MULTIPLEXED ISOFORM QUANTIFICATION IN HER2-POSITIVE BREAST CANCER

虽然靶向治疗提高了HER2阳性乳腺癌患者的总体存活率，但大多数患者 由于对最初成功的治疗(基于曲妥珠单抗)产生耐药性，将出现复发。因此， 有一种未得到满足的、由患者驱动的需求，即了解和规避乳腺癌耐药性，甚至 有针对性的治疗。在细胞水平上，细胞间的变异(异质性)是癌症的一个标志。也许吧 令人惊讶的是，分子异质性也是HER2+乳腺癌的一个标志。横跨外部 在癌细胞的膜上，大蛋白HER2显示一个胞外结构域，这是 “靶向治疗”(与化疗或放射治疗相比)。这些HER2+乳腺癌靶向治疗包括 标志性药物Herceptin®(曲妥珠单抗)。但HER2蛋白表现为一个蛋白质家族(称为 蛋白质异构体)，而不仅仅是单一的分子形式。令人遗憾的是，许多HER2亚型缺乏 全长HER2蛋白的胞外区，使细胞无反应，否则抗- HER2靶向治疗。这些较小的HER2亚型被称为‘截断亚型’，具有95 kDa的形式 ‘P95HER2’耐药能力特别强。直到我们之前的R01研究，辨别能力 截短的P95HER2亚型的全长HER2在相同的细胞分辨率下是不可能的。 因此，为了提高我们对抗HER2靶向治疗的耐药性的了解，我们建议建立 关于我们团队将P95HER2与其他HER2蛋白形式精确区分开来以仔细研究其作用的能力 P95HER2在：(1)存在于每个乳腺癌表面的强大的、信号激活的HER2二聚体 细胞和(2)显示P95HER2蛋白的潜在超耐药乳腺癌细胞亚群 异构体和抗药性驱动的DNA突变(PIK3CA)。这是两种细胞模式(P95HER2 人/异二聚体；P95HER2和PIK3CA突变的共表达)，这是其他工具无法直接和 在微小的组织样本中同时测量高特异性，低至单细胞分辨率。我们的临床， 生物统计和生物工程团队将进行研究，以生产能够执行这些异构体的工具- 涉及HER2阳性乳腺癌患者活检组织和细胞的多模式分析(Stanford 乳腺组织银行)，在对特征良好的乳腺癌细胞株进行早期开发后。这个 在有限的可用乳房中直接测量截短的HER2亚型和相互作用模式的能力 活检组织和单细胞分辨率应该会对理解产生巨大的优势，然后 评估抗药性的可能性。这些研究将使我们能够描绘出细胞和分子 HER2的异质性促进对持续性乳腺癌抗HER2耐药的理解 有针对性的治疗，并最终确定减少或消除复发的方法。

While targeted therapy increases overall survival rates in HER2-positive breast cancer patients, most patients will experience recurrence due to resistance to initially successful therapy (trastuzumab-based). Accordingly, there is an unmet, patient-driven need to understand and circumvent breast cancer resistance, to even targeted therapies. At the cellular level, cell-to-cell variation (heterogeneity) is a hallmark of cancer. Perhaps surprisingly, molecular heterogeneity is also a hallmark of HER2+ breast cancer. Spanning the outer membrane of a cancer cell, the large protein HER2 displays an extracellular domain, which is the target of ‘targeted therapies’ (vs. chemo- or radiation therapies). These HER2+ breast cancer targeted therapies include the landmark drug Herceptin® (trastuzumab). But the HER2 protein manifests as a family of proteins (called protein isoforms), not just a single molecular form. Regrettably, numerous of these HER2 isoforms lack the extracellular domain of the full-length HER2 protein, making the cell non-responsive otherwise powerful anti- HER2 targeted therapies. These smaller HER2 isoforms are known as ‘truncated isoforms’, with a 95 kDa form ‘P95HER2’ being especially potent in drug resistance. Until our previous R01 research, the ability to discern full-length HER2 from the truncated P95HER2 isoform was not readily possible with same-cell resolution. Consequently, to advance our knowledge of resistance to anti-HER2 targeted therapies, we propose to build on our team’s capacity to precisely distinguish P95HER2 from other HER2 protein forms to scrutinize the role of P95HER2 in: (1) the potent, signal-activating HER2 dimers that reside on the surface of each breast cancer cell and (2) potentially ultra-resistant breast cancer cell subpopulations that exhibit both the P95HER2 protein isoform and the resistance-driving DNA mutation (PIK3CA). These are two cellular ‘modes’ (P95HER2 homo/heterodimers; co-expression of P95HER2 and PIK3CA mutation) that no other tools can directly and with high-specificity concurrently measure in minute tissue samples, down to single-cell resolution. Our clinical, biostatistics, and bioengineering team will conduct research to yield tools that can perform these isoform- involved multimodal assays in tissues and cells from HER2-positive breast cancer patient biopsies (Stanford Breast Tissue Bank), after performing early development on well-characterized breast cancer cell lines. The ability to directly measure the truncated HER2 isoforms and interaction modes in sparingly available breast biopsy tissues and with single-cell resolution should yield a tremendous advantage for understanding and then assessing the potential for drug resistance. These studies will allow us to profile the cellular and molecular heterogeneity of HER2 to advance understanding of persistent breast cancer resistance to anti-HER2 targeted treatment and, ultimately, to identify approaches to reduce or eliminate recurrence.