Genentech Inc, South San Francisco, CA 94080, USA.
J Transl Med. 2013 Mar 23;11:76. doi: 10.1186/1479-5876-11-76.
The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.
We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.
The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.
We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.
磷脂酰肌醇 3-激酶（PI3K）通路在多发性骨髓瘤（MM）中发挥着重要作用，MM 是一种与骨髓浆细胞不受控制增殖相关的血液癌。本研究旨在开发一种稳健的临床药效动力学（PD）检测方法，以测量选择性 PI3K 抑制剂 GDC-0941 在 MM 患者中的靶标 PD 效应。
我们进行了一项体外药物洗脱研究，以评估生化方法在测量 S6 核糖体蛋白（S6）磷酸化方面的可行性，S6 磷酸化是 PI3K 通路抑制的常用 PD 标志物之一。然后，我们开发了一种 7 色磷酸特异性流式细胞术检测方法，或磷酸流式检测方法，以测量骨髓抽吸物（BMA）和外周血（PB）中细胞内 S6 的磷酸化状态。整合平均荧光强度（iMFI）用于计算磷酸化的倍数变化。通过比较磷酸流式与 Meso Scale Discovery（MSD）和免疫组织化学（IHC）检测方法，评估检测方法的灵敏度。最后，开发了一种样品处理方法，以在样品运输和储存过程中保持磷酸信号的完整性，确保临床应用。
磷酸流式检测方法使用固定的 BMA 和 PB 提供了肿瘤和替代细胞中 S6 磷酸化的单细胞 PD 监测，评估了 GDC-0941 对通路调节的反应，其灵敏度与 MSD 检测方法相似。本文所述的单次样品固定和处理方案在蛋白磷酸化方面表现出出色的保存效果。相比之下，免疫组化检测方法在信号定量方面的灵敏度较低，而生化方法（MSD）由于细胞分离对肿瘤细胞中蛋白磷酸化的不利影响，不太适合评估 PD 活性。
我们开发了一种用于 MM 中 PI3K 抑制剂临床评估的稳健 PD 生物标志物检测方法，使人们能够破译相关细胞群体中的 PD 反应。据我们所知，这是第一项易于实施的临床 PD 检测方法的报告，该方法包含一个无偏见的单次样品处理方案、所有（染色）-在一个（管）的磷酸流式染色方案，以及一个用于激酶抑制剂在 BMA 和 PB 中相关细胞群体的 PD 监测的综合改良数据分析。这里描述的方法确保了实时、可靠和可重复的 PD 读数，可为剂量选择提供信息，并有助于在早期临床试验中确定靶向药物的最佳组合。
