The Effect of ADAR1 Expression Level on Total Neoadjuvant Treatment Response in Locally Advanced Rectal Cancer
Conditions: Rectum Cancer, Adenocarcinoma
Sponsor: Necmettin Erbakan University

Study and Statistical Analysis Plan (SAP): (Protocol 24948/2025/5785) 
 
Official  Title: The Effect of ADAR1 Expression Level on Total Neoadjuvant Treatment Response in 
Locally Advanced Rectal Cancer 
Protocol Number: 24948/2025/5785 
NCT:  
Document date: July 25, 2025  
Sponsor: Necmettin Erbakan University 
Principal Investigator: Mehmet Artac, MD 
Affiliation: Necmettin Erbakan University Faculty of Medicine 
E-mail: mehmetartac@yahoo.com  
Location: Necmettin Erbakan University Faculty of Medicine 
Center Phone: +90 332 223 60 00  
Submission Format: PDF/A 
 
 
 
 
 
 
Confidentiality Statement: This document contains confidential information and is intended solely for authorized personnel, 
investigators, and regulatory authorities. Unauthorized reproduction or distribution is prohibited. No participant names or 
identifiable information are included. 
 

Unique Protocol ID:  24948/2025/5785 
Brief Title: 
ADAR1 Expression Level in Rectal Cancer (ADAR1TNT)   
Official Title: 
The Effect of ADAR1 Expression Level on Total Neoadjuvant Treatment 
Response in Locally Advanced Rectal Cancer   
Secondary IDs: 
Turkish Society of Med. Onc.   [Turkish Society of Medical Oncology] 
 
Study Start: 
June 1, 2025    
Primary Completion: July 1, 2026    
Study Completion: 
September 1, 2026   
 
 
Sponsor: 
Necmettin Erbakan University   
Responsible Party: Principal Investigator 
Investigator: Mehmet Artac [MArtac] 
Official Title: MD 
Affiliation: Necmettin Erbakan University 
 
Title 
The effect of ADAR1 expression level on total neoadjuvant treatment response in locally advanced 
rectal cancer 
Summary  
 
Total neoadjuvant therapy (TNT) is currently the standard treatment for locally/locally advanced rectal 
cancer due to better response and fewer distant metastases. In TNT, sequential chemotherapy (CT) and 
chemoradiotherapy (CRT) are planned and depending on the treatment response, either surgery is 
performed or a watch and wait approach is applied. Depending on tumor localization and patient 
performance status, CT is planned as induction or consolidation. Most of the time, mFOLFOX6 and 
CAPOX are preferred as CT regimen. In proximal rectal cancer, surgery can be performed without 
CRT and only after CT. In locally/locally advanced rectal cancer, the aim is to avoid surgery as much 
as possible or to perform sphincter-sparing surgery if possible. Colonoscopy and pelvic MR at the time 
of diagnosis are the most important steps in staging, treatment selection and decision making. These 
two diagnostic methods should be repeated especially for watch and wait decision and for response 
evaluation after TNT. 
ADAR 1 (Adenosine deaminase acting on RNA1) is an RNA editing enzyme that catalyzes the 
deamination of adenosine to inosine (A-to-I), a dynamic modification that can lead to a diverse 
transcriptome in a combinatorial manner. A defect in ADAR1-mediated RNA modification results in 
abnormal regulation of substrates that can affect phenotypic changes in cancer. This phenomenon of 
over-regulation is seen in many cancers such as colon, liver, lung, breast and esophageal cancers and 
in many cases promotes tumor progression. In studies, increased ADAR1 expression has been 
associated with lower survival and worse prognosis, especially in metastatic colon and gastric cancer. 
ADAR1 is also predicted to increase proliferation through both the AKT pathway and the mTOR 
pathway and therefore may be targeted in the near future. 
ADAR1 expression is monitored by RNA-based real time PCR. In order to demonstrate increased 
expression, biopsies should be taken from the malignant tissue and the intact tissue of the patient and 

the biopsy should be stored under -80 C conditions immediately after biopsy to prevent RNA 
degradation. The tissue will not come into contact with nitrogen or formaldehyde. 
In our study, sufficient biopsies from cancerous and intact tissue will be taken from patients with 
suspected rectal cancer, confirmed by pelvic MRI and consent for participation in the study, and fresh 
tissue will be stored at -80 C in the genetics laboratory. After the TNT plan is made by us and the 
treatment is completed, both pelvic MRI and control rectoscopy will be performed for preoperative 
evaluation. Again, biopsies will be taken from diseased and healthy tissue and ADAR1 expression will 
be evaluated. The study is planned to include 50 patients and a period of one year is foreseen for tissue 
procurement/storage. 
Our aim in this study will be to determine whether ADAR1 expression level changes after TNT, 
whether this predicts clinical and pathological response, whether responses change according to the 
selected CT, whether there is a difference between CT induction-consolidation/RT short or long 
course, and the relationship between tumor DNA mismatch repair enzyme status and ADAR1 level. 
Our primary endpoint will be the effect of the change in ADAR1 expression level on the response 
after TNT (ORR). Our secondary endpoints will be recurrence-free survival (RFS) and overall survival 
(OS). 
 
