiate tumor survival and therapeutic resistance. These results suggest several potential molecular bypass mechanisms facing proposed angiogenic strategies. Evaluation of resistant phenotypes provides insight on the adaptive complexity of glioblastoma and offers potential for targeting recurrent disease.
Early detection of colorectal cancer from patient blood plasma using microRNA-based q-rt-PCR
Authors: Nielsen Søren J. , Jacobsen Nana , Fog Jacob U. , Willenbrock Hanni , Baker Adam , Blondal Thorarinn , Vincent Martin , Oerntoft Torben , Nielsen Hans Joergen , Christensen Ib J. , Andersen Claus L. , Møller Søren , Stenvang Jan , Brünner Nils
Published: May 2015

microRNAs (miRNAs) constitute a recently discovered class of small RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, and that each miRNA potentially regulates hundreds of different mRNAs. As a consequence, miRNAs have been proposed to be master regulators of cellular state. This hypothesis has been borne out by a large number of studies demonstrating a causal link between miRNA dys-regulation and numerous disease states, including a diverse array of human cancers. Furthermore, the high stability of miRNA in common clinical source materials (e.g. FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications.

Colorectal cancer (CRC) ranks 4th in terms of prevalence and second in numbers of deaths among cancers of the western world. Although early detection of CRC leads to a favorable prognosis, and though CRC can easily be detected in the locally restricted state using a variety of diagnostic procedures, frequent late diagnosis means that CRC is still a leading cause of cancer mortality worldwide. Current procedures suffer from one or more disadvantages within areas such as cost, safety, inconvenience to the patient with the consequence of low compliance, lack of trained personnel, sensitivity etc, thereby precluding their adoption as a population screening tool. There is therefore an unmet need for a generally acceptable CRC screening assay.

To facilitate discovery and clinical transfer of miRNA-based diagnostic markers, we developed a genome-wide LNA™-based miRNA q-rt-PCR platform with unparalleled sensitivity and robustness. The platform uses a single RT reaction to profile >700 human miRNAs from 2 predefined 384 well plates and thus allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled a large number of plasma samples from localized and regional CRC patients, and from matched healthy controls. An extensive QC system has been implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We will present our approaches to data normalization and the results of signature development using linear classification methods. We show that we can detect the majority of cancer cases with good specificity, using a cross-validation approach. In summary, our results show that minimally invasive early detection of CRC using a clinically viable approach is feasible.
Using SELDI-TOF MS to identify serum biomarkers in NSCLC
Authors: Baykara Onur , Buyru Nur , Ulutin Turgut , Dalay Nejat
Published: May 2015

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide with poor differential diagnosis of the disease and with low response rates to standard chemotherapy. Individuals classified as Stage I by the present clinical-based scoring system may display survival characteristics ranging from 10 to 90 %. Identification of reliable biomarkers to detect NSCLC at early stages is therefore a subject of extensive research.

Surface-enhanced laser-desorption-ionization time-of-flight mass spectrophotometry (SELDI-TOF-MS) combines chromatographic surfaces with solid-state TOF-MS for the high throughput analysis of proteins in diverse clinical samples such as serum, urine, cerebrospinal fluids and has overcome the limitations of earlier proteomic approaches. The method has been widely used for biomarker discovery. Therefore, in this study we used the SELDI-TOF protein Chip system to achieve a stratification based on the protein expression profile and to identify differentially expressed proteins in sera from 50 patients with NSCLC and 50 healthy controls. IMAC 30-Cu protein chips were used to fractionate the proteins according to their metal binding affinity. The arrays were analyzed using the Ciphergen Protein Chip Reader PB IIa. The spectra were normalized to the total ion current of the mass to charge between 3000 — 10 000 Da. Protein peak clustering and classification analysis were performed using the Express 3.0 Biomarker Pattern Analysis Software.

