Rare Protein Detection on Cell Surfaces: Ultrasensitive Detection of B-Cell Chron

描述(申请人提供)：ROR1细胞表面生物标记物在B细胞慢性淋巴细胞白血病(B-CLL)细胞上表达，但在B-CLL患者的健康B细胞或其他白细胞(WBC)上不表达。我们建议开发一种廉价、易用、快速、超灵敏的ROR1特异性检测方法，用于检测和监测一滴血中的B-CLL。我们的目标是达到比目前可用的方法更好的检测水平，即最灵敏的流式细胞术(MRD-FC)或相当于可用于检测微小残留病(MRD)的聚合酶链式反应(ASO-PCR)方法。早期发现MRD对于管理治疗和检测耐药恶性肿瘤的出现至关重要。目前的方法在仪器、试剂和对训练有素的技术人员的需求方面都很昂贵。此外，FC方法不能很好地区分B-CLL和其他克隆性B细胞恶性肿瘤，如套细胞淋巴瘤和脾边缘带淋巴瘤。目前的检测需要静脉穿刺法采集1-3mls的血液，FC需要数小时，ASO-PCR需要一周。JBI拥有一种平台技术来解决这些缺点。JBI首次使用其技术导致了ZivaTM超灵敏的BrdU细胞增殖试验。只用了50&lt；L的样本，Ziva就在105个非增殖细胞中检测到1-4个增殖细胞，这是其他人从未接近过的说法。将这种灵敏度应用于B-CLL的检测，将使其成为最敏感的ASO-PCR方法的一个有吸引力的竞争对手。然而，与ASO-PCR需要一周的时间和非常昂贵的不同，Ziva检测可以在1小时内从一滴血液中进行，而且价格低廉。结合使用ROR1，一种特定的生物标记物或B-CLL，如果成功，所产生的检测结果可以区分B-CLL和其他克隆性B细胞恶性肿瘤。初步数据显示，对我们的技术进行早期调整后，感光度几乎是4色FC的2倍。使用我们的检测开发策略，我们预计会有进一步的改进。当监测疾病进展的标准FC方法需要静脉注射采集1-3毫升血液时，JBI的检测方法建议使用手指刺法采集血液样本，这是一种更方便的样本采集方法，用于对患者进行常规监测。如果成功，建议的测试将比FC更敏感和特异，并减少患者管理的总体成本负担。我们的方法集中于三个特定的目标：目标1：利用已知表面标记的细胞开发细胞表面分析模型系统。目的：建立和优化ROR1/CLL细胞系中试分析方法。目的：利用临床相关标本，初步评价ROR1/CLL检测方法的性能特点。 公共卫生相关性：这项赠款建议开发一种超敏感、廉价、易于使用的快速诊断方法，专门用于检测B-CLL细胞上的ROR1细胞表面生物标记物，用于使用一滴血检测和监测B-细胞慢性淋巴细胞白血病(B-CLL)。开发快速、廉价和易于使用的诊断工具，在灵敏度上优于最灵敏的流式细胞术或聚合酶链式反应方法，只需一滴血，就能为世界各地的基础癌症研究和患者管理带来非常广泛的公共健康益处。此外，该项目的成功完成有望带来一种更有用的方法来鉴别诊断B-CLL与其他克隆性B细胞恶性肿瘤，如套细胞淋巴瘤和脾边缘带淋巴瘤。这项提案中要求的SBIR资金将加速引进一项重要的新技术，用于检测研究和医学界使用的稀有细胞表面标记。

DESCRIPTION (provided by applicant): The ROR1 cell surface biomarker is expressed on B-cell Chronic Lymphocytic Leukemia (B-CLL) cells but not on healthy B-cells or other white blood cells (WBCs) in B-CLL patients. We propose to develop an inexpensive, easy-to-use, rapid, ultrasensitive assay specific for ROR1 for the detection and monitoring of B- CLL from a single drop of blood. Our goal is to attain detection levels better than currently available methods, the most sensitive flow cytometry (MRD-FC) or equal to polymerase chain reaction (ASO-PCR) methods available to detect Minimal Residual Disease (MRD). Early detection of MRD is critical in managing therapy and detecting the emergence of drug resistant malignancies. Current methods are expensive with regard to instruments, reagents, and the need for highly trained technicians. Moreover, FC methods do not adequately differentiate between B-CLL and other clonal B-cell malignancies such as mantle cell lymphoma and splenic marginal zone lymphoma. Current tests require venipuncture to collect 1-3 mLs of blood and the time to results are hours for FC and a week for ASO-PCR. JBI possesses a platform technology to address these shortcomings. JBI's first use of its technology resulted in the ZivaTM Ultrasensitive BrdU Cell Proliferation assay. Using only 50 <L of sample, Ziva detected 1-4 proliferating cells among 105 non-proliferating cells, a claim others have not approached. Applying this level of sensitivity to the detection of B-CLL would make it an attractive competitor to ASO-PCR, the most sensitive method. However, unlike ASO-PCR that takes one week to perform and is very expensive, the Ziva assay can be performed in 1 hour from a single drop of blood and is inexpensive. Together with the use of ROR1, a specific biomarker or B-CLL, if successful the resulting assay could differentiate between B-CLL and other clonal B-cell malignancies. Preliminary data show that an early-stage adaptation of our technology yielded an almost 2-fold better sensitivity than 4-color FC. We anticipate further improvements using our assay development strategies. Where standard FC methods for monitoring disease progression require 1-3 mL of blood collected by venipunture, JBI's assay proposes using a finger prick to collect a blood sample which represents a far more convenient method of sample collection for routine monitoring of patients. If successful the proposed test will be more sensitive and specific than FC and reduce the overall cost burden of patient management. Our approach focuses on three specific aims: AIM 1: To develop cell surface assay model systems using cells with known surface markers. AIM 2: To develop and optimize the analytical performance of the ROR1/CLL pilot assay using cell lines. AIM 3: To preliminarily evaluate the ROR1/CLL assay performance characteristics using clinically relevant samples. PUBLIC HEALTH RELEVANCE: This grant proposes to develop an ultrasensitive, inexpensive, easy-to-use, rapid diagnostic assay, that is specific for the detection of the ROR1 cell surface biomarker on B-CLL cells, for the detection and monitoring of B-cell Chronic Lymphocytic Leukemia (B-CLL) using a single drop of blood. Developing rapid, inexpensive and easy-to-use diagnostic tools that are better in sensitivity than the most sensitive flow cytometry or PCR methods, using a single drop of blood, presents a very broad public health benefit; for basic cancer research and for patient management worldwide. Furthermore, successful completion of this project is expected to result in a more useful method to differentially diagnose B-CLL from other clonal B cell malignancies such as mantle cell lymphoma and splenic marginal zone lymphoma. The SBIR funds requested in this proposal will accelerate the introduction of an important new technology for the detection of rare cell surface markers for use in both the research and medical communities.