bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.