2Department of General Surgery, Government Medical College Srinagar and Associated Hospitals, Srinagar-India
3Department of Biochemistry, Government Medical College Baramulla and Associated Hospitals, Kanth Bagh-India DOI : 10.5505/tjo.2025.4512
Summary
OBJECTIVEARID1A has emerged as a pivotal tumour suppressor gene, essential to chromatin remodelling and gene regulation. In colorectal cancer (CRC), dysregulated ARID1A gene expression triggers uncontrolled cellular growth and chemoresistance. This study investigates the spectrum of ARID1A variants in exon- 9, its mRNA expression in CRC tissues and potential correlations with clinicopathological parameters.
METHODS
The present study was a cross-sectional study conducted on 87 histopathologically confirmed CRC and
their adjacent normal tissue samples, collected from patients attending SMHS Hospital in Kashmir, India.
Sequence elucidation of Exon-9 was done by Di-deoxy Sequencing. mRNA expression of ARID1A
gene was carried out using quantitative real-time PCR (qRT-PCR).
RESULTS
Genomic variants of ARID1A were identified in 19.5% of CRC cases, with a predominant heterozygous
condition. In silico analysis of the Pro916His variant indicated a deleterious effect on protein function
and stability. The mRNA expression of ARID1A was significantly reduced in 17.2% of CRC tissues compared
to their adjacent normal tissue samples (p<0.001), with a 1.43-fold decrease observed.
CONCLUSION
Our study highlights the prevalence of ARID1A variants in CRC among Kashmiri population. This
study proposes that ARID1A under-expression might be relevant in CRC development.
Introduction
CRC is one of the most common cancers and ranks as the second leading cause of cancer-related deaths worldwide.[1] While CRC has historically been more common in individuals aged 50 and older, the incidence of early-onset CRC has been rising in both males and females. This shift in age distribution suggests that younger adults may be increasingly exposed to potential risk factors, leading to various genetic alterations.[2,3] CRC develops through a multistep process, beginning with the transformation of normal colorectal epithelium into an adenoma, and eventually progressing into an invasive and metastatic tumor.[4] Tumor suppressor genes (TSGs) play a crucial role in regulating key cellular functions, including DNA replication, repair, and transcription during cell division.[5] The loss of function in TSGs is a common mechanism that drives the initiation and progression of CRC.[6] ARID1A has emerged as a significant tumor suppressor, regulating the expression of multiple genes involved in cell division, DNA damage repair, and apoptosis.[7,8] It is typically expressed at low levels throughout the cell cycle and is degraded during mitosis, but it accumulates during the G0/G1 phase to induce cell cycle arrest.[9,10] Recent studies have revealed that ARID1A influences cancer-related signaling pathways by regulating the transcriptional activities of various proteins and nuclear hormone receptors.[11]Mutations and genetic variants are key hallmarks of cancer, playing a pivotal role in cancer initiation and progression.[12] In 2010, two studies using nextgeneration sequencing revealed a high frequency of inactivating ARID1A variants in highly aggressive ovarian cancers, prompting further investigation into its function as a tumor suppressor gene across various cancers.[13,14] More than 97% of cancer-related ARID1A variants are inactivating, often resulting from nonsense or frameshift mutations that lead to partial or complete loss of functional ARID1A protein.[15] Most cancer-associated ARID1A variants are heterozygous and correlate with reduced protein expression, suggesting that haploinsufficiency may contribute to cancer development.[16] The reduction or loss of ARID1A expression has been linked to tumorigenesis in a wide range of cancers, including gastrointestinal carcinoma. [17] Significant efforts have been made to identify molecular markers that enable personalized therapeutic strategies to improve disease diagnosis and treatment. Recently, there has been growing interest in developing cancer therapies tailored to the ARID1A variant status. This study aims to explore the spectrum of ARID1A mutations in exon 9 and their expression in colorectal cancer (CRC). We shall also examine the potential relationship between decreased ARID1A expression and various clinicopathological characteristics.
