Page 131 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

deaths worldwide. Aldolase A (ALDOA) has been linked to CRC progression by influencing cell proliferation and metastasis.
Recently, noncoding transcripts of protein-coding genes have been reported as promising diagnostic and prognostic
CRC biomarkers. The aim of this study was to obtain a transcriptional profile of ALDOA-212 (ENST00000566130.1),
a noncoding transcript with a retained intron, in malignant and non-malignant cell lines and their subcellular
compartments (nucleus, cytoplasm and exosomes), as well as in patients’ tumorous and healthy epithelial tissues.
Patients and Methods: Malignant HCT116, DLD-1, and SW620 and non-malignant human colon epithelial cell line
HCEC-1CT were cultivated in 3D as spheroids for 7 days. Tumorous tissues and samples of healthy mucosa from 5 CRC
patients were collected at the Clinic for Digestive Surgery, University Clinical Centre of Serbia. Total and compartmental
RNA was extracted using commercial kits. RNA sequencing was performed on ribosomal-depleted total RNA from cell
lines using Illumina's NovaSeq6000 platform. Patients’ total RNA and compartmental RNA from cell lines were analyzed
by qPCR. ALDOA-212 expression levels were also examined using available human databases via UCSC Xena Functional
Genomics Explorer, comparing primary tumor (TGCA Colon Adenocarcinoma database) with normal tissue (GTEx Colon
database; total of 596 samples), as well as with solid normal tissue (TGCA Solid Tissue Normal database; total of 329
samples). Additionally, AnnoLnc2 and lncLocator tools were used to assess the potential subcellular localization.
Results and Conclusions: ALDOA-212 was upregulated in all three malignant cell lines compared to non-malignant cell
line, while it was marginally downregulated in patient’s tumorous tissue in comparison to healthy mucosa (p=0.06).
Comparisons among larger available datasets showed no statistically significant difference between malignant
and normal tissues. ALDOA-212 was predicted to be localized in the nucleus or cytosol. However, we detected this
transcript in all examined compartments in HCEC-1CT and HCT116 cell lines. In DLD-1, it was detected in the nucleus
and exosomes, while in SW620, it was found exclusively in exosomes, suggesting its metastatic potential. CRC diagnostic
and prognostic significance of ALDOA-212 requires further confirmation and functional characterization.

Acknowledgments and funding: This work was funded by the Ministry of Science, Technological Development and
Innovation of the Republic of Serbia (Contract No. 451-03-136/2025-03/200042), and by the Science Fund of the
Republic of Serbia (PROMIS, #6052315, SENSOGENE).

P37

Analysis of Variants in Cytochrome P450 Superfamily Genes as Predictive Markers for Neoadjuvant
Chemoradiotherapy in Rectal Cancer

Anastasija Bubanja, Aleksandra Nikolic

Institute of molecular genetics and genetic engineering, University of Belgrade, Belgrade, Serbia

Keywords: biomarkers, genes, rectal cancer, SNPs

Background: Patients with locally advanced rectal cancer (LARC) often receive neoadjuvant chemoradiotherapy (nCRT)
based on 5-fluorouracil (5-FU). Cytochrome P450 (CYP) enzymes, involved in drug metabolism and carcinogenesis,
may influence response to therapy. Variability in CYP gene expression makes them promising biomarkers for predicting
treatment outcomes in rectal cancer.
Material and methods: Genomic DNA from 38 LARC patients treated with standard nCRT (fluorouracil + leucovorin
+ radiotherapy) was analyzed. Clinical response was assessed by tumor regression grade (TRG). Seven patients with
extreme responses – good (3 TRG1 and 2 TRG2) and poor (2 TRG5) were selected for whole-exome sequencing (WES).
Candidate predictive variants were identified based on their differential presence between responder and non-
responder groups and biological relevance (localization in coding region or regulatory elements and enzyme-altering
effects). Selected variants were validated in 29 moderate-response patients (15 TRG3 and 14 TRG4) by targeted
sequencing.
Results: Two genetic variants were selected according to the criteria outlined above: rs149012039, which is located
within the CYP2D7 pseudogene and represents a frameshift variant, and rs3093200, which is located in the first exon
of the CYP4F2 gene and has a damaging effect on the protein. Validation in the remaining samples showed that neither
variant was present in patients with a moderate response to therapy.
Conclusions: The presence of variants rs149012039 and rs3093200 in individuals with a good clinical response and
their absence in individuals with a moderate or poor response supports their potential as predictive biological markers
for response to nCRT in rectal cancer.

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