Molecular Kits and Software for Lymphoid Malignancy Work-ups

描述(由申请人提供)：这项第二阶段SBIR建议的具体目标是开发四种仅供研究使用(RUO)的多路分子试剂盒，用于全面的淋巴系统恶性肿瘤病理检查。多重扩增结合下一代测序(NGS)在淋巴系统恶性肿瘤的病理学方面有很大的等待市场，与目前的方法(Dr.Kerschmann，Zehnder和Negrin Los)相比，显著降低了材料和劳动力成本。例如，目前针对白血病少克隆的研究需要费力的定制，每个受试者的成本高达5000美元，而且需要几周的周转时间。每年约有17.5万例新的淋巴系统恶性肿瘤，每次检查费用为1000美元，我们估计仅在美国，我们试剂盒的年市场就约为1.75亿美元。第一阶段SBIR项目产生的技术创新(Johnson等人，2012年)是一个生物信息学系统，用于“清理”困扰多重谱系扩增的非代表性扩增(Robins等人，2012年)。这一拟议的第二阶段SBIR项目带来的市场创新将是为使用免疫谱系分析(“GigaMune(R)hemeOnc”)的淋巴恶性肿瘤检查提供一个更快、更便宜的系统。在第二阶段，我们将采取以下步骤来开发GigaMune(R)hemeOnc：(I)生产四个原始设备制造商(OEM)分子试剂盒，其中包括扩增主混合物、扩增引物、清理试剂和阳性对照混合物；(Ii)为四个分子试剂盒实施基于网络的分析、质量控制和质量保证软件；以及(Iii)使用美国病理学家学会(CAP)的参考样本来量化试剂盒和软件的分析有效性。如果我们实现以下里程碑，我们将成功完成我们的第二阶段项目：(I)在GigaGen的10次重复运行中，原始设备制造商试剂盒应该扩增出质粒阳性对照混合，使得低至0.1%的克隆类型的平均变异系数(CV)为&lt；20%(POWER=0.8，�=0.05)，并且接收器操作员特征曲线下的面积大于0.8(POWER=0.8，�=0.05)；以及(Ii)在三个测试点的15次重复运行中，原始设备制造商试剂盒应扩增参考样本，使得出现低至0.1%的克隆类型的平均变异系数为20%(POWER=0.8，�=0.05)，AUC值大于0.8(POWER=0.8，�=0.05)，故障率为2%。第二阶段完成后，我们将向世界各地的病理实验室销售Ruo GigaMune(注册商标)HemeOnc试剂盒和软件。这些试剂盒和软件将是同类中最好的，有助于CLIA实验室开发测试(LDT)的临床研究和开发。在未来，我们将探索将GigaMune(R)HemeOnc构建成FDA批准的体外诊断(IVD)试剂盒的优势。目前，IVD路线是不可能的，因为目前还没有FDA批准的NGS系统可以与GigaMune(R)HemeOnc一起使用。

DESCRIPTION (provided by applicant): 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 non-representative 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.