Biofluid Biopsies Agenda

Liquid Biopsies in the Management of Cancer
Ellen Beasley, Senior Vice President, Genomic Health Inc, United States of America

Solid tumors that originate in internal organs such as the breast, lung, or bladder often release DNA and tumor cells into the bloodstream or urine. When the presence of tumor-derived DNA in blood is high and persists or increases over time, the cancer is likely growing and a new course of treatment may be appropriate.  The ability to reliably measure tumor burden in blood and the evolution of tumor biology over time it will have a transformational impact on the management of cancer.  Patients could forego more invasive procedures like tissue biopsies and would not have to wait months to do screenings like PET scans. In real-time management of cancer patients, similar to cholesterol monitoring, we will have the opportunity to guide patients to the right treatment with the right dose more quickly, improving patient outcomes and potentially avoiding unnecessary side effects and cost.

Liquid Biopsies and the Potential to Make Tissue Less of an Issue
Walter Koch, Vice President, Roche Molecular Systems, United States of America

Blood-based testing can reduce test turnaround time and potentially serve as a surrogate for tissue for biopsy ineligible or fragile patients, for whom a tissue sample is not available.   Potential uses being explored in clinical studies and trials include 1) identification of genetic determinants that direct targeted therapies (eg EGFR activating mutations in NSCLC for several tyrosine kinase inhibitors), 2) evaluation of early treatment responses, 3) monitoring for minimal residual disease and 4) assessment of disease progression and resistance development.   Several technologies are in development for blood based analytes including circulating tumor cells, contents of exosomes released by cancer cells, and cell free circulating tumor DNA (ctDNA).  This presentation will exemplify the potential power of Liquid Biopsy approaches using ctDNA analyses with data from several clinical studies involving NSCLC patients undergoing targeted therapy treatment.

Saliva-Based Detection of Actionable EGFR Mutation in Lung Cancer Patients
David Wong, Felix and Mildred Yip Endowed Chair in Dentistry; Director for UCLA Center for Oral/Head & Neck Oncology Research, University of California-Los Angeles, United States of America

Constitutive activation of epithelial growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small-cell lung carcinoma (NSCLC). EGFR mutation predicts sensitivity to EGFR-targeted therapy and mutation detection is mainly based on tissue biopsy, which is invasive, expensive and time-consuming. Non-invasive, real-time, point-of-care, inexpensive detection and monitoring of EGFR mutations in NSCLC patients is highly desirable. We developed a novel core technology, Electric Field-Induced Release and Measurement (EFIRM), relying on a multiplexible electrochemical sensor that can detect EGFR mutations directly from body fluids. EFIRM for EGFR mutation detection was established in vitro, and correlated with tumor size from xenografted mice. In clinical application, we demonstrated that EFIRM can detect EGFR mutations from saliva and serum of 22 NSCLC patients. And finally, a blinded test was performed on saliva from 40 NSCLC patient saliva samples. The receiver operating characteristic analysis indicated that EFIRM detected the exon 19 deletion with an area under the curve (AUC) of 0.94 and the L858R mutation with an AUC of 0.96. Our data indicate that EFIRM is effective, accurate, rapid, user-friendly, and cost effective for the detection of EGFR mutations in saliva of NSCLC patients.

Plasma Extracellular Vesicle Content for Diagnosis and Prognosis of Cardiovascular Disease
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Cardiovascular Disease (CVD) is with the cardiovascular events of Ischemic Heart Disease and Stroke, the number 1 and 2 cause of death in the world and expect to increase especially in Asia. Atherosclerosis is the underlying syndrome for CVD and atherosclerotic plaques are detectable already in teenagers. Therefore for cardiovascular disease, we have to determine who is at high risk for a cardiovascular event in a high-risk background of the atherosclerotic syndrome without the possibility of taking a plaque biopsy. The risk for Cardiovascular events is region-dependent so tailored plasma-based biomarkers are essential to guide prevention and treatment of billions. Collecting plasma extracellular vesicles is like taking a liquid biopsy from the pathological tissue that can be used for diagnosis and prognosis of the disease. We show that plasma extracellular vesicle protein content especially when collected from different plasma subfractions can be used as an accurate source for diagnosis and prognosis of cardiovascular disease.

The Evil Little Things About Cancer: EVs as Infiltrators and Informants
Xandra Breakefield, Professor, Mass General Hospital (MGH)/Harvard Medical School, United States of America

Tumor cells release an abundance of extracellular vesicles which contain protein, RNA and DNA cargo. Studies are actively ongoing to determine the different types of tumor-derived vesicles and the function of their cargo, which appears to be designed to change the phenotype of many types of normal cells in favor of tumor progression. These evil-intentioned vesicles have two possible Achilles’ heels. In the first case, they can be isolated from biofluids of the cancer patient and used to inform on the mutational stratagem of the tumors and hence provide clues as to effective treatment strategies. In the second case, vesicles may be transformed into therapeutic vehicles to “silently” attack the tumors.

The Utility of Blood Exosomes from Cancer Patients as Biomarkers for Early Detection
Raghu Kalluri, Professor and Chairman, Department of Cancer Biology, University of Texas MD Anderson Cancer Center, United States of America

Ultrasensitive Detection of Circulating Tumor DNA by Deep Sequencing
Maximilian Diehn, Assistant Professor, Stanford University School of Medicine, United States of America

I will describe recent technical and clinical results from analyses of circulating tumor DNA using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq).

