The AACR 2013 in Washington D.C.

The American Association for Cancer Research (AACR) is the first and largest cancer research organization dedicated to accelerating the conquest of cancer. The stated purpose of the organization when it was founded 115 years ago was “to further the investigation and spread the knowledge of cancer.” Since that time, the AACR has been the driving force to eradicate cancer. Publication of research findings has always been critical to this endeavor, with the 11 founders recognizing the importance of “sharing observations” and the need for a journal to “collect under one cover such contributions as bear in any way upon the general problems of oncology.”The AACR launched its first peer-reviewed scientific journal, The Journal of Cancer Research, in 1916. At the time, it was the only English-language cancer journal in the world. Since then, the AACR portfolio of journals has grown to 10. These journals cover the full spectrum of cancer science and medicine and together, over the years, have published over 100,000 articles and over 130,000 meeting abstracts. This is an immense contribution to the body of knowledge on cancer and has ignited communication among cancer scientists and physicians, helping to catalyze a revolution in the field.We are honored to serve as the current Editors-in-Chief of the AACR journals. Each of the 10 journals was launched by the AACR to meet the scientific information needs of its membership and all those engaged in cancer research. Under our leadership, the journals engage closely and collaboratively with the community to facilitate the presentation and wide dissemination of research findings. Importantly, the AACR is a not-for-profit publisher. This means that any surplus from the AACR journals program is used to help support the cancer research community and the mission of the AACR to prevent and cure all cancers by contributing to funding for AACR grants, scientific conferences, and science policy and advocacy efforts. Here, we highlight the unique roles of our journals within the family of AACR journals and how they serve to advance the mission of the AACR.At the inception of the AACR, there was a clear need for journals focused on cancer research to assemble key studies to help guide the field. Cancer Research was launched in 1941 as the flagship journal of the AACR from a rich ancestry that started with its predecessors: The Journal of Cancer Research (1916–1930) and The American Journal of Cancer Research (1931–1940). From the very beginning, Cancer Research has published landmark papers (https://aacrjournals.org/cancerres/pages/landmark_articles) that have enriched our knowledge of cancer biology and treatment and have provided the foundation upon which major breakthroughs have been built. In the first year of publication, Cancer Research featured seminal studies on the effect of castration on prostate cancer and the mechanism of carcinogenesis that shaped the field (https://www.aacr.org/wp-content/uploads/2019/11/CancerResearch_75Anniversary_1941-2016.pdf). Cancer Research has continued to publish articles that cover the full range of research, spanning from investigations on drug discovery, cancer molecular mechanisms, and the tumor microenvironment to clinical and population studies. As cancer research has evolved over the past century, new AACR journals have emerged to embrace and support expanding fields, providing specialized venues for studies that deepen our understanding of areas that were historically covered by Cancer Research. In this regard, the “pedigree” of AACR journals stemmed from the principles set by Cancer Research, which remains a venue for publishing fundamental studies in all areas of cancer research, including emerging areas such as cancer data science and mathematics. As a society journal, Cancer Research serves the community through its editorial process that is guided by the judgment of international academic editors who provide rich expertise in various areas as practicing researchers. Further, the Journal embraces early career investigators and has recognized these researchers with the prestigious Cancer Research Early Career Award. Guided by its extensive history, Cancer Research hopes to motivate and support the next generation of scientists to lessen the burden of cancer through their innovative research.As cancer science and medicine entered the molecular biology era, it became clear that a communication channel was needed to emphasize basic science studies in oncology—studies that report significant mechanistic findings at the molecular or cellular level. Originally published under the title Cell Growth & Differentiation from 1990 to 2002, Molecular Cancer Research (MCR) was launched as the AACR was beginning to build its now-renowned focus on molecular biology and genetics. The Journal served to help emphasize this focus and attract more scientists into the AACR's membership. As part of this mission, MCR features studies exploring the molecular underpinnings of each of the hallmarks of cancer, with a particular emphasis on studies elucidating oncogenic alterations in metabolism. Cancer metabolism is increasingly recognized as a key vulnerability of tumor biology. MCR is committed to recruiting and disseminating studies featuring novel metabolomic analyses, mass spectrometry, metabolic imaging and tracing, and other approaches that clarify links between metabolism and cancer, with the goal of identifying metabolic vulnerabilities and adding to the armamentarium of anticancer interventions. In addition to publishing impactful basic cancer research studies, the Journal aims to promote diversity of thought and perspective within the basic cancer research community. To achieve this goal, MCR has launched an initiative in which senior investigators are paired with rising stars in their fields to write and edit review articles jointly. This initiative encourages collaboration among researchers by integrating historical perspectives with new directions to provide novel, actionable insights for the field.Cancer Epidemiology, Biomarkers & Prevention (CEBP) is the leading subspecialty journal for fundamental and applied population science research describing the burden of cancer; uncovering possible causes of cancer and its progression; and informing and evaluating strategies for cancer prevention, early detection, cancer survivorship, and closing the cancer disparities gap. CEBP is unique among AACR journals in focusing on populations at risk for cancer, with cancer, and surviving cancer, especially U.S. and global populations who experience health inequities. Among its many contributions, the Journal has advanced the field of molecular epidemiology of cancer by providing expert peer review and a highly regarded publications forum and has helped launch the careers of many investigators who conduct population science research on cancer. CEBP plays an active role in informing public health practice, policy, and recommendations by publishing articles that are cited as part of the evidence base, including by the FDA, the Centers for Disease Control and Prevention, the U.S. Preventive Services Task Force, and the World Health Organization. Recognizing the societal need to enhance the translation of population science evidence for cancer prevention and control today and in the future, the Journal recently expanded its scope to include cancer care delivery and implementation science research. Like the other AACR journals, CEBP serves the research community well through its editorial rigor and publication of innovative and impactful research addressing contemporary and emerging cancer problems.As biologic insights into cancer rapidly accelerated in the decades after the discovery of the structure of DNA and the armamenta­rium of anticancer agents began to expand, the AACR identified the need for a communication outlet focused on the clinical applications of these discoveries; Clinical Cancer Research