PROJECT SUMMARY Project Title: Molecular Kits and Software for Lymphoid Malignancy Work-ups Organization: GigaGen Inc. PI: David S. Johnson, Ph.D. The Specific Aim of this Phase II SBIR proposal is to develop four research use only (RUO) multiplexed molecular kits for comprehensive lymphoid malignancy pathology work-ups. Multiplexed amplification coupled with next generation sequencing (NGS) has a large awaiting market for pathology of lymphoid malignancy, significantly decreasing material and labor costs compared with current methods (Drs. Kerschmann, Zehnder, & Negrin LOS). For example, current work-ups for leukemia oligoclonality require laborious customization, cost up to ~$5000 per subject, and have a turnaround time of several weeks. With around 175,000 new lymphoid malignancies per year, and $1000 per work-up, we estimate that the annual market for our kits in the US alone is ~$175 million. The technical innovation resulting from the Phase I SBIR project (Johnson et al., 2012) was a bioinformatics system for cleaning the nonrepresentative amplification that plagues multiplexed repertoire amplification (Robins et al., 2012). The market innovation resulting from this proposed Phase II SBIR project would be to provide a faster and cheaper system for lymphoid malignancy work-ups that use immune repertoire analysis (GigaMune(R) HemeOnc). In Phase II, we will take the following steps to develop GigaMune(R) HemeOnc: (i) manufacture four original equipment manufacturer (OEM) molecular kits that include amplification master mix, amplification primers, clean-up reagents, and positive control mixes; (ii) implement web-based analysis, QC, and QA software for the four molecular kits; and (iii) use reference samples from the College of American Pathologists (CAP) to quantify the analytical validity of the kits and software. We will be successful in our Phase II project if we achieve the following milestones: (i) across 10 replicate runs at GigaGen, the OEM kits should amplify plasmid positive control mixes such that clonotypes present as low as 0.1% have an average coefficient of variation (CV) of <20% (power=0.8, =0.05) and with an area under the receiver operator characteristic curve (AUC) greater than 0.8 (power=0.8, =0.05); and (ii) across 15 replicate runs at three testing sites, the OEM kits should amplify reference samples such that clonotypes present as low as 0.1% have an average CV of <20% (power=0.8, =0.05) and with an AUC greater than 0.8 (power=0.8, =0.05) and a failure rate <2%. After Phase II is complete, we will market the RUO GigaMune(R) HemeOnc kits and software to pathology labs worldwide. These kits and software will be best in class and facilitate clinical research and development of CLIA laboratory-developed tests (LDTs). In the future, we will explore the advantages of building GigaMune(R) HemeOnc into an FDA-cleared in vitro diagnostic (IVD) kit. For now, the IVD route is not possible, because there are currently no FDA-cleared NGS systems that could be used in conjunction with GigaMune(R) HemeOnc.