Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
Mol Oncol. 2010 Feb;4(1):65-89. doi: 10.1016/j.molonc.2009.11.003. Epub 2009 Nov 23.
Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.
乳腺癌是目前最常见的癌症诊断形式，也是导致女性癌症死亡的主要原因。临床上有用的生物标志物可以早期发现乳腺癌，从而显著降低死亡率。在这里，我们描述了一项详细的分析，使用基于凝胶的蛋白质组学结合质谱和免疫组织化学（IHC）分析从 69 名前瞻性乳腺癌患者收集的肿瘤间质液（TIF）和正常间质液（NIF）。本研究的目的是鉴定丰富的癌症上调蛋白，这些蛋白是由肿瘤微环境中的细胞外化的，如果不是所有这些病变，那么这些蛋白在大多数病变中都是外部化的。为此，我们应用了分阶段的生物标志物发现研究策略来分析这些样本，而不是相互比较所有样本，因为存在内在的样本间和样本内变异性问题。为此，我们选择使用来自单个肿瘤/良性组织对（患者 46，三阴性肿瘤）的样本，就上皮细胞数量而言，这些样本具有很好的匹配性，为生成初始数据集。在第一阶段，我们发现 110 种蛋白质在 TIF 中上调了 2 倍或更多，其中一些通过 IHC 得到了证实。在第二阶段，我们对其余 68 个 TIF 样本的 2D 凝胶进行了系统的计算机辅助分析，以鉴定在 90%或更多可用 TIF 中失调的 TIF 46 上调蛋白，从而代表常见的乳腺癌标志物。第二阶段确定了一组 26 个乳腺癌标志物，其中大多数标志物也通过使用 LC-MS/MS 的互补分析得到了鉴定。钙网蛋白、细胞视黄醇结合蛋白 II、氯离子细胞内通道蛋白 1、EF-1-β、半乳糖凝集素 1、过氧化物酶-2、血小板衍生的内皮细胞生长因子、蛋白质二硫键异构酶和泛素羧基末端水解酶 5 的表达使用包含 70 个不同异型程度的恶性乳腺癌的组织微阵列进一步验证。这些蛋白中的许多蛋白已经被其他人在血液/血浆/分泌组中检测到。接下来的步骤包括基于这些标志物的层次评估进行生物标志物优先级排序、抗体和抗原开发、测定开发、分析验证以及在健康和乳腺癌患者的血液中进行初步测试，这些都将在讨论中进行。