Original Value and Hypothesis of the Study 
Colorectal cancer is the third most common cancer in both sexes, but it ranks second in mortality after 
lung cancer, with one in ten cancer deaths attributed to colorectal cancer[1]. Total neoadjuvant therapy 
(TNT) is currently considered the standard of care for the treatment of locally advanced rectal 
cancer[2]. In this approach, all non-surgical interventions, including multi-agent chemotherapy and 
concurrent chemoradiotherapy (CRT), are administered prior to surgical resection or the decision to 
opt for non-operative treatment. Accumulating evidence suggests that TNT improves survival in 
patients with locally advanced rectal cancer and is expected to reduce distant metastasis through 
systemic chemotherapy, thereby preventing the onset of micrometastases[3]. TNT has also been 
associated with improved compliance, reduced toxicity, decreased need for and duration of ileostomy, 
and improved anal sphincter preservation rates following a watch-and-wait strategy, along with 
increased complete clinical response[4, 5]. Studies have shown that the rate of pCR in patients 
receiving TNT therapy is approximately 20–40%[6, 7]. Although numerous randomized controlled 
trials have evaluated different TNT strategies compared to standard KRT treatment in terms of KRT 
sequencing, systematic chemotherapy, radiotherapy modality, and TNT intensity, it is known that the 
TNT approach demonstrates superior survival benefit compared to adjuvant chemotherapy following 
neoadjuvant KRT. However, TNT theoretically offers several surgical advantages, including an 
increased likelihood of sphincter-preserving surgery and a reduced need for ileostomy; however, 
results across studies are inconsistent[8]. 
ADAR 1 (Adenosine deaminase acting on RNA1) catalyzes the C6 deamination of adenosine (A) to 
produce inosine (I) in RNA regions characterized by a double-stranded structure, a process known as 
adenosine (A) to inosine (I) RNA editing (ATIRE)[9]. This process is crucial for altering RNA 
structures and sequences in both coding and non-coding RNA, influencing tumor characteristics, 
tumor stage, drug responses, and patient survival, thereby significantly contributing to cancer 
progression[10]. The role of ADAR1 in promoting tumorigenesis via the ATIRE pathway is becoming 
increasingly evident in various cancers, including stomach cancer, esophageal squamous cell 
carcinoma, breast cancer, hepatocellular carcinoma, and CRC[11-14]. 
ADAR1, ADAR2, and ADAR3 are members of the ADAR family; while ADAR1 and ADAR2 are 
widely expressed, ADAR3 is primarily expressed in the brain and lacks catalytic activity. ADAR1 
contains RNA-binding domains (RBDs) and Zα domains, whereas ADAR2 contains only RBDs. Zα 
domains enable ADAR1 to bind to newly synthesized RNA and inhibit the activation of pathogenic 

interferons (IFNs)[15]. ADAR1 has two isoforms: an IFN-inducible form (p150) and a constitutively 
expressed form (p110). It has been found that ADAR1-p110 reduces the chemotactic potential of 
melanoma cells and promotes immune exclusion [16]. Loss of ADAR1 function results in unregulated 
RNA accumulation, which can be mistakenly recognized as foreign by RIG-I (Retinoic acid-inducible 
gene I), thereby triggering its activation and subsequent IFN response. Additionally, DNA released 
from damaged cells can activate the cGAS (GMP–AMP synthase)-STING (stimulator of interferon 
genes) pathway, leading to the production of type I IFNs through both pathways. The interaction 
between ADAR1 and the cGAS-STING and RIG-I pathways highlights a crucial balance in immune 
regulation[17, 18]. ADAR1's ability to interact with IFNs, regulate IFN production, and its complex 
roles in cancer therapy, coupled with its higher expression in MSI-H (microsatellite instability-high) 
patients, its positive correlation with high TMB (tumor mutation burden), PD-1/PD-L1 levels, naive B 
cells, active memory CD4 T lymphocytes, and M1 macrophage cells suggests its potential for 
modulating responses to cancer therapies [19, 20]. A potential mechanism has been proposed whereby 
increased ADAR1 expression in gastric cancer leads to proliferation and migration of various diseases 
through the mTOR/p70S6 kinase/S6 ribosomal protein pathway. To investigate the functional 
relationship between ADAR1 and mTOR signaling, the mTOR kinase inhibitor rapamycin was used to 
treat gastric cancer cells and observe its effects on ADAR1-overexpressed cell proliferation and 
migration. The results showed that ADAR1 overexpression significantly promoted cell proliferation 
and migration as expected, but these effects were significantly attenuated in cells treated with 
rapamycin. This suggests that rapamycin may block the effects of ADAR1 overexpression on gastric 
cancer cell growth and migration. These results indicate that the mTOR signaling pathway plays an 
important role in ADAR1-mediated gastric cancer progression[21]. 
Recently, the effect of increased ADAR1 expression on survival in metastatic colon cancer was 
investigated. Both overall survival (OS) and relapse-free survival (RFS) were significantly reduced in 
the group with increased ADAR1 expression; however, the study did not include patients with early-
stage rectal cancer[20]. For all these reasons, the prognostic value of ADAR1 expression levels and/or 
changes in locally advanced rectal cancer patients for TNT response will be investigated. 
Purpose and Objectives 
1. To investigate the relationship between ADAR1 expression levels and pathological and clinical 
response to TNT 
2. To evaluate the relationship between potential differences in KT and RT administered in TNT and 
ADAR1 expression levels 
3. To determine the relationship between changes in ADAR1 expression and RFS and OS in patients 
undergoing surgery or watch and wait after TNT 
 