Several proteins with masses ranging from 2000-20000 Da were differentially expressed between NSCLC patients and healthy controls. Among the peak clusters revealing differences we selected two specific clusters as prospective candidate biomarkers in sera of the patients. High expression of one peak and underexpression of the other with respect to the healthy control sera were evaluated in a blind test in terms of the ability to classify a separate group of patients and controls.
Phosphorylation and novel mutations of β-catenin in colon tumors
Authors: Dalay Nejat , Akisik Elif , Yamaner Sumer , Bugra Dursun
Published: May 2015

A significant proportion of colon tumors have defects in the Wnt signaling pathway indicating that wnt signaling plays a major role in carcinogenesis. Mutations and aberrations in this pathway may result in constitutive activation. The β-catenin molecule is a key component of this pathway and in absence of wnt signaling the cellular β—catenin level is kept low through degradation of the protein. Disruption of the β-catenin regulation plays a critical rol ein tumor development. Four critical serine/threonine residues on the protein form the recognition site for the proteasomic degradation complex. Sequential phosphorylation of β —catenin at these sites regulates the degradation rate of the protein. Mutations in this region may block phosphorylation and the mutant protein can escape from destruction. In the present study we investigated β—catenin expression and phosphorylation of the protein at the Ser33, Ser37, Thr 41 and Ser45 sites in tumors and matched normal tissue samples from 77 patients with colon cancer. β—catenin protein expression and and phosphorylation were analyzed by Western Blotting. The phospho-β—catenin expressing SW 480 cell line was used as the normal control. β—catenin protein expression was higher in tumor tissue in % 57.1 of the patients but the difference was not significant. Likewise, no significant difference was observed between phosphorylation levels of the different sites in the tumor and normal tissue. However, phosphorylation of the Ser 33/37/Thr41 and the Ser45 sites were significantly correlated.

In the samples displaying differences in phosphorylation between the tumor and the corresponding normal tissue samples the β—catenin gene was analyzed for mutations. We observed a single nucleotide change which has not been described previously in three (5.8 %) patients. This mutation results from a A G transition and leads to the substitution of glutamic acid by lysine. DNA from matched normal colon tissue and peripheral blood of the patients was also analyzed. In none of the patients the mutation was present in the somatic cells.

Our data indicate that among the molecular alterations affecting the β—catenin molecule mutations rather than phosphorylation of the protein play a major role. Although not described so far, the mutation presented in this study may constitute a more common variant than some of the previously reported alterations.
CR-UK biomarker roadmaps
Authors: Lioumi Maria , Newell David
Published: May 2015

A clear consensus is developing in all organizations involved in cancer research and treatment — academic, commercial and governmental — that the effective management of the cancer problem will require a move towards personalized medicine, based on the use of targeted therapies guided by the results of biomarker studies.

Recognizing the importance of biomarkers, Cancer Research UK (CR-UK) undertook a strategic Review in Biomarkers in 2008. A major finding was that there is currently no well defined structure or common terminology for the biomarker development process as, for example, there is for the development of a new drug. Thus despite increasing investment in basic research to identify potential biomarkers and a demonstrable enthusiasm from clinicians for access to new biomarkers, the number of biomarkers that have been delivered into clinical practice is very small. As a result, the priority recommendation arising from the review was that “Working with national and international academic and commercial partners, CR-UK should develop a pathway and roadmap for biomarker and imaging discovery and development research.” CR-UK has developed biomarker roadmaps for risk/predisposition, diagnostic, prognostic and predictive, and pharmacological biomarkers. These roadmaps describe the process from initial biomarker discovery, assay development, initial correlation to clinical outcome and clinical qualification.
A novel method for mutation analyses using genomic DNA obtained from IHC-stained sections
Authors: Desai Rupal , Amler Lukas , Patel Rajesh , Stern Howard , Raja Rajiv
Published: May 2015

Biopsies obtained from patients are often limited in size. The ability to perform multiple diagnostic assays is dependent on the quantity and quality of tissue samples available from patients. Over the last few years, our laboratory has focused our efforts on developing novel methodologies for increased efficiency in use of tissue samples. Here we describe a method for performing immunohistochemistry (IHC) and genotyping using the same tissue section. We isolated genomic DNA (gDNA) from IHC-stained and unstained formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from the same paraffin block. Quality and quantity of gDNA obtained from either section were compared. We demonstrate that quality of gDNA obtained is acceptable for down-stream analysis using PCR techniques. We also show comparable specificity and sensitivity in detecting mutations using either sources of gDNA. This method has proven highly valuable in performing mutation analysis in certain tissue types such as lung cancer where tissue can be extremely limited.
Optimization of a method for preserving nucleic acids in tissue lysates from archival formalin-fixed tissue samples
Authors: Fu Ling , Desai Rupal , Holcomb Thomas , Raja Rajiv
Published: May 2015

