Zahnd Stefan, Braga-Lagache Sophie, Buchs Natasha, Lugli Alessandro, Dawson Heather, Heller Manfred, Zlobec Inti
Institute of Pathology, University of Bern, Bern, Switzerland.
Department for BioMedical Research, Proteomics and Mass Spectrometry Core Facility, University of Bern, Bern, Switzerland.
J Pathol Inform. 2019 Dec 12;10:40. doi: 10.4103/jpi.jpi_65_18. eCollection 2019.
Biomarkers in colorectal cancer are scarce, especially for patients with Stage 2 disease. The aim of our study was to identify potential prognostic biomarkers from colorectal cancers using a novel combination of approaches, whereby digital pathology is coupled to shotgun proteomics followed by validation of candidates by immunohistochemistry (IHC) using digital image analysis (DIA).
Tissue cores were punched from formalin-fixed paraffin-embedded colorectal cancers from patients with Stage 2 and 3 disease ( = 26, each). Protein extraction and liquid chromatography-mass spectrometry (MS) followed by analysis using three different methods were performed. Fold changes were evaluated. The candidate biomarker was validated by IHC on a series of 413 colorectal cancers from surgically treated patients using a next-generation tissue microarray. DIA was performed by using a pan-cytokeratin serial alignment and quantifying staining within the tumor and normal tissue epithelium. Analysis was done in QuPath and Brightness_Max scores were used for statistical analysis and clinicopathological associations. MS identified 1947 proteins with at least two unique peptides. To reinforce the validity of the biomarker candidates, only proteins showing a significant ( < 0.05) fold-change using all three analysis methods were considered. Eight were identified, and of these, cathepsin B was selected for further validation. DIA revealed strong associations between higher cathepsin B expression and less aggressive tumor features, including tumor node metastasis stage and lymphatic vessel and venous vessel invasion ( < 0.001, all). Cathepsin B was associated with more favorable survival in univariate analysis only.
Our results present a novel approach to biomarker discovery that includes MS and digital pathology. Cathepsin B expression analyzed by DIA within the tumor epithelial compartment was identified as a strong feature of less aggressive tumor behavior and favorable outcome, a finding that should be further investigated on a more functional level.
结直肠癌中的生物标志物稀缺，尤其是对于Ⅱ期疾病患者。我们研究的目的是使用一种新的方法组合从结直肠癌中识别潜在的预后生物标志物，即将数字病理学与鸟枪法蛋白质组学相结合，随后通过使用数字图像分析（DIA）的免疫组织化学（IHC）对候选物进行验证。
从患有Ⅱ期和Ⅲ期疾病的患者（各26例）的福尔马林固定石蜡包埋的结直肠癌中获取组织芯。进行蛋白质提取和液相色谱 - 质谱（MS）分析，随后使用三种不同方法进行分析。评估倍数变化。使用下一代组织芯片通过IHC在一系列413例手术治疗患者的结直肠癌上验证候选生物标志物。通过使用全细胞角蛋白序列比对并量化肿瘤和正常组织上皮内的染色来进行DIA。在QuPath中进行分析，并使用亮度最大值分数进行统计分析和临床病理关联分析。MS鉴定出1947种具有至少两个独特肽段的蛋白质。为了加强候选生物标志物的有效性，仅考虑使用所有三种分析方法均显示出显著（<0.05）倍数变化的蛋白质。鉴定出8种，其中组织蛋白酶B被选用于进一步验证。DIA显示组织蛋白酶B表达较高与侵袭性较低的肿瘤特征之间存在强关联，包括肿瘤淋巴结转移分期以及淋巴管和静脉血管侵犯（均<0.001）。仅在单变量分析中，组织蛋白酶B与更有利的生存率相关。
我们的结果提出了一种包括MS和数字病理学的生物标志物发现新方法。通过DIA在肿瘤上皮区室中分析的组织蛋白酶B表达被确定为侵袭性较低的肿瘤行为和良好预后的一个显著特征，这一发现应在更功能层面上进一步研究。