was launched to provide this much-needed outlet. Early therapeutic development has since transitioned from rigidly divided trial phases into an adaptive process that encompasses investigation of safety, pharmacokinetics, proof of mechanism, and first evidence of clinical activity. These same trials now frequently serve as the basis for first regulatory approval. The Journal bridges the transition from late preclinical development, when a new therapy is being paired with insights into the molecular features that predict responsiveness to therapy, to clinical validation of those hypotheses, and the Journal has evolved into a premier venue for phase I/II trials. As the only AACR journal with clinical research of all types, including clinical trials, as its sole purview, Clinical Cancer Research has taken the charge of being the journal that accounts for what happens to the discoveries described in basic and translational cancer research when they are tested prospectively for their potential impact on the outcomes of patients with cancer. Since its launch, Clinical Cancer Research has also played a distinct educational role among clinical oncology journals, with a robust collection of review articles to benefit basic scientists with clinical interests and clinical investigators studying new agents with unique biologic mechanisms of action. Clinical Cancer Research has also become an important forum for critical discussions in clinical oncology. Field-leading experts are invited to deliberate on the progress made in a given therapeutic area, focusing on the lessons to be learned from recent successes and failures. Authors from the FDA provide detailed insights into the data that drive regulatory decision-making for each successful therapy. Clinical Cancer Research continues to demonstrate the AACR's long-standing commitment to accelerating research that leads to potential interventions for prevention, early detection, and treatment of cancer and particularly to showcasing clinical trials.Improving and expanding the arsenal of anticancer therapies is an essential goal in our shared mission to prevent and cure all cancers. From before the humble beginnings of nitrogen mustards and vinca alkaloids to the advent of immuno-oncology and rationally designed targeted therapy, the last 115 years have seen quantum leaps in therapeutic strategies and clinical options to combat cancer. Molecular Cancer Therapeutics has been at the forefront of this effort for more than 20 years, publishing top-tier science in the design, synthesis, discovery, and preclinical study of novel therapeutic agents for the treatment and prevention of cancer. The Journal's scope covers anticancer therapeutics broadly, including precision medicine therapies of all types: cell therapies, gene therapies, RNA therapeutics, oncolytic viruses, and vaccines in addition to established regimens such as mole­cular and biologic agents, chemotherapy, and radiotherapy. Among these efforts, the Journal provides a specialized venue for the first disclosure of experimental therapeutics advancing toward clinical trials, providing a platform with international reach to authors. The crucial role of Molecular Cancer Therapeutics among the AACR journals—and, indeed, among all oncology journals—cannot be overstated. The Journal's laser focus on disseminating rigorously vetted research with near-term clinical benefit has enormous potential to impact the entire cancer research community and to improve, extend, and defend the lives of patients with cancer. To that end, Molecular Cancer Therapeutics continues to provide a vital forum for the communication of important discoveries in cancer therapeutics research to the global research community.Effective global cancer control must emphasize efforts to reduce cancer incidence rather than concentrating solely on cancer treatment. To help catalyze such efforts, the AACR launched Cancer Prevention Research (CaPR) in 2008 to be devoted excl­usively to cancer prevention research. From its inception, CaPR has endeavored to be a primary resource for original articles, scholarly reviews, and timely perspectives regarding basic, translational, clinical, and population science investigations related to cancer risk reduction. Five years ago, CaPR published the landmark perspective, “Transforming Cancer Prevention through Precision Medicine and Immune-oncology,” which proposed a Pre-Cancer Atlas and recognized unprecedented opportunities to interrogate the biology of premalignancy (1). The subsequent development of the Pre-Cancer Atlas, together with other advances, represents a “moonshot” opportunity for the field of cancer prevention. CaPR will continue to cultivate high-caliber content from pioneers in fields such as carcinogenesis, the biology of premalignancy, cancer risk assessment, screening, and policy implementation that will transform the way we detect and, ultimately, intercept and prevent cancer. The Journal's mission to reflect the current state of cancer prevention and to catalyze the development of the field supports the AACR mission to prevent and cure all cancers. The community of researchers, educators, advocates, and funders that works together under the umbrella of the AACR and its family of journals is a critical part of the cancer prevention ecosystem. Together, we are publishing research that defines the field of cancer prevention, building a translational bridge between bench and community, and developing the next generation of cancer prevention researchers.In 2011, recognizing the cancer research community's need for a journal that published the most impactful research while placing a premium on streamlined editorial processes and service to authors, the AACR launched Cancer Discovery. Bringing together basic scientists with detailed knowledge of cancer biology and physicians at the vanguard of science-driven clinical trial design, Cancer Discovery immediately changed the landscape of scientific publishing in the field, with many other journals now trying to emulate the unique mix of basic, translational, and clinical research that the Journal pioneered. Cancer Discovery has been at the forefront of the most important developments in cancer over the past decade, from targeted therapy and immunotherapy to technological advances in single-cell sequencing and liquid biopsy that have fundamentally changed our understanding of cancer biology and response to treatment. Preclinical and clinical studies published in Cancer Discovery have laid the foundation for numerous FDA approvals within a short period, and influential commentaries and reviews have galvanized the field and guided policy decisions. The unique editorial team consisting of leading researchers and experienced professional editors works collaboratively with the community to quickly publish and disseminate the strongest, immediately impactful science to support the AACR mission to prevent and cure all cancers.The first cancer immunotherapeutic is famously considered to have been administered in 1891 by William B. Coley, one of the 11 founders of the AACR, when he used a mixture of heat-killed bacteria (subsequently known as Coley's toxins) to create an inflammatory response that could treat a patient with sarcoma. However, it took more than 100 years before the field of cancer immunology and immunotherapy research gained meaningful traction among those working in the broader discipline of cancer science and medicine. The rapid expansion of the field began in the early 2000s as a result of increasing evidence linking the immune system with cancer development and demonstrations that the immune system might be harnessed to treat cancer, and it has continued at an even more remarkable pace since the FDA approval of the first immune checkpoint inhibitor, the CTLA4-specific blocking antibody ipilimumab, in 2011. Recognizing the need to further enhance interactions between researchers in the realm of cancer immunology and immunotherapy