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Posters & White Papers

Development and Characterization of a Novel Liquid Biopsy Reference Material (ctDNA) Based on CRISPR/Cas9-Engineered Cell Lines Mixed at Low Allelic Frequency

Authors:Qi Zheng, Saranya Chandrasekar, Saman Setoodeh, Andrew Hilmer, Deepak Joshi, Gianluca Roma, and Sonika Saddar

Recent advances in Next-Generation Sequencing (NGS) and digital PCR (dPCR) technologies have enabled low allele frequency detection with greater precision, allowing for non-invasive detection of cancer-associated somatic variants from circulating tumor DNA (ctDNA) in blood or plasma (i.e. liquid biopsy). Due to high heterogeneity in cancer cells and the low prevalence of ctDNA in blood and plasma, there is an immediate need for a reproducible source of ctDNA-mimetic reference material for use in assay development, limit-of-detection (LOD) assessment, quality assurance, and proficiency testing. Current commercially available ctDNA reference standards are developed using two principal methods, each with its own limitations. Sonication of cancer cell line DNA does not mimic naturallyoccurring ctDNA fragment properties...

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Development and Characterization of a Multigene Molecular Cytopathology Cell Slides as a Fine Needle Aspirate (FNA)-Mimetic Specimen for Proficiency Testing

Authors:Gianluca Roma, Qi Zheng, and Sonika Saddar

In the era of personalized medicine, with an increasing need for molecular testing, cytologic specimens comprise a crucial component in providing prognostic & predictive information for clinical management of lung cancer patients. Fine needle aspirate (FNA) of the lung is the most common sampling method which provides good quality nucleic acid for downstream molecular testing. However, due to the lack of reference standards mimicking FNA specimens, there is a lack of validation of different assays & standardization of the specimen across laboratories. Cytopathology reference material can be employed for assay development, quality assurance & proficiency panels to validate assay performance & understand cross-site and inter-operator variabilities across different laboratories..

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Engineering of Isogenic Cell Lines Using the CRISPR/Cas9 Technology, And Precise Characterization of Low Allelic Frequency FFPE Cell Line Blocks for Use as Molecular Reference Standards

Authors:Gianluca Roma, Kevin Yoon, Andrew Hilmer, Saranya Chandrasekar, Sonika Saddar, Qi Zheng

1. In the era of personalized medicine, precision diagnostics and molecular profiling significantly influence clinical decisions in therapeutic treatment. 2. It has been established that 3-35% of cancer-related premature deaths can be prevented with better screening methods. 3. Cell line-based reference standards provide a renewable reproducible, and therefore reliable source of control or reference materials.

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Engineering of Isogenic Cell Lines Using the CRISPR/Cas9 Technology, and PreciseCharacterization of Low Allelic Frequency FFPE Cell Line Blocksfor Use as Molecular Reference Standards

Authors:Gianluca Roma, Kevin Yoon, Andrew Hilmer, Sonika Saddar

Cancer is a heterogeneous disease in which hundreds of genes, and many thousands of mutations, have been implicated in oncogenesis. As we enter an era of precision tumor profiling, there is a significant need for molecular reference standards that can be employed for assay development and quality assurance in order to validate assay performance, and understand cross-site and inter-operator reproducibility. Cell-line based reference standards are ideal for this application, since they represent a biologically-relevant, reproducible, and renewable source of control materials. With this in mind, we have engineered a reference bank of cell lines that contain over 100 oncogenic point mutations, insertions, and deletions...

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Engineering of Isogenic Cell Lines & Molecular Reference Standards Using CRISPR/Cas9

Authors: Qi Zheng, Xin Chen, Nga Nguyen, Andrew Hilmer, Yuan Yuan, Jinling Li, Ruby Yanru Chen-Tsai, and Ling-jie Kong

1. In the era of personalized medicine, precision diagnostics and molecular profiling significantly influence clinical decisions in therapeutic treatment. 2. It has been established that 3-35% of cancer-related premature deaths can be prevented with better screening methods. 3. Cell line-based reference standards provide a renewable reproducible, and therefore reliable source of control or reference materials.