Biomarker analysis of patient biopsies is essential for identifying biomarkers that have predictive or prognostic value. Such analyses not only have the potential for helping us understand the molecular mechanisms underlying response to therapy, but also in personalizing healthcare based on biomarker profiles in patient sub-populations. Patient biopsies are commonly preserved by formalin-fixing and paraffin-embedding (FFPE). Ability to evaluate nucleic-acid-based biomarkers in FFPE tissue is limited by the fact that formalin-fixing degrades the quality of nucleic acids. RNA and miRNA are further degraded in FFPE blocks as time passes. Hence, there is the basic need to find methods for stabilizing nucleic acids in FFPE tissues so that nucleic-acid-based biomarkers can be evaluated in archival tissue samples. We have evaluated various methods of tissue lysate preparation to identify ones that are optimal for stabilizing nucleic acids in storage, and for efficient recovery of DNA, RNA and miRNA. We also evaluated the quality and quantity of DNA and RNA recovered from these lysates across time. We will present a method that preserves quality of nucleic acids across time, and is compatible with protocols for DNA, RNA and miRNA isolation. This method is currently used in our laboratories to prepare tissue lysates from clinical trial samples and has enabled the development of molecular diagnostic assays.
A flow cytometry detection at a protein level of human papillomavirus
Authors: Kalodimou Vasiliki E. , Kontogiorgi Marina , Papalois Apostolos , Chandakas Stefanos
Published: May 2015

Introduction: Human papillomavirus (HPV) is one of the most common sexually transmitted infections and a necessary cause of cervical cancer. Fortunately about 90% of HPV infections clear within two years without treatment. Only in a small amount of the women infected with oncogenic types of HPV the infection will progress to high-grade lesions or even cervical cancer in the absence of treatment. During persistent infections the viral genomes often integrate into the host chromosome, which results as an over expression of the viral proteins E6 and E7. These oncoproteins induce immortalization and malignant transformation of cells conferring a certain growth advantage to the infected cells.

Method: A total of 100 women undergoing routine cervical cytology samples joined this cohort study. A Thin Prep Pap Test sample was taken following consent for study participation and was tested for HPV. Cytological screening of the samples was done by trained cytologists. After sample collection, a 1 ml aliquot was removed and prepared for flow cytometric analysis and the rest of the sample was sent for PCR analysis. The samples were run on a Beckman Coulter FC500 with a 488 nm argon laser, with forward (FS) and side (SS) scatter. The gating was fixed for all samples. The instrument was set to measure counts collected from the whole volume of the samples under investigation

Results: In the total of 100 cases, 4 specimens were characterized as unsatisfactory. For the rest 96 specimens, 82 specimens were found to be positive for the HPV virus and 14 negative. In the total of 100 cases the analysis of flow cytometry and PCR detection gives the same results in most cases. The cervical cells found in the liquid specimen are resolved in distinct populations using forward and side scatter of a flow cytometer. All samples in the study were analyzed using previously established gates for the presence of endocervical, ectocervical and polymorphonuclear cells. Each population was determined and the percentage of cells included was counted. The total time of analysis depended on the density of each specimen.