1. Study Design 
 
The study will include 50 patients diagnosed with locally advanced rectal cancer who applied to the 
General Surgery Department of Necmettin Erbakan University Faculty of Medicine. The control group 
will be formed with healthy rectal tissue samples taken from the same patients. Within the scope of the 
study, tissue samples will be collected from each patient at two different time points: before and after 
TNT. A total of 200 tissue samples will be examined, including 50 cancerous rectal tissue samples and 
50 healthy rectal tissue samples before TNT, and 50 cancerous rectal tissue samples and 50 healthy 
rectal tissue samples after TNT.  

 
 
 
Inclusion criteria 
Histological and stage diagnosis of local/locally advanced rectal cancer 
Age range between 18 and 90 
ECOG performance score between 0 and 2 
No contraindications for CT and/or RT 
 
Exclusion criteria 
Patients with metastatic rectal cancer 
Those with suspected rectal cancer 
Those who did not approve the informed consent form 
Those with contraindications for CT and/or RT 
 
2. Dermographic data collection 
Obtaining and recording dermographic data from patients and controls included in the study 
 
3. Storage and evaluation of biopsy materials 
Tissue samples will be stored at -80°C until the day of analysis in the Medical Genetics Department 
laboratory of Necmettin Erbakan University Faculty of Medicine and total RNA will be isolated from 
the tissue samples. During this process, 5-30 mg of tissue will be homogenized in a 1.5 mL 
homogenization tube, and extraction will be performed according to the kit manufacturer's instructions 
to obtain 30-50 μl of total RNA. cDNA will be obtained from the RNA samples whose quantities have 
been calculated. In the PCR protocol, primer binding will be performed at 25°C for 10 minutes, 
reverse transcription will be performed at 42°C for 15 minutes, and finally, enzyme inactivation will 
be performed at 85°C for 5 minutes. Gene expression levels will be determined by the real-time PCR 
method. The p150 isoform, which is larger in size and has been used in previous studies, will be used. 
The reaction will be performed in a real-time PCR device with an initial denaturation at 95°C for 5 
minutes, followed by 40 cycles of 95°C for 5 seconds, 55°C/59°C for 30 seconds (reading). The 
relative mRNA expression levels of the ADAR1 gene obtained via the device will be determined using 
the R package and the 2-ΔΔCt method. A total of 200 gene expression data obtained from 50 patients 
before and after treatment will be statistically compared. 

 
Data Collection and Statistical Analysis 
Clinical, pathological, and demographic characteristics, along with laboratory results, will be obtained 
from patient files and the hospital database. 
For descriptive statistics, the Chi-square test and Fisher's exact tests will be used. For measurable 
evaluations, if the distribution is normal, the Student's t-test will be performed using the mean and 
standard deviation; if the distribution is not normal, the Mann-Whitney U test will be performed using 
the median value. 
Non-parametric related tests will be used to compare two or more variables between non-parametric 
dependent variables. Kaplan-Meier survival curves will be used to calculate survival, and the log-rank 
test will be used to compare differences in survival times. All variables affecting prognosis and 
survival will first be evaluated using univariate Cox regression analysis, followed by multivariate Cox 
regression analysis. To determine the correlation between categorical variables, the chi-square test or 
Fisher's exact test will be used. All statistical analyses will be performed using IBM SPSS Statistics 
25.0. Two-sided p-values <0.05 will be considered statistically significant.