Digital PCR quantification of BCR-ABL for CML diagnosis and monitoring in a LMICs setting

工程摘要 我们建议开发一种低资源设置（LRS）的数字PCR（dPCR）仪器来检测BCR-ABL 从少量血液样本中提取转录本，以确定符合以下条件的慢性粒细胞白血病（CML）患者： 酪氨酸激酶抑制剂（TKI）治疗在低收入和中等收入国家（LMIC）。尽管成功的 靶向TKI Glivec（格列卫，伊马替尼）在发达国家治疗CML，世界上大多数 CML患者居住在LMIC，获得诊断检测和TKI治疗的机会有限。格里维茨 国际病人援助计划（GIPAP）是全球范围内最全面、影响最深远的计划之一。 诺华（Glivec的制造商）与Max合作设计的癌症访问计划 基金会（CDC）通过以下方式直接向合格患者提供和分发格列卫（伊马替尼） 他们的供应商。实施GIPAP计划的主要挑战之一是缺乏诊断 许多GIPAP国家的能力对于选择患者进行TKI治疗至关重要，因为只有 正确诊断为费城染色体阳性CML（Ph+ CML）的患者有资格 参加GIPAP计划。我们假设LRS dPCR仪器检测BCR-ABL 以高度敏感的方式进行记录将使更多的中低收入国家的卫生保健机构能够诊断 Ph+ CML，并参与GIPAP项目，以及监测正在接受TKI治疗的患者。 我们将采用我们实验室最近开发的技术开发LRS dPCR仪器 包括基于自数字化（SD）微流控芯片的新型数字核酸扩增平台， 其将水性样品分配成适于PCR的数万纳升体积，仪器 为了使用离心力有效地装载样品和使用光盘（OD，例如CD，DVD， 蓝光）风格的读者，这是高度兼容的LMIC设置。我们建议利用这些 创新技术，创造出匹配或超过灵敏度和特异性的LRS dPCR仪器 目前最先进的RT-PCR BCR-ABL融合转录本测定，样品制备量最少， 而不需要运行标准。这样一个工具将对改善慢性粒细胞白血病产生重大影响 LMIC患者的生存率。

Project Abstract We propose to develop a low resource setting (LRS) digital PCR (dPCR) instrument to detect BCR-ABL transcripts from small blood samples to identify chronic myeloid leukemia (CML) patients who are eligible for tyrosine kinase inhibitor (TKI) therapy in low- and middle- income countries (LMICs). Despite the success of the targeted TKI Glivec (Gleevec, imatinib) in treating CML in developed countries, the majority of the world's CML patients reside in LMICs with limited access to diagnostic testing and TKI treatment. The Glivec International Patient Assistance Program (GIPAP) is one of the most comprehensive and far-reaching global cancer access programs designed by the Novartis (the manufacturer of Glivec) in partnership with the Max Foundation (TMF) to facilitate access to and distribution of Glivec (imatinib) directly to eligible patients through their providers. One of the major challenges of the implementation of GIPAP program is the lack of diagnostic capabilities in many GIPAP countries which are essential for selecting patients for the TKI therapy, since only patients who are properly diagnosed with Philadelphia chromosome positive CML (Ph+ CML) are eligible to participate in the GIPAP program. We hypothesize that a LRS dPCR instrument to detect the BCR-ABL transcript in a highly sensitive manner will enable more health care institutes in LMICs capabilities to diagnose Ph+ CML and participate in the GIPAP program, as well as monitor patients who are being treated with TKI. We will develop the LRS dPCR instrument by adapting technologies recently developed in our laboratories including a novel digital nucleic acid amplification platform based on a self-digitization (SD) microfluidic chip, which partitions an aqueous sample into tens of thousands of nanoliter volumes suitable for PCR, instruments for efficient sample loading using centrifugal force and signal detection using optical disc (OD, e.g. CD, DVD, Blu-Ray)-styled readers which are highly compatible to the LMICs setting. We propose to leverage these innovative technologies to create a LRS dPCR instrument matching or exceeding the sensitivity and specificity of the current state of the art RT-PCR BCR-ABL fusion transcript assay, with minimal sample preparation and without the need of run to run standards. Such an instrument will have significant impact on improving CML patients' survival in LMICs.