Long Noncoding RNAs as Putative Biomarkers for Prostate Cancer Detection: Urine as a Biofluid for Liquid Biopsies
Ranjan Perera, Associate Professor, Scientific Director Genomics and Bioinformatics, Sanford Burnham Medical Research Institute, United States of America

Prostate cancer is one of the leading causes of mortality among US males. There is an urgent unmet need to develop sensitive and specific biomarkers for the early detection of prostate cancer to reduce overtreatment and accompanying morbidity. We identified a group of differentially expressed long noncoding RNAs in prostate cancer cell lines and patient samples and further characterized six long noncoding RNAs in prostatic adenocarcinoma tissue samples (Gleason score >6.0) and compared them with matched normal (healthy) tissues. Interestingly, these markers were also successfully detected in patient urine samples and were found to be up-regulated when compared with normal (healthy) urine.  Chromogenic in situ hybridization assay was developed to detect long noncoding RNAs in primary prostatic adenocarcinoma tissue samples, paving the way for clinical diagnostics. We believe that these results will set the stage for more extensive studies to develop novel long noncoding RNA-based diagnostic assays for early prostate cancer detection and will help to distinguish benign prostate cancer from precancerous lesions.

NanoVelcro-Embedded Microchips for Detection and Characterization of Circulating Tumor Cells
Hsian-Rong Tseng, Professor, Crump Institute for Molecular Imaging, California NanoSystems Institute, University of California-Los Angeles, United States of America

Inspired by the nanoscale interactions observed in the tissue microenvironment, our research team at UCLA pioneered a unique concept of “NanoVelcro” cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of VelcroTM – when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding.  Through continuous evolution, 3 generations of NanoVelcro CTC Chips have been established to achieve different clinical utilities. My presentation will summarize the continuous evolution of NanoVelcro CTC Assays from the emerge of the original idea all the way to their applications in cancer research and diagnosis. We envision that NanoVelcro CTC Assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.

Circulating Tumor Cells: From Enumeration to Molecular Characterization
Nicholas Dracopoli, Vice President/Head, Johnson & Johnson, United States of America

Profiling CTC Heterogeneity in Patients Using Microfluidics
Shana Kelley, Professor, University of Toronto, Canada

The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management.  CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance.  Here, we present a new device for CTC spatial sorting and profiling that sequesters blood-borne tumor cells with different phenotypes into discrete spatial bins. Antibody-functionalized magnetic nanoparticles facilitate CTC sorting, and permit deconvolution of phenotypic subpopulations. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients.  Samples from patients undergoing treatment for prostate, breast, and renal cancers have been analyzed using this approach, and samples collected from xenograft cancer models have also been used for validation of the approach.

Circulating Tumor Cells: The Promise and Reality of the Liquid Biopsy
Minetta Liu, Associate Professor and Chair, Oncology Research, Mayo Clinic, United States of America

The presentation objectives are as follows:  To discuss the potential impact of CTC molecular characterization in clinical practice. To review CTC isolation platforms in the context of demonstrating clinical utility as a liquid biopsy.

Oral and Oropharyngeal Cancer Detection is Spitting Distance Away
Chamindie Punyadeera, Associate Professor, Institute of Health and Biomedical Innovation, Queensland University of Technology, Australia

Oral (OC) and oropharyngeal cancers (OPC) are significantly fatal; only approximately 50% of patients survive to 5 years. Surgery and chemoradiotherapy can leave survivors with pain, disfigurement and disability that further add to the burden of the disease. Unfortunately, more than half of these cancers are only diagnosed at an advanced stage, at which stage their 5-year survival rate plummets. Therefore, there is an urgent need for a reliable early diagnostic method. Current diagnosis is usually made after clinical presentation based on symptoms from the primary cancer and/or when metastatic cervical lymph nodes become obvious. Diagnosis involves biopsy identification of Human Papilloma Virus (HPV) status and histological classification. These diagnostic methods are subjective and become problematic when tumours are too small to be visualised or they are cryptic. These issues commonly result in misdiagnosis. The direct contact between saliva and OC and OPC lesions make the measurement of tumour markers in saliva an attractive alternative to serum and tumour tissue testing.

OncomiRs as Functional Cancer Biomarkers
Frank Slack, Director, Institute for RNA Medicine, Beth Israel Deaconess Medical Center Cancer Center/Harvard Medical School, United States of America

Liquid Biopsies in NSCLC
Oscar Puig, Biomarker Leader, Hoffmann La Roche, United States of America

ALK inhibitors have shown efficacy in Non-Small Cell Lung cancer (NSCLC) patients whose tumors have activating rearrangements comprising ALK kinase. ALK rearrangements are routinely assessed in formalin-fixed samples by FISH, IHC or NGS. Unfortunately, patients progress after therapy with ALK inhibitors because of the appearance of secondary activating mutations in ALK and other escape pathways, which leads to acquired resistance. Quantitative assessment of dynamic changes of ALK rearrangement in plasma could be used as an indicator of initial treatment response, and appearance of secondary mutations could indicate development of acquired resistance.  We will present data from plasma samples of patients enrolled in phase 2 studies treated with the ALK inhibitor alectinib. These results suggest that cell free DNA in plasma is a useful method to assess response and resistance in NSCLC.