Harrington Sean C, Weroha S John, Reynolds Carol, Suman Vera J, Lingle Wilma L, Haluska Paul
Department of Oncology, Mayo Clinic, Rochester, MN 55905, United States.
Growth Horm IGF Res. 2012 Jun-Aug;22(3-4):108-15. doi: 10.1016/j.ghir.2012.04.001. Epub 2012 Apr 30.
The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER)+and ER- breast cancer tumors.
FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n=83) and ER-(n=64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n=61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors.
All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER- and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p<0.05, HR ER+ p<0.0005) and both groups had greater InsR-A expression when compared to ER- tumors (ER+ p<0.0005, HR ER+ p<0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p<0.0005) but higher than InsR-B (p<0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p<0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p<0.0005).
The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.
胰岛素样生长因子（IGF）靶向抗癌疗法预测性生物标志物的开发仍然是一个关键的未满足需求。胰岛素受体A亚型（InsR-A）已被确定为一种可能的生物标志物候选物，但在广泛使用的福尔马林固定石蜡包埋（FFPE）组织中对InsR-A进行定量分析较为复杂，因为它与代谢信号传导胰岛素受体亚型B（InsR-B）相似。在本研究中，开发了用于FFPE组织的针对InsR-A、InsR-B和IGF-1R的基于定量聚合酶链反应（qPCR）的检测方法，并在临床存档的FFPE雌激素受体（ER）阳性和ER阴性乳腺癌肿瘤中进行了可行性测试。
设计了与FFPE兼容的引物组，扩增子大小小于60个碱基对，并对其进行了靶标特异性、检测重复性和扩增效率的验证。确定了来自ER阳性（n = 83）和ER阴性（n = 64）原发性未治疗乳腺癌的FFPE肿瘤，以及ER阳性激素难治性（HR ER+）（n = 61）乳腺癌用于可行性测试。使用所有肿瘤组测试InsR-A和InsR-B qPCR的可行性，并使用HR ER+肿瘤确定IGF-1R qPCR的可行性。
所有qPCR检测方法均具有高度可重复性，在6个对数范围内扩增效率为96 - 104%，每个反应的检测限为4或5个拷贝。使用InsR-A、InsR-B或IGF-1R qPCR引物组成功扩增了超过90%的样本，超过88%的测试样本同时扩增了两种InsR亚型或两种亚型以及IGF-1R。InsR-A是82%的ER阳性、68%的ER阴性和100%的HR ER+乳腺癌中的主要亚型。探索性分析表明，与InsR-B相比，ER阳性和HR ER+组中InsR-A的表达明显更多（ER阳性p < 0.05，HR ER+ p < 0.0005），并且与ER阴性肿瘤相比，两组的InsR-A表达更高（ER阳性p < 0.0005，HR ER+ p < 0.05）。HR ER+肿瘤的IGF-1R表达低于InsR-A（p < 0.0005）但高于InsR-B（p < 0.0005）。与其他肿瘤亚组（ER阳性和ER阴性，p < 0.0005）相比，HR ER+肿瘤的InsR-B表达显著降低，并导致HR ER+的InsR-A:InsR-B比值显著升高（ER阳性和ER阴性，p < 0.0005）。
本文介绍的经过验证的、高度灵敏的基于InsR-A和InsR-B qPCR的检测方法首次证明了在FFPE组织中对InsR亚型进行可行的扩增。该可行性研究产生的定量数据表明，InsR-A在乳腺癌中比InsR-B更占主导地位，支持使用这些检测方法进一步研究InsR-A和InsR-B作为IGF靶向治疗的预测性生物标志物。