Conclusion: HPV analysis by flow cytometry takes advantage of the fact that those oncogenic genotypes over express E6 and E7 mRNA following integration of HPV into genomic DNA. If the percentage is over 2% the result is positive, and if is lower is a negative result. With a negative result the risk of having a cervical cancer in the future is almost null. It can identify persistent HPV infections that can progress to high-grade lesions, detects the oncogenic activity, analyzing cervical cells that over express oncoproteins E6 and E7, and can be used at any time of woman life because there is no low age limit. The specificity and positive predictive value was higher than PCR analysis. This might be very useful in screening young women, a cohort in which the prevalence of HPV DNA positivity may be as high as 20%.
microRNAs as clinical biomarkers for lung cancer classification
Authors: Edmonston Tina B. , Kushnir Michal , Aharonov Ranit , Yanai Gila Lithwick , Benjamin Hila , Bibbo Marluce , Thurm Craig , Horowitz Laurie , Huang Yajue , Gilad Shlomit , Chajut Ayelet
Published: May 2015

Background: Lung cancers have been traditionally subdivided into two main groups: neuroendocrine and non-small cell lung cancer (NSCLC). In 20-30% of lung cancer cases it is difficult to make a definitive diagnosis of the tumor subtype based on the cytological specimen (e.g., fine-needle aspirates). In general, the classification of lung cancers poses a diagnostic challenge and there is lack of standardized techniques for cancer sub-typing. For patients with lung carcinoma, accurate determination of tumor type significantly influences treatment decision. MicroRNA expression profiling is a promising new strategy for the precise subclassification of lung cancers. We have previously described a microRNA-based assay which accurately differentiates between squamous and non-squamous NSCLC in FFPE derived from resections as well as from cytological samples. We have also used microRNA microarray data generated from over a hundred formalin-fixed, paraffin-embedded (FFPE) samples of primary lung cancer, to identify microRNA expression profiles that differ significantly for various sub-types of lung cancer samples. Based on these findings, we present here the development of a microRNA-based assay that is able to differentiate primary lung cancers into neuroendocrine vs. NSCLC and then further sub-classify NSCLC into squamous vs. non-squamous and neuroendocrine into SCLC vs. carcinoid using pathology and cytology samples.

Methods: FFPE of resections and cell blocks from different histological subtypes of lung cancer, taken from primary lung cancer resection or pre-operative diagnostic procedures, were collected. High-quality RNA, including the well-preserved microRNA fraction, was extracted from the samples using proprietary protocols. Expression levels of potential microRNA biomarkers were profiled using a sensitive and specific qRT-PCR platform.

Results: We find that combinations of small numbers of microRNAs can successfully differentiate between the four different histological types of lung cancers, with very high sensitivity and specificity. These microRNAs are able to subclassify lung cancer FFPE samples derived from pathological and cytological samples.

Conclusion: We identified microRNAs that represent excellent biomarkers for the classification of lung primary cancers. These findings are the basis for the development of a simple and reliable qRT-PCR based diagnostic assay which will offer an accurate and standardized tool for primary lung cancer pathological and cytological samples, including those that presently fail pathological evaluation.
MLH1 promoter methylation in suspected Estonian Lynch syndrome families
Authors: Rebane Egle , Kask Martin , Kostina Olga , Anderson Wally
Published: May 2015

Background: Microsatellite instability (MSI) occurs in 10-20% of colorectal cancer (CRC), and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. Inactivation of MLH1 due to promoter hypermethylation strongly suggests a sporadic origin, providing exclusion criteria for Lynch syndrome.

The aim of the study is to show the need to perform MLH1 methylation analysis in all MSI-H tumors before sequencing of MLH1 gene in Estonian clinical practice.

Methods: The study group included 50 tumors DNA from colorectal cancer patients who fulfill the Amsterdam criteria or met at least one of the revised Bethesda criteria. MSI and activating mutations of BRAF and KRAS were identified in all tumor samples. Methylation of the 5’ regulatory MLH1 region C at −197 to −295 bp (from the transcriptional start site) was analyzed by high-resolution melting (HRM) and also was performed methylation specific-MLPA (MS-MLPA) in all MSI-H cases.

Results: The average age of colorectal patients was 46 (29 to 79) years. MSI-H, BRAF and KRAS mutations were observed in 16, 1 and 14 out of all cases, respectively. Tumor localization was right-side in 9 of the MSI-H cases. Hypermethylation of the MLH1 promoter was found in 2 MSI-H tumors with KRAS mutation and right-side tumor localization.