Methods
This study received approval from the Institutional Ethics Committee of Government Medical College Srinagar (No: 66/ETH/GMC, Date: 30/03/2017) and conducted according to the Declaration of Helsinki.
Study Design
This cross-sectional study was conducted by the Department
of Biochemistry and General Surgery, Shri
Maharaja Hari Singh and Super Specialty Hospital, associated with Government Medical College Srinagar,
North India.
Study Subjects and Sample Collection
The study included eighty-six (n=87) histopathologically
diagnosed CRC patients.
Inclusion and Exclusion Criteria
All enrolled patients were newly diagnosed cases, and
none had undergone chemotherapy or radiation. The
individuals did not have any genetic disorder
Sample Collection
Colorectal tumor tissue samples and their neighboring
normal tissue was taken from each CRC patient being
operated upon in the Department of Surgery, Shri Maharaja
Hari Singh (SMHS) and Superspeciality Hospital,
Srinagar. The patients' clinicopathological data was
supplied by the Medical Record Department. One aliquot
of each tissue sample was immediately stored in
RNA-later (Sigma-Aldrich, USA) at 4°C overnight to
achieve full solution penetration before being frozen at
-80°C for RNA extraction. Another aliquot was frozen
immediately and kept at -80°C for DNA analysis.
DNA Isolation
Genomic DNA was extracted from tissue samples using
the QIAamp DNA Mini Kit (Qiagen, Germany) in
accordance with the protocol. The purity of the isolated
DNA was validated using 1% agarose gel electrophoresis.
The purity and concentration were evaluated using
the NanoDrop 2000c spectrophotometer (Thermo Scientific,
USA). DNA samples with high molecular mass,
little fragmentation or shearing, and an A260/280 ratio
of 1.7-1.9 were employed for molecular analysis.
Polymerase Chain Reaction (PCR)
The primers used to amplify the exon-9 of ARID1A
gene were: Forward 5´-CACAGCACTATTTGGCTCCAG-
3´; reverse 5´-ATCATCTCTGGGCTGGCTG-3´
(Eurofins Genomics, Germany). The PCR reaction
was performed in 50µl of reaction mixture containing
12.5µl of 2X PCR master mix (3B BlackBio, Biotech,
India), 50-150 ng of genomic DNA, and 0.2 mM of
each primer. The reaction cycle included an initial denaturation
at 94°C for 5 minutes, followed by 35 cycles
of denaturation at 94°C for 20 sec, annealing at 58°C
for 30 sec, extension at 72°C for 30 sec, with a final extension
at 72°C for 7 minutes. The 343 bp PCR product
was run on 2.5% agarose gel and visualized using
Omega Lum G Gel Documentation System (Aplegen).
DNA Sequencing
The PCR products were sequenced using the ABI
prism 310 automated DNA sequencer (ThermoFisher
Scientific, USA) using the Sangar dideoxy technique.
RNa Isolation Followed by cDNA Synthesis
Total RNA was extracted from tissue samples using
the TRIzol reagent (ThermoFisher Scientific, USA)
and quantified. The RNA purity and concentration
were determined using a NanoDrop 2000c spectrophotometer
(ThermoScientific, USA), and an A260/
A280 ratio of 1.8-2.0 was judged suitable for further
analysis. A 1.5% agarose gel revealed the presence of
28S, 18S, and 5S rRNA bands, confirming RNA integrity.
cDNA was produced using the RevertAid First
Strand cDNA Synthesis Kit (ThermoFisher Scientific,
USA) according to the manufacturer"s instructions,
with the reactions incubated at 37°C for 60 minutes,
followed by 10 minutes at 95°C.
Quantitative real-time PCR for (qRT-PCR) for
relative mRNA expression of ARID1A
The relative mRNA expression of ARID1A in colorectal
tumor tissue samples and their adjacent normal
tissues was measured by using qRT PCR (7500 Real-
Time PCR system (ThermoFisher Scientific, USA).