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To Obtain More With Less: Cytologic Samples With Ancillary Molecular Techniques-The Useful Role of Liquid-Based Cytology

Authors:Maurizio Martini, Sara Capodimonti, Tonia Cenci, Mirna Bilotta, Guido Fadda, Luigi Maria Larocca, and Esther Diana Rossi

Fine-needle aspiration cytology has been increasingly used as the first tool in the evaluation of several diseases. Although cytology has a relevant role in the discrimination between benign and malignant lesions, conventional slides cannot lead to 100% conclusive results. It was hoped that the introduction of liquid-based cytology (LBC) would improve the efficacy of cytology through standardization, quality improvement, and the possibility of carrying out ancillary techniques on the residual stored material. In recent decades, the application of genomic alterations has been studied on cytologic samples with feasible and reliable results. The molecular analysis offers a powerful aid to define the best clinical or surgical approaches and follow-up for patients. In recent years, the application of different ancillary techniques has been carried out on conventional slides even though LBC represents a useful additional and alternative method for molecular testing.

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Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling

Authors:Sinchita Roy-Chowdhuri, Meenakshi Mehrotra, Ana Maria Bolivar, Rashmi Kanagal-Shamanna, Bedia A. Barkoh, Brette Hannigan, Stephanie Zalles, Wenrui Ye, Dzifa Duose, Russell Broaddus, Gregg Staerkel, Ignacio Wistuba, L. Jeffrey Medeiros & Rajyalakshmi Luthra

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling.

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Cytology Smears in the Era of Molecular Biomarkers in Non-Small Cell Lung Cancer: Doing More With Less

Authors:Lozano MD, Echeveste JI, Abengozar M, Mejías LD, Idoate MA, Calvo A, de Andrea CE1.

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling.

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Molecular Testing of Cytology Specimens: Are We Ready for the New Era?

Authors:Priya S. Vishnubhotla

Our understanding of cellular targets for cancer treatment and biomarkers for cancer detection has expanded exponentially over the last decade. Cytopathology sampling is frequently used in the diagnosis of non-small cell lung cancer (NSCLC),and there are several challenges in procuring and analyzing such specimens for molecular testing. This article explores these challenges.

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Acquired Resistance to Tyrosine Kinase Inhibitors in Non–Small Cell Lung Cancers: The Role of Next-Generation Sequencing on Endobronchial Ultrasound–Guided Transbronchial Needle Aspiration Samples

Authors:Elena Guerini-Rocco, Antonio Passaro, Chiara Casadio, Valeria Midolo De Luca, Juliana Guarize, Filippo de Marinis, Davide Vacirca, & Massimo Barberis

Molecular testing is essential for the diagnostic workup of patients with advanced non–small cell lung cancers. Cytology specimens from minimally invasive procedures, such as endobronchial ultrasound–guided transbronchial needle aspiration, are often the only available samples for these patients. The implementation of molecular diagnostic testing, and in particular next-generation sequencing–based testing, on these cytologic specimens is currently an evolving field for lung cytopathology. The application of these molecular analyses on tyrosine kinase inhibitor–resistant non–small cell lung cancers raises unique technical, biologic, and clinical challenges.

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Precise Characterization of a FFPE Cell Block Developed Using CRISPR/Cas9 Engineered Cell Lines for Use as a Molecular Reference Standard

Authors:Vigneshwaran Mani, Gianluca Roma, Qi Zheng, Debapriya Sengupta, Sravanthi Kondapalli, Suhani Thakker, and Sonika Saddar


: Development and Characterization of EML4-ALK and KIF5B-ALK Gene Fusion NSCLC Cell Line Using CRISPR/Cas9 Technology as a Reference Material for Use with Next-Generation Sequencing Platforms

Authors:Qi Zheng, Gianluca Roma, Debapriya Sengupta, Saman Setoodeh, Nikita Gupta, Suhani Thakker, Vigneshwaran Mani, and Sonika Saddar


Novel Synthetic Plasma Liquid Biopsy (ctDNA) Reference Material

Authors:Qi Zheng, Gianluca Roma and Sonika Saddar


Novel EGFR Liquid Biopsy (ctDNA) Reference Material Development and Characterization using CRISPR/Cas9-Engineered Cell Lines

Authors:Suhani Thakker, Vigneshwaran Mani, Jieyun Cao, Shravanthi Kondapalli, Gianluca Roma, Qi Zheng, and Sonika Saddar


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