Conclusion: The patients selection for the study is ongoing. Methylation analysis of MLH1 can give better performance characteristics in the selection of patients for sequencing of MLH1.
Companion diagnostic development and commercialization: The good, the bad, and the ugly
Authors: Jackson David
Published: May 2015

QIAGEN (formerly DxS Ltd) has established itself as the market leader in the successful co-development of drug-diagnostic solutions with pharmaceutical partners. QIAGEN has a considerable portfolio of over 15 ongoing collaborations with drug giants such as Amgen, AstraZeneca, Bristol-Myers Squibb and ImClone Systems, Pfizer and Boehringer Ingelheim. This presentation will outline the current state of companion diagnostics as a driver of personalized healthcare; investigate the regulatory and commercial hurdles involved in bringing a companion diagnostic to market and the importance of pharma partnering during clinical development as a way to overcome these hurdles, as well as discuss some of the challenges inherent to companion diagnostic partnerships.

Focusing on two of QIAGEN's key experiences with companion diagnostic development for cancer, the presentation will look first at the KRAS story and the challenge of producing the first companion diagnostic of its kind — the TheraScreen®: K-RAS Mutation Kit to predict patient response to metastatic colorectal cancer therapies Vectibix® (Amgen) and Erbitux® (BMS/Imclone Systems) based on the mutation status of the KRAS oncogene. Next, it will examine the importance of another companion diagnostic, the TheraScreen: EGFR29 Mutation Test Kit and its role in facilitating the marketing approval of AstraZeneca's non-small cell lung cancer drug IRESSA® in Europe for EGFR mutation positive patients.

With the global perspective on personalized healthcare shifting and pharma recognizing the importance of independent partners for the co-development of drug and diagnostic, the obstacles to bringing companion diagnostic tests to market have shifted. While victories have been won winning the hearts and minds of drug companies, regulators and physicians in key markets like oncology, we are now fighting for value-based reimbursement so that predictive tests can be made available patients and expand the utility of companion diagnostics to new therapeutic areas. The personalized healthcare landscape is set to continue its rapid evolution to meet these new challenges.
A systems approach to identification and validation of biomarkers
Authors: Mills Gordon B. , D UT M.
Published: May 2015

The realization of the promise of personalized molecular medicine will require the efficient development and implementation of novel targeted therapeutics. The goal will be to deliver the right drug to the right patient at the right time at the right dose. This effort will require an integration of information from the DNA, RNA, and protein level into predictors of which patients are likely to benefit from particular therapies. The overall likelihood of response to particular drugs represents the interaction between predictors of sensitivity with predictors of resistance. This session will use examples with predictors from the DNA, RNA, and protein level to illustrate some of the opportunities and challenges to the implementation of personalized medicine. It will demonstrate the need to integrate information from multiple technological platforms into visualizable pathways and networks as a key step in the process.
Modeling drug sensitivity and resistance in human tumor-derived cell lines
Authors: Settleman Jeffrey
Published: May 2015

Accumulating evidence points to the importance of genomic heterogeneity across human tumors as a critical determinant of the variable clinical response to molecularly targeted cancer therapies. We have used cancer cell lines as a model system for examining the relationship between underlying genotypes and sensitivity to candidate anticancer agents. These studies have revealed the utility of large panels of cancer cell lines to capture the genomic heterogeneity of human cancer and to identify clinically relevant genotype-phenotype relationships. These studies have begun to shed light on mechanisms underlying sensitivity as well as de novo and acquired resistance to a variety of small molecule inhibitors. Using this same platform, we have also identified a mechanism of reversible drug “tolerance” that involves a distinct chromatin state displayed transiently by small subpopulations of cancer cells. This drug-tolerant state can be disrupted by chromatin-modifying agents, potentially yielding a therapeutic opportunity to prevent or delay the development of more stable genetically conferred drug resistance mechanisms.
Fluid phase of solid tumors, circulating tumor cells in high definition
Authors: Kuhn Peter
Published: May 2015