Each sample was analyzed in triplicate and expression
levels were normalized using GAPDH as an internal
control. The primers used were as follows: Forward
5´-CTTCAACCTCAGTCAGCTCCCA-3´ reverse:
5´GGTCACCCACCTCATACTCCTTT-3´; and GAPDH
forward 5´-TTTGGCTACAGCAACAGGGT-3´
and GAPDH reverse 5´-TCCTCTTGTGCTCTTGCTGG-
3´. The PCR reaction mixture consists of 10µl
of KAPA SYBR®FAST master mix (Sigma-Aldrich,
USA), 0.4µl of cDNA from each sample, 0.2µM of each
forward and reverse primers, in a total volume of 20µl.
The PCR condition included an initial 10-minute preheat
at 95°C, followed by 40 cycles of denaturation at
95°C for 30 sec, annealing at 60°C for 1 minute, extension
at 72°C for 35 sec, and a final extension at 72°C for
10 minutes. Specificity of the PCR was confirmed using
melting curve analysis (58-95°C). ARID1A mRNA was
quantified based on the cycle threshold (Ct) value. Relative
expression was calculated using the 2-ΔΔCt method.
[18] The fold change in expression between tumor and
adjacent normal tissue was determined as 2-ΔΔCt.
Statistical Analysis
The data was analyzed using SPSS software version 23.0
(SPSS Inc., Chicago, IL, USA). Continuous variables
were analyzed using independent and paired t-tests, whilst categorical/dichotomous variables were tested
using Pearson's χ2 test, Fisher's exact test, or χ2 test. Logistic
regression was used to compute odds ratios (ORs)
and 95% confidence intervals (CIs). Statistical significance
was assessed using a two-sided p-value<0.05.
Results
Patient CharacteristicsIn this study, histologically confirmed colorectal cancer (CRC) cases (n=87) and their adjacent normal tissues were analyzed for ARID1A variants and mRNA expression. The mean age of CRC patients was 52.7±12.2 years. CRC was more prevalent in males (64.3%) than in females (35.6%). Notably, 56.3% of patients had BMI out of the normal range. 15.5% of CRC patients had a positive family history of gastrointestinal cancer. Table 1 provides a detailed breakdown of the socio-demographic and clinicopathological characteristics of enrolled CRC cases.
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Table 1. Demographic and clinicopathological parameters of the study subjects
Analysis of ARID1A Variants
The CRC samples were screened for the presence of
ARID1A variants at exon-9 of the ARID1A gene. Partial
electrophoretograms (forward) of ARID1A exon-9
in CRC patients is shown in Figure 1 (a-d). The detected
variants were present in 17 cases (19.5%). Out of 17 cases, 9 (53.0%) were mutually exclusive (only
one variant) whereas, 6 (35.3%) harboured at least two
variants and 2 (11.7%) of them carried three variants.
The heterozygous condition was predominant. The
nonsynonymous variants were rare than synonymous
type (Table 2). The high frequency of variants (70.3%)
was observed in the age group of ≥50 years.
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Table 2. ARID1A variants at exon-9 of colorectal tumor tissue samples
In Silico Analysis of Pro916His
The nonsynonymous missense Pro916His was submitted
to SIFT and PROVEN computational tools
to predict the effect of amino acid substitution on protein function. Both software tool predicted the
deleterious effect with the Sift score of ≤0.05 and
PROVEN score of -3.839. The disease-associated impact
of histidine substitution was analyzed by Mut-
Pred2 & GO software. The amino acid substitution
was predicted as neutral by both tools (score>0.5).
I-mutant 2.0 and MU pro were used to examine the
effect of ARID1A variant on the structural stability
of proteins. I-mutant 2.0 detected decreased protein
stability with reliability index 5, whereas the MU pro
predicted decreased protein stability with ΔΔG=-0.5.
Furthermore, to study the effect of the variant on
protein structure, the Hope project revealed substitution
of proline to histidine disturbs the backbone
conformation of the resultant protein.