and those in all other areas of the cancer research community, and to provide a forum for the presentation of advances in the rapidly evolving field of cancer immunology science, the AACR collaborated with the Cancer Research Institute to launch Cancer Immunology Research in 2013. Since its inaugural issue, the Journal has disseminated exciting discoveries and developments in the field of cancer immunology and immunotherapy, and successfully introduced the central principles of immunology to cancer biologists and clinical investigators. As a journal devoted to the science underlying some of the most transformative therapeutics to have entered the clinic for the treatment of cancer in recent years, Cancer Immunology Research has played a unique role in advancing the mission of the AACR.Unprecedented progress in understanding the pathogenesis of hematologic malignancies and in translating these insights into improved clinical outcomes has created a demand for a high-profile outlet for publishing these discoveries. To address this need and to emphasize the AACR's commitment to the prevention and cure of all types of cancer, Blood Cancer Discovery was launched in 2020 to inspire, facilitate, and broadly disseminate important discoveries about hematologic malignancies. The Journal's model of operation replicates Cancer Discovery's success in creating synergies between the expertise of leaders in the field and the dedication of in-house editorial and publishing teams, enabling rigorous, rapid, and transparent editorial processes. Blood Cancer Discovery has published cutting-edge research articles on a broad spectrum of topics encompassing leukemia, lymphoma, myeloma, and other blood cancer subtypes that also have profound implications for our understanding of solid tumors. The Journal has also published all the phases of clinical research: drug development in preclinical disease models, clinical trials, molecular events underlying clinical responses and resistance to therapies, and real-world epidemiology. As a forum for diverse ideas shaping future research directions, Blood Cancer Discovery has published incisive commentaries and has partnered with the AACR's hematologic malignancy-focused Special Conferences, quickly emerging as a thought leader in blood cancer research.The landscape of cancer science and medicine continues to change and accelerate, becoming ever more multifaceted. Launched in October 2021, Cancer Research Communications was designed with these considerations in mind. Most crucially, the Journal is fully open access and boasts the broadest and most flexible scope of any AACR title to date, providing a pathway to publication for authors working within funder mandates or whose work falls outside the scope of other titles in the AACR portfolio. The Journal's nine sections offer a comprehensive view of the spectrum of oncology research. With an editorial ethos centered on accessibility, reproducibility, and value to the field, the Journal is inclusive of a wide variety of important research findings that may be beyond the scope of other journals in the program. Cancer Research Communications is committed to catalyzing the rapid dissemination of rigorously peer-reviewed scientific findings in diversified areas of study, including but not limited to physics, engineering, mathematics, and computational biology. This interdisciplinary approach stimulates the cross-pollination of insight and innovation among cancer researchers. Moreover, the Journal emphasizes flourishing areas of research—such as precision medicine, immuno-oncology, and disparities in cancer health outcomes—that will be foundational to the next 115 years of progress against cancer. As a thoroughly modern platform designed to meet the needs of our authors and readers while complementing the other AACR journals, Cancer Research Communications is poised to stimulate the efficient and dynamic exchange of knowledge, actively sparking new ideas and discoveries to prevent and cure all cancers.Since the very first issue of The Journal of Cancer Research was published in 1916, the AACR journals have been a critical resource for cancer researchers and physicians, providing a forum for disseminating groundbreaking discoveries in cancer science and medicine. As the current Editors-in-Chief of the AACR journals, we are proud that this tradition of publishing seminal, rigorously vetted research that can change the trajectory of the field has established the AACR journals as a trusted partner of the research community. Importantly, we would like to take this opportunity to thank our authors, editorial teams, reviewers, and readers for their invaluable contributions to this endeavor. The tireless efforts of this community provide a beacon of hope to patients and their loved ones.As we look to the future, we continue to collaborate with the research community to ensure that the journals operate with a forward-thinking mentality that adapts to the evolving needs of all stakeholders. In recent years, we have seen critical scientific and technological innovations propel extraordinary progress against cancer. We are confident that this progress will continue, and we remain dedicated to ensuring that the AACR journals continue to provide a platform for the communication of impactful research and support the AACR mission to prevent and cure all cancers.L. Cantley consulted for Petra Pharmaceuticals, Agios Pharmaceuticals, EIP Pharmaceuticals, Volastra, Larkspur, and Cell Signaling Technologies over the past year. He also received grant support from the NIH/NCI, Gray Foundation, The Mark Foundation, the Breast Cancer Research Foundation, and Stand Up To Cancer/American Association for Cancer Research over the past year, and is a stockholder in Agios Pharmaceuticals, EIP Pharmaceuticals, Cell Signaling Technologies, Volastra, and Larkspur. R. Dalla-Favera is a consultant for NeoGenomics and AstraZeneca and receives research funds from AstraZeneca. L.A. Diaz is a member of the board of directors of Jounce Therapeutics and Epitope and is a compensated consultant to PetDx, Innovatus CP, Se'er, Delfi, Blackstone, Kinnate, and Neophore. He is an inventor of multiple licensed patents related to technology for circulating tumor DNA analyses and mismatch repair deficiency for diagnosis and therapy; some of these licenses and relationships are associated with equity or royalty payments directly to the inventors. He holds equity in Epitope, Jounce Therapeutics, PetDx, Se'er, Delfi, Kinnate, and Neophore; he divested his equity in Personal Genome Diagnostics to LabCorp in February 2022 and he divested his equity in Thrive Earlier Detection to Exact Biosciences in January 2021. His spouse holds equity in Amgen. The terms of all these arrangements are being managed by Memorial Sloan Kettering in accordance with their conflict of interest policies. K.T. Flaherty reports personal fees from Clovis Oncology, Checkmate Pharmaceuticals, Strata Oncology, Kinnate Biopharma, Scorpion Therapeutics, PIC Therapeutics, Apricity, Tvardi, ALX Oncology, xCures, Monopteros, Vibliome, Soley Therapeutics, Quanta Therapeutics, Nextech, Takeda, Novartis, OMRx, and Transcode Therapeutics and other support from Roche/Genentech outside the submitted work. P.D. Greenberg reports grants and personal fees from Juno Therapeutics; grants from Lonza; personal fees and other support from Earli, Metagenomi, Elpiscience, Rapt Therapeutics, and and grants, personal and other support from outside the submitted work. reports personal fees from reports personal fees from Oncology, Memorial Sloan Kettering Cancer The Institute for AACR, and Cancer Research grants, personal and support from and grants and personal fees from and Jounce Therapeutics; and personal fees and support from outside the submitted work. reports personal fees from and Therapeutics; grants from Novartis, Therapeutics, and and personal fees and grants from and outside the submitted work. were by the other authors.

