Page 108 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

underwent long-course nCRT. Radiation therapy was delivered using volumetric modulated arc therapy with a
simultaneous integrated boost (VMAT-SIB), combined with concomitant chemotherapy (5-fluorouracil and leucovorin)
administered during the first and fifth weeks of treatment. Treatment response was evaluated eight weeks after the
completion of nCRT. Patients who achieved a cCR and had tumors located distally (not amenable to sphincter-sparing
surgery) were not immediately referred for radical surgery but were instead enrolled in a strict follow-up program
(i.e., the “watch and wait” approach). In cases of tumor regrowth, salvage surgery was performed. Following surgery,
the follow-up protocol included a first surveillance colonoscopy at 12 months post-treatment, then every 3–5 years
depending on findings. Chest, abdominal, and pelvic CT scans were performed every 6 months for the first 3 years,
and annually thereafter. Clinical evaluations and tumor markers (CEA, CA 19-9)were measured every 3–6 months in
the first 3 years, and then every 6–12 months Overall survival (OS) was defined as the time from diagnosis to the date
of last clinical follow-up or death. Disease-free interval (DFI) was calculated from the date of surgery or, for patients in
the “watch and wait” group without relapse, from the control MRI until disease progression, death, or last follow-up.
For patients included in the “watch and wait” protocol who experienced tumor regrowth during follow-up, DFI was
calculated from the date of salvage surgery until disease progression, death, or last follow-up. Gene expression levels
(IL6, CXCL9, CYBB, IDO1) were measured using qRT-PCR in pretreatment FFPE samples. Patients were divided into two
groups based on DFI status (with or without disease relapse), and gene expression levels were compared between the
groups.
Results: Of the 82 enrolled patients, 65 (79.3%) underwent surgery, while 17 (20.7%) were managed with the “watch
and wait” strategy. Among the “watch and wait” cohort, 8 patients experienced tumor regrowth; 7 of these (87.5%)
underwent successful salvage surgery. One patient developed both local and distant relapse and was not operated
on. The median disease-free interval (DFI) was 36 months (range: 1–54 months), with a 3-year DFI rate of 74.8%.
Median overall survival (OS) was 46 months (range: 12–58 months), with a 3-year OS rate of 88.9%. When comparing
gene expression between patients with and without disease relapse, statistically significant differences were observed
for CYBB and IDO1 (p < 0.05). Receiver operating characteristic (ROC) curve analysis was performed to determine
the optimal cut-off values for these genes. While the ROC analysis itself did not show statistical significance (CYBB:
p = 0.744, AUC = 0.670; IDO1: p = 0.544, AUC = 0.679), when the derived cut-off values were applied, higher CYBB
expression (cut-off value 0.447) was significantly associated with better DFI, while higher IDO1 expression (cut-off
value 1.471) was significantly associated with better both DFI and OS (p < 0.05 for both).
Conclusions: This prospective study evaluated the prognostic significance of selected immune-related genes in patients
with locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy. While initial in silico findings did
not correlate with treatment response, further analysis revealed that high expression levels of CYBB and IDO1 were
significantly associated with disease-free and overall survival. These results suggest that CYBB and IDO1 may serve
as valuable prognostic biomarkers, supporting their potential role in risk stratification and individualized patient
management using a multigene prognostic panel. Further validation in larger, independent cohorts is warranted.

References:

1. Marinkovic, M.; Stojanovic-Rundic, S.; Stanojevic, A.; Ostojic, M.; Gavrilovic, D.; Jankovic, R.; Maksimovic, N.;
Stroggilos, R.; Zoidakis, J.; Castellví-Bel, S.; et al. Exploring novel genetic and hematological predictors of response
to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Front. Genet. 2023, 14, doi:10.3389/
fgene.2023.1245594.
2. Chen, B.; Alvarado, D.M.; Iticovici, M.; Kau, N.S.; Park, H.; Parikh, P.J.; Thotala, D.; Ciorba, M.A. Interferon-Induced
IDO1 Mediates Radiation Resistance and Is a Therapeutic Target in Colorectal Cancer. Cancer Immunol. Res. 2020,
8, 451–464, doi:10.1158/2326-6066.CIR-19-0282.
3. Takasu, C.; Nishi, M.; Yoshikawa, K.; Tokunaga, T.; Nakao, T.; Kashihara, H.; Wada, Y.; Yoshimoto, T.; Okikawa, S.;
Yamashita, S.; et al. Role of IDO expression in patients with locally advanced rectal cancer treated with preoperative
chemoradiotherapy. BMC Cancer 2022, 22, 1263, doi:10.1186/s12885-022-10357-1.
4. Uhlén, M.; Björling, E.; Agaton, C.; Szigyarto, C.A.-K.; Amini, B.; Andersen, E.; Andersson, A.-C.; Angelidou, P.;
Asplund, A.; Asplund, C.; et al. A human protein atlas for normal and cancer tissues based on antibody proteomics.
Mol. Cell. Proteomics 2005, 4, 1920–1932, doi:10.1074/mcp.M500279-MCP200.
5. Ortega, M.A.; Fraile-Martinez, O.; Pekarek, L.; García-Montero, C.; Alvarez-Mon, M.A.; Castellanos, A.J.; García-
Honduvilla, N.; Buján, J.; Alvarez-Mon, M.; Sáez, M.A.; et al. Oxidative Stress Markers Are Associated with a Poor
Prognosis in Patients with Pancreatic Cancer. Antioxidants 2022, 11.
6. Wu, Z.; Huang, X.; Han, X.; Li, Z.; Zhu, Q.; Yan, J.; Yu, S.; Jin, Z.; Wang, Z.; Zheng, Q.; et al. The chemokine CXCL9
expression is associated with better prognosis for colorectal carcinoma patients. Biomed. Pharmacother. 2016, 78,
8–13, doi:10.1016/j.biopha.2015.12.021.
7. Turano, M.; Cammarota, F.; Duraturo, F.; Izzo, P.; De Rosa, M. A Potential Role of IL-6/IL-6R in the Development and

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