We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) in the context of peripheral blood leukocytes, with no protein-based enrichment, and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. We refer to this technique as the HD-CTC assay and report on the use of this methodology to characterize the incidence of HD-CTCs in peripheral blood samples from a small cohort of metastatic cancer patients, including those with prostate cancer, breast cancer, pancreatic cancer, along with set of normal controls. Importantly, we find HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of the cancer patients in this cohort. As a case study, one patient was followed over time and both cytology from a solid tumor metastasis and the HD-CTCs were quantitatively characterized. This initial validation of an enrichment-free assay demonstrates the ability to identify significant numbers of HD-CTCs in a majority of patients in prostate, breast and pancreatic cancers. Due to the enrichment free approach, the HD-CTC assay has significant implications for determining the biology of cancer spread and therapy response in both the HD-CTC population and the populations of rare events that are outside of the strict HD-CTC definition but are still fully characterized and tracked in this assay. This fluid biopsy can then be used for routine pathology evaluation and additional molecular characterization. Future studies will investigate the clinical utility of this methodology.
From heat maps to clinical tests: Why so few regulatory approved molecular profiles?
Authors: Dracopoli Nicholas C.
Published: May 2015

Currently, only about 10% of oncology compounds entering Phase I clinical trials make it all the way to regulatory approval. The main goals of biomarker research in the pharmaceutical industry are to increase efficiency of the drug development process by improving understanding of the mechanism of action, deeper exploration of PK-PD interactions and predicting response to novel therapies in clinical development. The development of novel biomarkers by analysis of multidimensional clinical and biological data has enormous potential to impact drug development and improve patient outcomes. However, no complex molecular or protein profiles have been approved by the FDA to drive therapeutic use of any drug. Predictive markers are described in the labels for only a minority of the oncology drugs approved in the US since Herceptin in 1998. These markers all measure the status of the drug target or pathway and do not involve complex molecular or protein profiles. This presentation will discuss why this is the case and show how the nature of the clinical trial process, the limited numbers of patients enrolled in clinical trials, and the lack of long-term outcome data have severely limited the impact of molecular profiling in the drug approval process and ultimately clinical practice.
Targeting an embryonic signaling pathway to suppress the metastatic phenotype
Authors: Hendrix Mary J.C. , Seftor Elisabeth A. , Lynne-Marie Postovit , Margaryan Naira V. , Katharine Hardy , Richard Seftor E.B. , Kirschmann Dawn A. , Costa Fabricio F. , Bischof Jared M. , Maria de Fatima Bonaldo , Soares Marcelo B. , Strizzi Luigi
Published: May 2015

The diagnosis of melanoma is becoming more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity, phenotypic plasticity and the expression of embryonic related molecules and signaling pathways that are important for self renewal and pluripotency, present a challenge in targeting the most aggressive tumor cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways. In particular, the embryonic morphogen Nodal, belonging to the TGF-β superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is also expressed in aggressive melanoma, although unlike embryonic stem cells, melanoma does not express Lefty, a natural inhibitor of Nodal, thus allowing Nodal to be expressed in an unregulated manner. Further analysis revealed that Lefty is silenced in these tumor cells by DNA methylation. Down-regulation of Nodal expression in melanoma cells results in a significant reduction in clonogenicity and suppression of tumorigenesis. Additional translational studies indicate that antibodies against Nodal are capable of targeting and inducing apoptosis in human melanoma tumors lodged in the lungs of nude mice. Furthermore, our data show another stem cell signaling pathway, Notch, is expressed in these aggressive melanoma cells, and there is direct evidence demonstrating the molecular cross-talk between Nodal and Notch. Thus, these two prominent stem cell pathways contributing to melanoma cell plasticity may serve as promising dual therapeutic targets for clinical intervention.
Circulating free DNA as a surrogate for tumor material for EGFR and KRAS analysis
Authors: Ellison Gillian , Sherwood James , Gokhale Carolyn , Wallace Andrew , Elles Robert , Ryan Anderson
Published: May 2015

The analysis of a patient's tumor mutation status has become increasingly important. Pharmaceuticals such as gefitinib require that a mutation test is performed prior to treatment to help predict a positive outcome to the patient before the drug is administered. However, obtaining a suitable tumor sample for analysis is not always possible and therefore some patients that may benefit will not receive the drug. The use of easily obtainable samples such as plasma or serum would be ideal if tumor derived material could be readily extracted and contained the same predictive mutations as the source tumor.