*These authors contributed to the devlopment and review of this manuscript but are unable to endorse the request for NIH funding. On Sept. 20, 2011, the American Association for Cancer Research (AACR) released its inaugural AACR Cancer Progress Report to commemorate the

Background: Presence of EpCAM+ cytokeratin+ nucleated circulating tumor cells (CTC) in metastatic carcinoma patients is associated with poor survival and may be used to guide treatment. Assessment of treatment targets on these CTC holds the promise of a liquid biopsy. However the proportion of patients in which a sufficient number of CTC are detected in 7.5 mL of blood is not sufficient. In this study we estimate the number of CTC in blood of patients with metastatic disease, explore the relationship with survival and determine whether assay modifications and/or increases in sample volume are needed to achieve the required increase in number of CTC detected. Methods: EpCAM+CK+DNA+CD45- CTC enumeration was performed with the CellSearch® system in 7.5 mL of blood of 836 patients with metastatic breast, colorectal and prostate cancer patients (Cristofanilli, NEJM 2004, Cohen, JCO 2008, De Bono, CCR 2008). EpCAM+, CD45-, Nucleic acid+ CTC were enumerated in 100ul of NH4CL lysed blood from 140 metatatic cancer patients and compared to CTC as enumerated with the CellSearch system. Image analysis for automated CTC enumeration and modeling of CTC frequencies was performed in Matlab. Results: The median number of CTC in 7.5mL of blood metastatic breast, colorectal and prostate is 5 and in 39% of patients no CTC are detected. The best fit of the CTC frequency distribution was used to extrapolate the sample volume to 5 liters of blood and predicted that 99% (95% confidence interval, CI, 95-99.8%) of patients had at least 1 CTC before initiation of therapy, which decreased to 97% (95% CI 87-99.5%) after the first cycles of therapy. One hundred CTC per liter of blood are present in ∼80% of patients, 1 CTC per ml of blood in ∼40% of patients and 10 CTC per ml of blood in ∼20% of patients. The median survival of patients with CTC is reduced by 6.6 months for each tenfold CTC increase. CTC definitions that do not include a requirement for presence of DNA, EpCAM, cytokeratin 8,18,19 (CK) or absence of CD45 are less predictive of survival, than the strictest definition requiring a CTC to be EpCAM+CK+DNA+CD45-. Comparison of CTC detected by flow cytometry to the CellSearch CTC method on 140 patients shows that improvements in EpCAM recovery may increase the average number of detected CTC by 6.5 fold (95% CI 5.6-7.3), yet the fits predict that this will only reduce the number of patients with 0 CTC from 39% to 21% (95% CI 13-31%). Conclusions: EpCAM+CK+DNA+CD45- CTC are present in blood of all patients with metastatic breast, colorectal and prostate cancer. The predicted strong relation with survival suggests the importance of this phenotype for metastasis. To use CTC as a liquid biopsy for the majority of patients, the CTC yield needs to be improved 100-1000 fold. This requires a dramatic increase in sample volume which may be achieved by in vivo flow cytometry, or through processing an apheresis product. 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 2391. doi:1538-7445.AM2012-2391

