Fernando Feliciano López Calderón¹, María del Carmen Turpín Sevilla², Silvia Montoro García³, Begoña Alburquerque González⁴, Ginés Luengo Gil⁵, Victoria Pérez Parra⁶, Irene Pérez Saura⁶, JOSE GARCIA SOLANO⁵, Pablo Conesa⁵

(1) Universidad Católica San Antonio de Murcia, 30204, España (Región de Murcia)
(2) Unviersidad Francisco de Vitoria, España (Comunidad de Madrid)
(3) UCAM, 3007, España (Región de Murcia)
(4) UCAM, 30007, España (Región de Murcia)
(5) PATOLOGÍA MOLECULAR Y FARMACOGENÉTICA, IMIB-Arrixaca, España
(6) Servicio de Anatomía Patolñogica, HGUSL, Cartagena, 30202, España (Región de Murcia)

Colon cancer is one of the most prevalent in the overall population, scoring the third in incidence for men and women, just below lung and nbeast1. Colon cancer is usually classified as conventional carcinoma (CC), serrated adenocarcinoma (SAC) or genetically unstable carcinoma (MSI-H). Among these types, CS is the one that has the worst prognosis, with a higher risk of metastasis and treatment failure as conventional carcinoma. Immune response in the tumoral microenvironment has been studied thoroughly in recent years, due to its importance in prognosis, survival, progression and treatment response2,3. In this study, we are focused on the expression of different biomarkers that have been described as modulators of the immune response and its relationship with the different types of colorectal cancer. Our results using both RT-qPCR and IHC show a stronger modulation of the immune response in serrated adenocarcinomas, than conventional carcinomas. This partially explains the bad prognosis, low survival rate, high metastasis risk and few therapeutic successes dealing with this type of tumor, especially when compared with conventional colon cancer.

In this study, a total of 180 randomized samples of tumors where selected, ranging from 2001 to 2019. Therse tissues belong to a cohort of 180 individuals, ranging from 37 to 89 years old, with an average of 67. The FFPE tissues where cut and s10 different TMAs were done accordingly. The slides were stained using a Ventana System (Roche). Each primary antibody has its own protocol, following manufacturer's especifications. From each tumor, 10 5um-thick sections were cut and RNA extracted using RNeasy extractkion kit (Qiagen). The quality and concentration of RNA extracted his way was measured using a Nanodrop device. Following this path, the RT reaction was performed using 100ug of RNA and a Maxima First Strand kit (Thermoscientific). Then, the RT-PCR was done in a 7500 Real-Time PCR System, of Applied Biosystems and SYBR master mix was used to the RT-PCR (Qiagen). The statistical analyisis was performed using Graph Pad Prism v8.0.

A total of 10 genes were analyzed via RT-PCR, but only 4 of them with statistical significance. CTLA-4 is overexpressed in SAC compared to CC and MSI-H. This also true for PD-L1. In addition to this, B2M is overexpressed in MSI-H compared to both SAC and CC. IDO-1 is low in CC compared to SAC and MSI-H. The staining revealed that IDO-2, paradoxically, was overepressed in CC, while the rest of the genes where mostly overexpressed in MSI-H, this includes FOXP-3, CTLA-4, CD14, CD163, CD3 and CD8.

These results, while provisional, and incomplete., suggest that there is a different tumor microenvironment in the three main kinds of colorectal cancer. Both innate and specific immune responses are disrupted in the tumors. This, in addition to taking clinical data could partially explain the differemt outcomes of the same therapy depending on the tumor. And, overall, the lack of a good Immunotherapy against any colon cancer. In order to clarify the discrepancies between RT-PCR and immunohistochemistry methods, this tumors should be reorganized following the latest molecular classification of colorectal cancer by the WHO.