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SDIRSACR Oncology Insights

10. Ashour AA et al. Elongation factor-2 kinase regulates metastasis and epithelial to mesenchymal transition of
pancreatic cancer cells. J Biol Chem. 2014.

P09

From in silico to clinical validation: CYBB and IDO1 as potential prognostic markers in rectal cancer

Mladen Marinković , Aleksandra Stanojević , Suzana Stojanović-Rundić , Dušica Gavrilović , Radmila Janković ,
1
1,2
1,2
1
1
Aleksandar Tomašević , Milan Kocić , Jerome Zoidakis , Sergi Castellvi-Bel , Remond J.A. Fijneman , Milena Čavić 1
6
5
3,4
1
1,2
1Institute for Oncology and Radiology of Serbia, Belgrade, Serbia
2Faculty of Medicine, University of Belgrade, Belgrade, Serbia
3Department of Biotechnology, Biomedical Research Foundation, Academy of Athens, Athens, Greece
4Department of Biology, National and Kapodistrian University of Athens, Athens, Greece
5Gastroenterology Department, Fundació Recerca Clínic Barcelona-Institut d’Investigacions Biomèdiques August Pi i Sunyer,
Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Clínic Barcelona, University of Barcelona,
Barcelona, Spain
6 Department of Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands
Keywords: locally advanced rectal cancer; neoadjuvant chemoradiotherapy; prognostic biomarkers; CYBB; IDO1;
disease-free interval
Background: Locally advanced rectal carcinoma (LARC) is typically treated with neoadjuvant chemoradiotherapy
(nCRT) followed by surgery. Identifying predictive and prognostic biomarkers is crucial for selecting patients who will
benefit the most from neoadjuvant treatment. In our previous study, an in silico approach was used to select candidate
genes associated with response to nCRT [1]. Three genes involved in the Hallmark inflammatory response pathway and
associated with immune evasion (IL6, CXCL9, and CYBB) were selected for further validation, along with IDO1, a gene
identified through literature review as having potential relevance [2,3]. The expression of these genes was measured
using quantitative real-time PCR (qRT-PCR) in pretreatment formalin-fixed paraffin-embedded (FFPE) tissue samples,
using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. However, in a prospective cohort from the
Institute for Oncology and Radiology of Serbia, no significant correlation was observed between the expression of the
selected genes (IL6, CYBB, CXCL9, IDO1) and overall treatment response [1]. Patients were divided into responders—
those who achieved a clinical complete response (cCR) and were managed with a “watch and wait” approach, as well
as those with tumor regression grade (TRG) 1 or 2 according to Mandard, in patients who underwent surgery. The non-
responder group included patients with TRG 3 or 4. In the subgroup of patients who underwent surgery, a comparison
between the best responders (TRG1) and those with the poorest response (TRG4) revealed a statistically significant
difference (p<0.05) in the expression of the IDO1 gene [1]. Higher expression of IDO1 was associated with a better
treatment response [1]. The CYBB gene encodes the gp91-phox subunit of the nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase complex, which is primarily recognized for its role in the oxidative burst of the immune
system. A literature review of the CYBB gene revealed a statistically significant association between its expression levels
and survival in patients with LARC, with higher expression linked to improved overall survival (p = 0.02) [4]. However,
emerging studies suggest that CYBB activity is also associated with oxidative stress and the response to radiotherapy
in cancer treatment [5]. Elevated oxidative stress levels have been linked to increased radiosensitivity of cancer cells.
The prognostic significance of IDO1 has been demonstrated in several malignancies. In renal cell carcinoma, high
IDO1 expression correlates with poorer outcomes, whereas in ovarian cancer and melanoma, it has been identified
as a favorable prognostic marker [4]. Similarly, CXCL9 has shown prognostic relevance in renal, endometrial, breast,
and ovarian cancers, where its elevated expression is associated with better survival [4]. In colorectal cancer (CRC)
specifically, increased CXCL9 expression has been linked to a favorable prognosis in a cohort of 326 patients [6].
Regarding IL6, literature data highlight its involvement in the development and progression of CRC [7], as well as its
potential as a prognostic biomarker. While some studies have reported that elevated plasma IL6 levels are associated
with poorer survival in CRC patients, these findings have not been consistently validated in subsequent research [8].
Although investigating genes associated with the inflammatory response holds considerable promise for understanding
therapy response in rectal cancer, the lack of validation in our study may be attributed to population-specific factors
and the limited sample size [1]. Since their predictive significance was not confirmed in previous research, this study
aimed to evaluate their prognostic role in our cohort of patients with LARC.
Patients and Methods: Between June 2020 and May 2022, we prospectively enrolled 82 patients with LARC who

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