Background: The concept of “liquid biopsy” refers to analysis of rare cells in the blood such as Circulating Tumor Cells (CTC) and represents a powerful tool for molecular characterization of tumors for which biopsies are not available. Because of a low frequency of CTCs, assay sensitivity for mutational analysis of CTCs needs to be below 1%. An activating mutation in the serine/threonine kinase BRAF (V600E) is present in up to 70% of melanomas and 10% of colon carcinomas. Herein, we developed and validated a highly-sensitive allele-specific PCR (AS-PCR) assay to detect the presence of the BRAFV600E mutation in CTCs. Methods: Genomic DNA isolation was optimized for rare cell analysis. AS-PCR for detection of BRAFV600E mutation was developed and tested, using cell spiking experiments to define assay sensitivity, limit of detection, assay precision and specificity. Use of the CTC BRAFV600E AS-PCR assay was demonstrated in melanoma and colon cancer patient blood. Results: Performance of the BRAFV600E AS-PCR was tested using gDNA extracted from HT-29 colon cancer cells heterozygous for BRAFV600E, and BRAFwild-type MDA-MB-231 breast cancer cells. Assay sensitivity and limit of detection for determination of BRAFV600E in a background of BRAFwt was tested by spiking HT-29 cells into 50,000 MDA-MB-231 cells at following ratios: 5000 (5% mutant DNA copies), 500 (0.5%), 50 (0.05%), 10 (0.01%), 5 (0.005%) and 0 (0%). The assay limit of detection was found to be 0.01% or 5 BRAFV600E mutant copies in a wt background. Precision was demonstrated by repeating assay sensitivity testing at the limit of detection on three different days. The performance and specificity of the BRAF AS-PCR assay in enriched CTCs was further verified using HT-29 and MDA-MB-231 cells spiked into donor blood and isolated by EpCAM-based CellSearch® CTC Profile kit, which we have previously shown to yield a higher number of CTCs than the standard CellSearch® CTC enumeration kit. Finally, CTC BRAFV600E AS-PCR assay performance was demonstrated in patients with colon cancer as well as melanoma, in which CTCs were isolated using a previously developed melanoma immunomagnetic enrichment panel. Conclusions: We have developed an AS-PCR assay for detection of the BRAFV600E mutation in CTCs. Validation testing demonstrated that the assay detects 0.01% or 5 copies of BRAFV600E DNA in a wild-type background. Testing melanoma and colon cancer patient blood specimens demonstrated feasibility of the BRAFV600E mutation analysis using liquid biopsy. Further concordance testing between tumor biopsies and blood specimens is ongoing and will establish whether liquid biopsies are a suitable alternative for identifying patients for whom a particular targeted therapy may or may not be effective based on genetic status. 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 3159. doi:10.1158/1538-7445.AM2011-3159

Head and neck squamous cell carcinoma (HNSCC) frequently involves metastasis at diagnosis. Circulating tumor cells (CTCs), captured as “liquid biopsy”, can provide real-time clinical information on prognosis, therapy choice and drug response and resistance. Cyclooxygenase 2 (COX-2), a key enzyme involved in prostaglandin production, participates in inflammatory and neoplastic processes. It is expressed by various tumours and contributes to carcinogenesis. Previous study showed that expression of COX2 in HNSCC tumor tissue samples is associated with clinical response to radiotherapy. The aim of study is to investigate whether expression of COX2 in CTCs could be used as a predict factor to clinical response to radiotherapy.An multiplex RNA in situ hybridization (RNA-ISH) method, named CanPatrol CTC enrichment technique, was used to isolate and categorize CTCs as epithelial, biophenotypic epithelial/mesenchyma, and mesenchymal phenotypes. A total of 51 pateints with HNSCC were enrolled into this study and assessed for CTC levels. Of these 51 pateints, 13 patients were evaluated for COX2 expression in CTCs before and after radiotherapy. The COX2 expression in one CTC or more was defined as positive presence. Primary end point was the overall objective response rate. At baseline, CTCs, ranging from 1 to 15, were identified from 36(70.59%) patients, and the median and mean values were 2 and 3(±3 SD) respectively. No significant correlations were observed between CTCs amounts and clinicopathological parameters. Thirteen (25.49%) patients had ≥2 CTCs both at the beginning and the end of treatment. COX2 expressions was identified in 6(46.15%) patients, which associated with ascending mesenchymal rate afterwards, and indicate a trend of less response to radiotherapy. Compared to 46.15% (6/13) of COX2-positive patients, disease control characterized as decreased CTCs/ mesenchymal CTCs after systemic radiotherapy was obtained in 53.85% (7/13) of COX2-negative (p< 0.05). In summary, COX2-positive CTCs is a promising predictive factor for response to radiotherapy. Citation Format: Feng Wang, Xiaoqi Xie, Zhen Wang, Ping Li. Correlations of COX2 expressions in circulating tumor cells (CTCs) and response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1811. doi:10.1158/1538-7445.AM2015-1811

Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having <75 CTCs. For samples with high CTC counts (>75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of “non-canonical” tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types. Citation Format: David Chianese, Mark Connelly, Carrie Morano, Thai Bui, Steven Gross, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2422.

Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was >30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (>50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2015-363

Introduction: Liquid biopsies and cell-based therapies are emerging approaches for the treatment of cancer. Accuracy of diagnosis using liquid biopsies and effectiveness of treatment using cell-based therapies will rely upon the preservation cells for both viability and the information contained in them. Experimental procedures: Biomatrica's proprietary chemical library was used to develop formulations that could preserve cells in multiple formats. Cell preservation was assessed by a variety of cell-based and molecular techniques such as microscopy, flow cytometry and real-time PCR. Results: Blood samples spiked with circulating tumor cells (CTCs) were preserved for several days at room temperature to allow for downstream morphological and transcriptomic analyses. CTC recoveries in preserved samples were as much as 4 times those in non-protected samples over 4 days. Changes in RNA expression of CTC-specific genes in preserved samples ranged from 0 to +/- 2 delta Ct values, while in non-protected samples, changes ranged from 0 to +/-5 delta Ct values over 4 days. In some formulations, CTC viabilities were as much as twice the viabilities of CTCs in non-protected samples. Supporting these viability results was a demonstrated culturability of isolated CTCs. Furthermore, cell-free DNA in plasma of preserved blood samples changed by less than 2 Ct values over 4 days, whereas in control samples, plasma DNA changed by as much as 10 Ct values. Hemolysis in preserved blood samples was also lower by as much as 69% compared to non-protected samples. In a separate set of experiments, activated T-cells were stored in the presence of common buffers (RPMI, PBS) or formulations at room temperature. T-cells cultured with formulations displayed greater 40 to 60% greater cell viability after 3 days compared to those stored in common buffers. Conclusions: Biomatrica has developed a library of cell preservatives that can be used to preserve cells in blood samples and in cell culture. The ability to recover cells in blood samples and determine their genetic information would enable monitoring of a patient's cancer status and personalizing drug treatments. In addition, cell preservation may allow further advances in cell-based therapies as cells could be more reliably recovered from patients, modified (e.g. CAR-T, stem cells), and administered to patients. Citation Format: Cecille Browne, Dan Lu, Daniela Roth, Vasco Liberal. Preservation of cells in blood and culture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 401.