We have evaluated circulating free (cf) DNA from plasma, and to a lesser extent serum, from lung cancer patients for EGFR and KRAS mutations using allele specific PCR tests (ARMSTM). The majority of the samples had no matched tumor sample available so the mutation detection rate was compared to the expected frequency of mutations in this tumor and population type. EGFR mutations were detected in 21 of 237 plasma cf DNA samples (8.9%) (expected: 10-15%). KRAS mutations were detected in 11 of 168 plasma cf DNA samples (6.6%) (expected: 17%). Where analysis was performed on the matched cf DNA extracted from serum the mutation detection rate was lower than in plasma. Interestingly the EGFR mutation frequency was higher than KRAS in the plasma samples, although the KRAS mutation frequency was expected to be higher than EGFR mutation frequency in the tumors. This could be due to the frequent amplification of mutant EGFR in lung tumors resulting in more target copies in the cf DNA, the EGFR assays could be more sensitive or perhaps the nature of EGFR mutation containing tumors is different making them shed more DNA or survive longer in the blood. In conclusion, cf DNA derived from plasma is a useful surrogate for mutation detection when no tumor sample is available, but the mutation detection rate is not equal for different mutations in different genes.
A prospective trial of plasma EBV DNA for the screening of nasopharyngeal carcinoma
Authors: Chan K.C. Allen , Hung Emily C.W. , Cheng Anita S.M. , Lai Franco P.T. , Hesselt Andrew C. Van , Woo John K.S. , Chan Paul K.S. , Leung S.F. , Chan Anthony T.C. , Lo Y.M. Dennis
Published: May 2015

Nasopharyngeal carcinoma (NPC) is common in the southern part of China. Making use of the close relationship between Epstein-Barr virus (EBV) infection and NPC, our group has established plasma EBV DNA as a tumor marker for NPC. In this prospective trial, we have investigated the efficacy of plasma EBV DNA analysis for identifying asymptomatic NPC. We screened 1318 subjects with no NPC symptoms with plasma EBV DNA analysis. At recruitment, 69 subjects (5.2%) had detectable EBV DNA in their plasma and 19 of these 69 subjects (28%) had persistently positive results after a median of 16 days. As a comparison, 86 (6.5%) were positive for IgA against EBV viral capsid antigen (VCA). Nasal endoscopy was carried out for all subjects with either a positive EBV DNA or VCA-IgA result. Three subjects were subsequently diagnosed of having NPC. All of them were positive for plasma EBV DNA at recruitment and on follow-up. In contrast, only one of them was positive for EBV VCA-IgA. Interestingly, in one patient, the cancer was too small to be visible in endoscopic examination but was identified in a subsequent magnetic resonance imaging (MRI) examination because of an increasing EBV DNA level. In summary, our results show that plasma EBV DNA is useful for the screening of NPC in asymptomatic individuals and the repetition of the analysis in 2 weeks is useful for identifying individuals with false positive results. This study is supported by the Michael and Betty Kadoorie Cancer Genetics Research Program. This talk is also presented as Poster B3.
Imaging endpoints to predict response to IGF1R/IR inhibition in CRC models
Authors: Serkova Natalie J. , Bradshaw-Pierce Erica L. , Hasebroock Kendra M. , Merz Andrea L. , Pitts Todd M. , Eckhardt Gail S.
Published: May 2015

Colorectal cancer (CRC) represents a major health burden and accounts for nearly 10% of all cancer-related deaths in the U.S. Among the many new pathways being exploited for cancer treatment is the insulin-like growth factor receptor (IGF1R) signaling pathway, which appears to be a robust target in CRC (e.g., hyperactivated in more than 50% of CRC patients). OSI-906 is a novel selective dual inhibitor IGF-1R and IR kinase activities. Despite advances in molecular predictive markers, a substantial proportion of patients selected for targeted therapy has nonresponsive tumors and require additional combination-based regimens. Unlike cytotoxic chemotherapeutic agents, most novel signal transduction modulators (STMs) induce only modest tumor regression, thus there is significant need for establishing functional pharmacodynamic (e.g., therapy response) endpoints for inhibition of signaling via particular pathways. In the present study we established noninvasive imaging-based endpoints for response to OSI-906 in CRC mouse models. Female nude mice were implanted with either of two human CRC cell lines (one sensitive and one resistant) or individual human surgical CRC explants (with unknown sensitivity). Metabolic changes (decreased glucose uptake and lactate production by FDG-PET and proton MRS, respectively) were apparent in OSI-906-sensitive xenografts as early as 2 days post-treatment when no changes in tumor size were yet detected. A significant decrease in tumor size and membrane choline-containing phospholipids by MRI and MRS, respectively, were seen after 27 days of treatment in OSI-906-sensitive xenografts. No changes in FDG-PET, MRS, or tumor progression were observed in OSI-906 resistant xenografts. Our data indicate that noninvasive functional imaging endpoints can provide early and sensitive markers for cytostatic action of IGF1R/IR inhibitors.