Relative mRNA Expression of ARID1A Gene
We used qRT-PCR to examine the expression of ARID1A
mRNA in 87 CRC tumor tissues and surrounding
normal tissue samples. Among the 87 CRC patients,
17.2% (15/87) exhibited under-expression of ARID1A mRNA compared to adjacent normal tissues. The average
fold change (2-ΔΔCt) found to be 0.70 exhibited a
statistically significant decrease of 1.43-fold (1/0.70) in
mRNA levels of the ARID1A gene (Table 3).
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Figure 2 shows a box and whisker plot of relative mRNA expression. The melt curve studies revealed no production of any non-specific product. Table 4 shows the association between ARID1A expression and other clinicopathological characteristics. The data revealed that there was no significant association between the decreased mRNA expression of the ARID1A with any of the socio-demographic and clinicopathological characteristics of CRC cases (p>0.05).
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Table 4. Correlation between ARID1A expression and clinicopathological variables of CRC
Discussion
CRC is one of the leading causes of cancer-related mortality in the world. The global trends of CRC are also heterogeneous, as it is manifested in differences in incidence and mortality rates influenced by factors such as age, gender, and ethnicity.[19]The underlying molecular mechanism of CRC is driven by the continuous acquisition of genetic and environmental risk factors, related to the suppression of tumor suppressor genes (TSGs).[6] ARID1A has emerged as a potent TSG due to its association with various types of cancer, including CRC.[20] ARID1A is an important subunit of the SWI/SNF chromatin remodelling complex, which plays a pivotal role in regulating gene expression and maintaining cellular homeostasis. Therefore, ARID1A is essential for a broad spectrum of cellular processes including proliferation, differentiation, DNA damage repair, and genomic stability.[8] Genetic variations leading to altered gene expression, disrupt these processes resulting in uncontrolled cell division and tumor formation.[4] Several studies have demonstrated that genetic variations in ARID1A often leads to haploinsufficiency in various types of cancers, which are linked to more aggressive characteristics and poor clinical outcome.[21] In the present study, we identified that 19.5% of the patients have variations in the ARID1A gene. This prevalence emphasizes the role of ARID1A in the molecular context of CRC. Majority of these variations were heterozygous as it was expected because earlier studies suggested that the haploinsufficiency of ARID1A is sufficient to cause tumorigenesis by disrupting normal chromatin remodelling structure and gene expression.[8] Therefore, ARID1A haploinsufficiency is associated with increased genomic instability. The Pro916His variation was predicted to be deleterious by the in-silico tools because it is a nonsynonymous missense variant that may affect the protein function and stability. This variation was demonstrated to change the protein's secondary structure which could alter the protein"s ability to correctly interact with the SWI/SNF complex possibly causing abnormal cell division and defective DNA repair mechanism.
Furthermore, according to the published data the loss or low expression of ARID1A has a significant role in the development and progression of CRC.[22] The reduced ARID1A could alter the antigen presentation of tumor, potentially leading to strong immune response in CRC. However, this can also prompt the tumor to develop mechanism to evade immune detection.[23]
Spontaneous or mutation-induced reduction in ARID1A expression can act as a tumorigenic trigger, contributing to both the initiation and progression of cancer. ARID1A serves a dual role: As a "gatekeeper" controlling cell proliferation, and as a "caretaker" tumour suppressor that preserves genomic integrity by preventing sequence mutations and chromosomal aberrations.[16] Studies by Wei et al.[24] and Erfani et al.[25] have demonstrated that decreased or lost ARID1A expression is significantly associated with distant metastasis, lymph node invasion, and consequently, poor prognosis in CRC.
Although statistically insignificant, the frequency of CRC among older individuals (≥50 years) was higher compared to younger individuals. The CRC risk increases significantly with age due to the combined effects of several risk factors, with over 70% of CRC patients being over 55 years old.[1] Nevertheless, early exposure to many of the recognized risk factors increases the likelihood of developing early-onset of CRC (EOCRC), which is a considerable cancer concern among young individuals.[3,26] The causes of EOCRC are not yet fully understood; however, factors such obesity, physical inactivity and diet have been linked to its development.[2]
Obesity is a well-established risk factor for colorectal cancer across all age groups. Elevated BMI, especially abdominal obesity, is closely associated with chronic low-grade inflammation and insulin resistance, both of which may contribute to the formation of neoplastic lesions in colorectal tissue.[27] In line with these findings, our study observed a higher incidence of CRC in patients with abnormal BMI compared to the control group, although the difference was not statistically significant (p<0.001).