Increasing evidence confirms the prognostic relevance of Circulating Tumor Cells (CTCs) in a variety of cancers including advanced breast cancer. Recent data also suggests that CTCs are a useful tool for monitoring treatment and identifying potential targets for therapeutic intervention. The objective of this study was to investigate technologies that can be used for defining the genomic landscape of CTCs in order to compare the derived genomic information (1) among CTCs to assess genetic heterogeneity and (2) with that derived from bone-marrow metastasis tissue biopsy (BMM) samples to assess how reflective the molecular profile from CTCs is to the metastasis. To evaluate these potential applications, positive CTCs were identified using the AccuCyte -CyteFinder (AC/CF) system (RareCyte, Seattle WA) from the blood of a patient with triple negative breast cancer (TNBC). Twenty CTCs and twenty white blood cells (WBCs) were picked from slides using the AC/CF system. To determine whether tumor specific genomic characteristics are reflected in the isolated cells, we used whole genome amplification (WGA) followed by next-generation sequencing to perform a comprehensive analysis using WBCs as genome controls. Whole genome, exome and targeted sequencing of known cancer-associated genes using Illumina and Life Tech panels identified mutations in TP53, PTEN, ERBB2, STK11, ABL1, HRAS, MLL2 and INPPL1 which were present in the CTCs alone and not in the WBCs. Further analysis comparing the results between CTCs revealed that the majority of the identified mutations were specific to individual CTCs revealing a high degree of genetic heterogeneity. Only 5% of mutations were shared between at least 40% of the CTCs examined and included mutations in ATM, ALK, BRAF, NOTCH1, ATR, JAK3, COL1A1 and XPC. Additionally, all of the profiled CTCs contained two novel mutations in LPP and HLA-A which were not present in the WBCs. Mutations in these genes have recently been associated with aggressive solid tumors. Genetic heterogeneity was also observed in the WBC population enabling the calculation and subsequent subtraction of background noise associated with WGA of single cells. Molecular information derived from the CTCs is being compared to multiple BMM samples from the same patient. Additional analyses of copy number and structural variations and transcriptomic analysis are being performed in order to gain further insights into the genetic heterogeneity of CTCs and identify genomic markers to establish the utility of CTCs as a non-invasive real-time liquid biopsy for breast cancer. Citation Format: Kellie Howard, Sharon Austin, Arturo Ramirez, Leila Ritter, Debbie Boles, James Cox, Fang Yin Lo, Kerry Deutsch, Christopher Subia, Tuuli Saloranta, Nicole Heying, Heather Collins, Amanda Leonti, Lindsey Maassel, Jackie Stilwell, Eric Kaldjian, Michael Dorschner, Anthony Blau, Marcia Eisenberg, Steven Anderson, Anup Madan. Comprehensive multi-omic analysis of circulating tumor cells isolated from a metastatic triple-negative breast cancer patient to identify pathogenic genomic aberrations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2015-1609

Introduction and objective: The presence of circulating tumor cells (CTC) is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. The aim of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by gene profiling using the NanoString low RNA input nCounter assay to achieve reliable CTC gene profiling. Methods: Blood samples were collected from 4 mPCa patients and 3 healthy donors. In the LP procedure, different blood elements were separated based on density. The mononuclear cell layer (presumed to contain any CTCs) was collected. The Alere™ CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method for isolation of EpCAM+ CTCs, followed by RT-qPCR for PSA, PSMA, and HOXB13 was used to detect CTCs from LP. The Ct value thresholds for positive CTC signal by RT-qPCR were set based on healthy donor LP subjected to AdnaTest as a negative control (PSA 36.3, PSMA 36.82, HOXB13 40). The cDNA of cells captured by EpCAM+ selection (including putative CTCs) and of the EpCAM+ depleted white blood cells (WBC) were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of cDNA and overnight hybridization with the PanCancer Progression panel. To validate the assay, healthy donor LPs were spiked with known numbers of PCa cell line LN95 cells. Results: For mPCa patients, the LP procedure took 136.2±10.6 min, with a screening volume of 10L of peripheral blood. The mean volume of mPCa LP product was 123.64±12.23 ml. The WBC concentration was 69.75±28.2×109/L. All samples were subjected to AdnaTest in triplicate and CTCs were identified in 3 out of 4 patients. The positive Ct values of PSA were 33.86±0.81, 32.14±0.31 and 33.09±0.34; PSMA were 35.55±1.02, 33.55±0.83 and 35.30±0.65; HOXB13 were 36.25±0.34, 37.15±0.22 and 37.09±0.33. To assess profiling of CTCs isolated from LP using the nCounter platform, 100, 500, and 1000 LN95 cells were spiked in healthy LPs and were analyzed with the AdnaTest. Within each spiking group, pre-spiking WBC, captured CTC, post-capture WBC, and LN95 cells in PBS alone were subjected to the Nanostring assay. Eisen Cluster analysis separated pre- and post- WBCs, captured CTCs, LN95 cells into three groups. The gene expression of captured CTCs correlated with those of LN95 cells, and the correlation increased with the spiking cancer cell number (R2=0.45, 0.57, and 0.70, respectively). The gene expression of pre-spiked WBCs were highly correlated with those of post-capture WBCs regardless of spiking cancer cell number (R2=0.83, 0.84 and 0.85). Conclusions: Analyzing CTCs in LP samples by AdnaTest is feasible and offers a promising method for liquid biopsy of mPCa patients. The NanoString low RNA input nCounter assay can provide reliable gene expression profiling of CTC. Citation Format: Liang Dong, Chung-Ying Huang, Changxue Lu, Diane Reyes, Sarah R. Amend, Jun Luo, Kenneth Pienta. Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5365.

Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis. CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Hyeun Joong Yoon, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Diane Simeone, Sunitha Nagrath. Application of label-free microfluidic technologies for the enrichment, expansion and characterization of circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1594. doi:10.1158/1538-7445.AM2015-1594

Introduction: Current CTC detection platforms require the processing of patient blood within 48 hrs after collection to preserve CTC integrity and biomarker expression. Here we demonstrate the recovery of CTCs from banked patient blood samples that have been cryopreserved for up to 7 years. Our results show that cryopreserved CTCs have equivalent morphology and protein expression patterns as CTCs in freshly processed blood and are suitable for genomic analyses. This process enables retrospective studies of banked patient blood samples inclusive of CTC enumeration, biomarker quantification, FISH and genomic analysis via the Epic Platform. Methods: 30 cryopreserved mCRPC patient blood samples were sent to Epic Sciences alongside 10 matched fresh blood draws (<48 h after draw). All nucleated cells from mCRPC patient samples or cell line-spiked healthy donor (HD) controls were plated onto glass slides and subjected to IF staining, CTC identification by fluorescent scanners and algorithmic analysis. Traditional CTCs (CK+, CD45-, intact DAPI nuclei and morphologically distinct) and CK- CTCs (CK-, CD45-, intact and distinct) were identified. Androgen receptor (AR) expression was assessed by IF staining and PTEN status determined by FISH. Relative CTC enumeration, morphology, protein expression and genomic status were compared between freshly processed and cryopreserved samples. Results: Initial testing in VCaP (AR+) and PC3 (AR-) cell line-spiked HD blood demonstrated preservation of cell morphology and biomarker expression. In VCaP, CK signal decreased 20% and AR decreased 6% after freezing, but CK and AR expression were 90- and 8-fold higher than negative controls, respectively. In patients, CTCs were detected in 7/10 freshly processed samples and in equal relative abundance in 6/10 matched frozen samples. Cryopreserved CTCs retain CK and AR expression as compared to freshly processed samples: there were no significant differences in mean CK or AR expression among all matched samples compared, and% AR+ CTCs correlated strongly between fresh and frozen samples (r2 = 0.96). Furthermore, PTEN FISH on cryopreserved CTCs resulted in a near-equal percentage of PTEN variant cells as compared to matched fresh draws. After concordance studies, an additional 20 frozen mCRPC patient samples were processed: CTCs were detected in 16/20 samples, with 11/20 harboring AR+ CTCs. Conclusion: Recovery of CTCs in cryopreserved patient blood samples is feasible using the Epic Platform. These data demonstrate concordance of CTC recovery, protein characterization and genomic analysis in an initial cohort of cryopreserved mCRPC samples. Further studies characterizing cryopreserved CTCs in mCRPC and in cancers of other tissues are warranted, with the goal of enabling retrospective protein and genomic characterization of banked patient samples. Citation Format: David Lu, Melissa Harvey, Ravi Madan, Christopher Heery, Jennifer Marte, Sharon Beasley, Mark Landers, Rachel Krupa, Jessica Louw, Justin Wahl, Natalee Bales, Dena Marrinucci, Jeffrey Schlom, James Gulley, Ryan Dittamore. Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2015-1586

Introduction: Monitoring CTCs from a blood draw could enable a minimally invasive, longitudinal observation of an evolving disease. Antibody drug conjugates (ADC) are a promising approach to deliver highly potent chemotherapeutics to cells expressing target proteins. An ADC that targets 5T4, an oncofetal antigen expressed on tumor initiating cells, is currently in clinical development. Patients with tumors or CTCs expressing 5T4 may benefit from this therapeutic. We sought to develop a 5T4 CTC test and to evaluate the incidence of 5T4 expressing CTCs in breast, ovarian, and lung cancer patients. Experimental Procedures: Blood samples from breast (n = 10), ovarian (n = 10) and NSCLC (n = 25) patients on active therapy were collected and shipped to Epic Sciences. Upon receipt, nucleated blood cells were plated onto glass microscope slides and stored at -80C. Two slides per patient were thawed and stained for cytokeratins (CK), CD45, 5T4, and DAPI. CTCs were detected using a combination of CD45 exclusion, CK and 5T4 expression, and morphology parameters. CTC enumeration and 5T4 expression were analyzed alongside pathology, staging and treatment information. Results: Assay Development: A novel CTC assay utilizing an antibody against 5T4 was developed. Dilutions of high, medium and low/negative 5T4 expressing cell lines were spiked into normal whole blood to assess the linearity (R2 = 0.99) of CTC detection. These cell lines as well as 5T4 high and 5T4 low patient samples were used to assess intra- and inter-assay reproducibility of% 5T4(+) CTCs. All patient and calibration control samples met acceptance criteria of ≤ 30% CV and ≤ 20% CV, respectively. Breast Cancer: 7 of 10 patients had traditional CTCs (median: 6/mL range: 0-23/mL). 9 of 10 patients had additional subpopulations of CK(-) CTCs, apoptotic CTCs, and CTC clusters. Among non-apoptotic CTCs, 5T4 positivity was detected in 6 of 10 patient samples, with the highest% positivity seen in patients with corresponding ER(+) primary tumors. Ovarian Cancer: 3 of 10 patients had traditional CTCs (median: 0/mL, range: 0-41/mL). Additional subpopulations were identified in the same 3 of 10 patients. NSCLC: 14 of 25 patients had traditional CTCs (median: 1/mL, range: 0-45/mL). 20 of 25 patients had additional subpopulations. Conclusions: We have developed a robust, sensitive and specific assay to quantify 5T4 expression on CTCs and tested this assay on small cohorts of breast, ovarian and lung cancer patients on active therapy. CTCs expressing 5T4 were detected in a subset of patients from each indication. Citation Format: Steven R. Pirie-Shepherd, Iman Jilani, Eric Tucker, David Valenta, Ryon Graf, Amanda Anderson, Dena Marrinucci, Puja Sapra, Eric L. Powell. Circulating tumor cell (CTC) detection and 5T4 characterization in breast, ovarian, and lung cancer patients on active therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2015-565