This talk is also presented as Poster A14.
Breast Cancer Patients' Fear of Treatment: Results from the Multicenter Longitudinal Study BRENDA II
Authors: Singer Susanne , Blettner Maria , Kreienberg Rolf , Janni Wolfgang , Wöckel Achim , Kühn Thorsten , Felberbaum Ricardo , Flock Felix , Schwentner Lukas ,   for the BRENDA II study group
Published: May 2015

Background: Fear of cancer treatment can become overwhelming. It is important to understand what patients are mainly afraid of and what factors are correlated with intense fear of treatment. Methods: Patients with primary breast cancer (n = 761) completed questionnaires about fear of treatment before surgery (t1), and before (t2) and after (t3) adjuvant treatment. Psychological co-morbidity was assessed using the Patient Health Questionnaire. Logistic regression identified predictors of intense fear of treatment. Results: Patients were most afraid of chemotherapy (mean score 3.5), and fear remained high throughout follow-up; fear of radiotherapy and of surgery was lower and decreased over time (from 2.7 to 2.2, p < 0.0001; and from 2.6 to 2.2, p < 0.0001, respectively). Patients with psychological co-morbidity (odds ratios (OR) 1.7-3.0) and those who had heard reports of negative experiences with cancer treatments from others (OR 3.8-16.2) were more likely to have intense fear of all the treatments. Patients with a previous cancer less often expressed fear of surgery (OR 0.6, 95% confidence interval 0.4-1.0). Conclusion: Fear of treatment, especially of chemotherapy, is prevalent in many patients with primary breast cancer. Patients with psychological co-morbidity and those who have heard reports of negative experiences with cancer treatment are at higher risk of experiencing intense fear.
O reforço do “homem cordial” nas conexões entre senadores e cidadãos nas redes sociais online
Authors: Tavares Daniel Nardin , Quiroga Tiago
Published: May 2015

O presente artigo busca contribuir com as análises das chamadas redes sociais online a partir do viés cultural, especificamente o brasileiro. Com as novas tecnologias de comunicação, abriu-se a possibilidade de ser estabelecido o contato direto e rápido entre representantes e representados no Congresso Nacional, desde que sejam usuários de uma mesma plataforma de comunicação. Com base em levantamento junto às assessorias de comunicação dos senadores da 54ª legislatura, verificamos que a substância cultural é um forte componente das trocas de mensagens presentes no aparato técnico propiciado pelo ambiente online, onde é marcante o reforço de traços do “homem cordial”, de Sérgio Buarque de Holanda, cujas características parecem ser renovadas ou mesmo aprofundadas.
Lâminas de Corte: sobre três estratégias para o encontro com o “humano”
Authors: Efrem Filho Roberto
Published: May 2015

Tomando como objeto de análise a peça teatral O Deus da Fortuna (uma parábola sobre a metafísica do capital), este ensaio procura compreender as intenções políticas e a narrativa dramatúrgica do espetáculo como uma oportunidade de olhar para as representações das relações sociais em que o Coletivo de Teatro Alfenim, autor da peça, intervém artisticamente. Este ensaio discute tais intervenções como estratégias de encontro com o “humano”, problematizando a noção de “humanidade” e explicitando as relações de classe, gênero e sexualidade nas quais os sujeitos criam, fazem-se e conjugam seus verbos.