Highly Multiplexed Analysis of Solid Tumors and Liquid Tissues by Cyclic Immunofluorescence

PROJECT SUMMARY The long term goal of this proposal is to developvalidate and market robustcost effective technologies for highly multiplexed imaging of cells in liquid and solid biopsies at sub cellular resolution as a means to diagnose diseaseidentify patients likely to benefit from specific anti cancer drugsand investigate mechanisms of action of cytoxictargeted and immuno oncology drugsThis will be accomplished by optimizing a recently developed methodtissue based Cyclic Immunofluorescencet CycIFthat enableschannel imaging of a wide range of tumor types and normal tissuestCycIF uses commercially available antibodies and wide field fluorescence imaging to obtain high resolution images ofiformalin fixedparaffin embeddedFFPEsamples as routinely used by pathologists for diagnosis of disease in routine practice and in clinical trials andiiliquid tissues prepared using the RareCyte AccuCyteblood separation systemIn contrast to other methods for high dimensional imaging of tissuest CycIF is purely optical and therefore has excellent resolutionIt is sufficiently robust and inexpensive that it can readily be implemented in translational research labs and clinical pathology coresThis STTR proposal focuses on turning an academic t CycIF protocol into an industrial grade standard operating procedureSOPon RareCyte s hardware and software platformThis will involve multiple rounds of method development and optimization jointly by RareCyte and HMS scientistsengineers and pathologists culminating in a milestone test on immune cells in solid tumors and human bloodThe test is expected to provide information about how best to combine small molecule and immunotherapy in treatment of melanomaa problem of outstanding interest in cancer biology and precision medicineAimfocuses on process optimizationtransfer of the t CycIF protocol to RareCyte and optimization of staining protocolsimaging procedures and data analysis algorithmspreliminary studies have established multiple ways in which methods used by RareCyte could substantially improve the HMS t CycIF protocolAimfocuses on validation of antibodies for immune profiling of human solid tumors and bloodincluding optimization of fluorophore combinationsthis work will pave the way for Aimand for creation by Rarecyte or its commercial partners of antibody kits for specific applicationsAimfocuses on a critical milestone test in which we will immune profile FFPE samples from multiple patients with metastatic melanoma before and after treatment with dabrafenib and trametinibtargeted MEK RAF inhibitors used in patients with BRAFVE K tumorsWe will quantify changes in tumorstromal and immune cells with therapy and disease progressionA second milestone study will demonstrate the use of CycIF on immune cells in liquid biopsies as a complement to multi parameter flow cytometryThe stretch goal of the current proposal is to match the very high multiplicity of imaging cytometry CyTOFchannelson tissues but using a purely optical method readily deployable in a clinical setting PROJECT NARRATIVE Histological analysis of patient biopsies is the primary tool for diagnosing cancer andincreasinglyfor understanding tumor immune system before and after use of immunotherapiesWe describe a new method for high dimensional histology of solid tissue and describe how the method will be testedimproved and commercialized using state of the art instrumentation and robust chemistry to create a powerful new tool for improving cancer outcomes