Head and neck squamous cell carcinoma is leading cancer in the Indian subcontinent with Oral squamous cell carcinoma (OSCC) as the most frequent subtype. Conventionally OSCC is classified as a locoregional disease and its increased frequency is attributed to lack of good biomarkers compared to other epithelial cancers. At the time of diagnosis, above 50% of cases present the manifestation of advanced-stage disease, and are predisposed to disease failure in spite of appropriate treatment. Thus, early diagnosis of OSCC can significantly reduce the disease burden. Here we describe a novel, regulatory approved method to establish Circulating tumor cells (CTCs) presence in peripheral blood of OSCC Indian patients and its positive correlation with various clinicopathological parameters, suggesting the potential use of CTCs as a significant parameter to stratify oral cancer with respect to the disease advancement. Methods: In a cross-sectional observational study, 230 OSCC patients at the different pathological stage of the disease and treatment mode were enrolled. CTCs were isolated using approved OncoDiscover liquid biopsy technology (Drug controller general of India approved)', platform technology based on immunomagnetic CTC enumeration. CTCs were detected for cytokeratin 18 (CK18 marker) presence and well-defined, DAPI-stained nuclei. Enumerated CTC subsequently analyzed for various clinic-pathological parameters such as pstage, extra-capsular spread (ECS), lymphovascular emboli (LVE), perineural invasion (PNI) and depth of invasion (DOI). CTC cut off values were obtained to differentiate early vs advanced stages with respect to different clinical stages and parameters.Results: CTCs obtained from peripheral blood of OSCC patients correlated positively with the cancer stages (clinical as well as pathological) as well as aggressive pathological features such as presence or absence of ECS, LVE, PNI and DOI. Interestingly, with the presence of aggressive pathological features that often suggest the poor outcome of the disease, we observed a 25-50 % increase in CTC number. On the other hand early stage, treatment naïve patients had a lower number of CTCs. Mean CTC number in advanced-stage patients was 50 % higher than early-stage OSCC patients. Conclusions: Efficient and rapid isolation of CTCs from peripheral blood of OSCC patients using proprietary technology suggested a positive correlation between CTC number and pathological stages of the disease. Considering a positive correlation of CTC number with various pathophysiological features, CTC can be contemplated as a reliable parameter to predict the disease outcome in oral cancer. The consistent presence of CTC across all disease stages also suggests a probable nature of OSCC as a biological systematic disease. Citation Format: Jayant Khandare, Burhanuddin Nuruddin Qayyumi, Atul Bharde, Gourishankar Aland, Sreeja Jayant, Swati Tripathi, Nitin Singh, Reecha Badwe, Alain D'Souza, Balram Singh, Smriti Arora, Narendra Kale, Aravindan Vasudevan, Kumar Prabhash, Pankaj Chaturvedi. CTCs positively correlate clinical manifestations in Indian oral cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-148.

Background: Recent reports suggest that circulating tumor cells (CTCs) mimic cancer progenitor cells, which are commonly resistant to various treatment modalities and are capable of invasion and tumor initiation at a single cell level. We hypothesized that CTCs isolated from metastatic breast cancer (MBC) patients can be propagated in 3-D in vitro systems, representing a clinically relevant model to study MBC biology and to test efficacy of multiple anti-cancer drugs. Methods: CTCs were isolated from MBC patient peripheral blood by depleting leucocytes and red blood cells. Enriched CTCs were immunostained for detection as CD45-negative (-) and nuclear counter stain-positive (+) cells, and for expression of estrogen receptor (ER) and HER-2. In order to obtain a large and continuous supply of CTCs for our culture studies, we screened 19 primary transplantable xenograft lines, established by directly transplanting human breast tumors into epithelium-free mammary fat pads of SCID/Beige mice, for their production of CTCs. The CTCs were isolated together with mouse mononuclear blood cells by either gradient centrifugation or red blood cell lysis, and identified by human pan-cytokeratin immunostaining. Viable CTCs isolated from both patients and xenograft mice were plated in mammosphere conditions either alone or in co-culture with mammary fibroblasts tagged with green fluorescent protein (GFP). the propagated CTCs were identified as GFP-, cytokeratin 19+ cells. Results: To date, we have collected and processed blood samples from 14 MBC patients. CTCs were detected in 5 out of the 8 samples (62%) already analyzed. Interestingly, in 2 out of 4 patients (50%) with positive CTC counts and ER+ primary tumor, all the isolated CTCs were ER-. Moreover, we found HER-2+ CTCs in 1 out of 4 patients (25%) with HER-2 negative disease. CTC count and characterization of the additional collected samples is ongoing and will be presented at the conference. In the mice bearing primary xenografts, we detected CTCs in 13 out of 19 lines (68%) and in 26 out of 54 mice (48%). When CTCs isolated from either human patients or primary xenografts were cultured alone, no mammospheres were observed. In contrast, when CTCs were cultured in direct contact with fibroblasts we observed the formation of mixed CTC/fibroblast spheres that may reproduce the tumor cell/stroma interaction observed in vivo. Moreover, in the presence of GFP+ fibroblasts, the numbers of GFP- cells and cell aggregates were clearly higher at all time points compared to the single culture. Conclusions: Our study suggests that viable CTCs isolated from human patients and primary xenograft bearing mice can be propagated in vitro in presence of mammary fibroblasts. The successful development of a CTC-based cell culture platform will provide a unique model to better understand the biological mechanisms of the metastatic process and to guide treatment decisions in breast cancer. 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 LB-305. doi:1538-7445.AM2012-LB-305