Montero-Calle Ana, Garranzo-Asensio María, Torrente-Rodríguez Rebeca M, Ruiz-Valdepeñas Montiel Víctor, Poves Carmen, Dziaková Jana, Sanz Rodrigo, Díaz Del Arco Cristina, Pingarrón José Manuel, Fernández-Aceñero María Jesús, Campuzano Susana, Barderas Rodrigo
Chronic Disease Programme (UFIEC), Instituto de Salud Carlos III, 28220 Madrid, Spain.
Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28014 Madrid, Spain.
Cancers (Basel). 2023 Mar 31;15(7):2102. doi: 10.3390/cancers15072102.
Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV ( = 31), individuals with premalignant lesions ( = 31), and healthy individuals ( = 48). p53γ, Δ40p53β, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms' seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.
结直肠癌（CRC）是全球第三大常见癌症，也是癌症相关死亡的第二大常见原因。通过液体活检已证明，检测血浆样本中针对特定肿瘤相关抗原的自身抗体有助于CRC的早期诊断。然而，需要开展与癌症体液免疫反应相关的新研究，以实现基于血液的疾病诊断。在此，我们的目的是表征与不同的p53和p63蛋白异构体相关的体液免疫反应，这些异构体源自可变剪接，先前已被描述为在CRC中异常表达。因此，我们通过发光HaloTag磁珠免疫测定法，研究了迄今为止所描述的12种p53蛋白异构体、8种p63蛋白异构体及其特定的N端和C端肽段的诊断能力。将全长蛋白异构体或特定肽段克隆为HaloTag融合蛋白，并使用I-IV期CRC患者（n = 31）、癌前病变个体（n = 31）和健康个体（n = 48）的血浆分析其血清反应性。发现p53γ、Δ40p53β、Δ40p53γ、Δ133p53γ、Δ160p53γ、TAp63α、TAp63δ、ΔNp63α和ΔNp63δ，以及特定的C端α和δ p63肽段，与健康个体相比，对CRC患者和/或癌前病变个体的血浆具有更高的血清反应性。此外，ROC（受试者工作特征）曲线显示，这些p53和p63蛋白异构体检测CRC和癌前病变个体的诊断能力较高（AUC高于85%）。最后，将电化学生物传感平台应用于类似即时检测（POC）的设备中，以研究其使用选定的p53和p63蛋白异构体检测CRC的效用。我们的结果不仅证明了这些生物传感器在同时分析蛋白异构体血清反应性方面的潜力，还证明了其在通过液体活检进行CRC临床检测中的便利性和多功能性。总之，我们在此表明，与对照相比，p53和p63蛋白异构体在CRC患者中具有不同的血清反应性，这与经典蛋白不同，这应能改进诊断面板，以获得用于CRC检测的基于血液的生物标志物特征。