As per previous studies, the significant loss of ARID1A expression in CRC patients has been recorded compared to controls.[25,28] Consistent with the above studies, Quantitative real-time PCR (qRTPCR) analysis revealed significant under-expression of ARID1A mRNA in 17.2% of the CRC cases compared to adjacent normal tissues. This reduction in mRNA levels indicates a potential loss of functional protein, corroborating the variational analysis. The fold change analysis exhibited a 1.43-fold decrease in ARID1A expression, suggesting that even partial loss of ARID1A function can contribute to CRC pathogenesis. Several studies have reported that the ARID1A variations and reduced expression levels were significantly correlated with tumor histological grade, advanced tumor depth, poor differentiation, lymphatic metastasis, primary tumor location and early tumor stage.[29] Notably, the study failed to establish any correlation of ARID1A mRNA expression with clinicopathological characteristics such as age, gender, and tumour stage. This fact indicates that ARID1A variation and altered expression can be involved in CRC development without consideration of other characteristics of patients.
The discovery of the presence of ARID1A variations and their effects on the function of the protein provides new possibilities of targeted treatment of CRC. Understanding the link between the ARID1A loss and immune activation can help to shape the development of effective therapies, such as tumors with different ARID1A variations might respond differently to the immunotherapies such as checkpoint inhibitors. By targeting the altered immune pathways or harnessing the increased immune activation found in ARID1A-deficient tumors, novel treatment approach can be developed to boost anti-tumor immunity and improve patient outcome. Furthermore, therapies that can either reverse the loss of ARID1A function or at least supply for it might be new treatment strategies for CRC patients. For example, drugs that act on epigenetic changes or those that boost DNA repair mechanisms could be used in combination with the ARID1A status to enhance the effectiveness of the treatment. The results obtained are in line with the prior literature that has established the involvement of ARID1A in several cancers such as ovarian, gastric, and endometrial cancers.[9]
Limitation of the Study
Considerable number of patients in this region were
diagnosed with advanced stage CRC and were in-operable.
For that reason, the tumor tissue could not be
collected from those very patients. Therefore, studies
are warranted to validate the results, especially in case
of CRC, due to relatively modest sample size.
Conclusion
In conclusion, the present study confirmed a high frequency of ARID1A variations and down-regulation of ARID1A protein in CRC tissues compared to the normal adjacent tissues. Our research has shown that ARID1A plays a significant role in colorectal tumorigenesis and may be used as a biomarker for CRC. Subsequent research with more participants and deeper assessment of the ARID1A-associated molecular processes are required to enhance the understanding of the gene's function in CRC and to evaluate the possibility of using it as a biomarker for individualized treatment.Ethics Committee Approval: The study was approved by the Institutional Ethics Committee of Government Medical College Srinagar (no: 66/ETH/GMC, date: 30/03/2017).
Informed Consent: Informed consent was obtained from all participants.
Conflict of Interest Statement: The authors have no conflicts of interest to declare.
Funding: The study was funded by the Department of Biochemistry, Govt. Medical College Srinagar and Associated Hospitals, 190010, J&K, India.
Use of AI for Writing Assistance: No AI technologies utilized.
Author Contributions: Conception: J.Q., M.S.K.; Design: J.Q.; Supervision: S.M., M.D.W.; Fundings: S.M.; Materials: S.M.; Data Collection and/or Processing: J.Q., M.S.K., S.S.L.; Analysis and/or interpretation: M.S.K.; Literature Review: J.Q.; Writer: M.S.K., J.Q.; Critical Review: M.S.K.
Acknowledgments: The authors express their gratitude for the valuable efforts undertaken by the research team in this research.
Peer-review: Externally peer-reviewed.
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