Timisnar - Biomarkers Substudy (Timisnar-mirna)

Study Overview
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Brief Summary
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Neoadjuvant chemoradiotherapy (nCHT) followed by surgery is the mainstay treatment for locally advanced rectal cancer, leading to significant decrease in tumor size (downsizing) and a shift towards earlier disease stage in the primary tumor and lymph nodes (downstaging). Extensive histopathological work-up of the tumor specimen after surgery including tumor regression grading (TRG) and lymph node status (ypN) helped to visualize individual tumor sensitivity to CRT retrospectively. Since the response to nCHT is heterogeneous, however, valid biomarkers are needed to monitor tumor response. A relevant number of studies aimed to identify molecular markers retrieved from tumor tissue while the relevance of blood-based biomarkers is less stringent assessed. As a potential alternative to CEA and ctDNA, microRNAs (miRNAs) are currently under investigation to serve as blood-based biomarkers. miRNAs are small, noncoding RNAs that regulate gene expression by post-transcriptional mRNA binding, which promotes the destabilization of target miRNAs. The target specificity of miRNAs is largely predetermined by their so-called seed-sequence (containing nucleotides at position 2-7 of the miRNA). They are highly conserved between species, stable and easy detectable even in small concentrations. They have been widely analyzed in physiological and pathological processes, and their expression is tissue specific.

Detailed Description
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Neoadjuvant chemoradiotherapy (nCHT) followed by surgery is the mainstay treatment for locally advanced rectal cancer, leading to significant decrease in tumor size (downsizing) and a shift towards earlier disease stage in the primary tumor and lymph nodes (downstaging). Extensive histopathological work-up of the tumor specimen after surgery including tumor regression grading (TRG) and lymph node status (ypN) helped to visualize individual tumor sensitivity to CRT retrospectively. Since the response to nCHT is heterogeneous, however, valid biomarkers are needed to monitor tumor response. A relevant number of studies aimed to identify molecular markers retrieved from tumor tissue while the relevance of blood-based biomarkers is less stringent assessed. Blood samples, i.e. Liquid Biopsy, however, offer several advantages: Taking blood samples is less invasive, less expensive, easy to schedule, and nearly without any severe complications. Blood samples are a source of fresh DNA and RNA, without modifications due to preservatives; especially in the case of rectal cancer, beyond intratumoral heterogeneity, tumor biopsies are in general accompanied by normal, adenomatous or stromal tissue. This contamination may affect results of molecular analyses Investigating blood from patients can account for molecular heterogeneity and surrogate for tumor burden since tumor-derived fragments or biomarkers are collected from all tumor cells in a patients' body through circulation. Liquid biopsy may offer both the possibility of dynamic monitoring under treatment and the possibility to assess disease activity even after pathologic complete response (pCR) or after resection of the tumor when no tissue is left for molecular analyses. In clinical routines, to date, carcinoembryonic antigen (CEA) is established as a colorectal cancer (CRC) related tumor marker but is not recommended as a screening test for colorectal cancer. First, normal levels of CEA do not exclude the possibility of a colorectal cancer. Second, an elevated CEA is not categorically associated with CRC, or in the period of follow-up with disease progression. Circulating tumor DNA (ctDNA) represents nowadays, the main approach to monitor tumor burden and therapy resistance, to evaluate the presence of residual disease after potentially curative treatment and to monitor disease recurrence with high sensitivity and specificity. As a potential alternative to CEA and ctDNA, microRNAs (miRNAs) are currently under investigation to serve as blood-based biomarkers. miRNAs are small, noncoding RNAs that regulate gene expression by post-transcriptional mRNA binding, which promotes the destabilization of target miRNAs. The target specificity of miRNAs is largely predetermined by their so-called seed-sequence (containing nucleotides at position 2-7 of the miRNA). They are highly conserved between species, stable and easy detectable even in small concentrations. They have been widely analyzed in physiological and pathological processes, and their expression is tissue specific. To date, no screening approach to identify relevant miRNAs as biomarkers in blood of patients with rectal cancer was undertaken.

Official Title
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Timing To Minimally Invasive Surgery After Neoadjuvant Chemoradiotherapy For Rectal Cancer: A Multicenter Randomized Controlled Trial - Biomarkers SubStudy

Conditions
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Rectal Cancer

Intervention / Treatment
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* Diagnostic Test: miRNA


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age >18 years cT3/4N0/+M0 confirmed on CT-scan, MRI (stratification for T3a-b-c-d) Histologically-proven adenocarcinoma of the rectum Eligible for a resective surgery with TME Eligible for chemoradiation treatment Exclusion Criteria: Metastatic disease Squamous carcinoma of the anal canal Unable to complete neoadjuvant treatment

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Intervention/Treatment |
| --- |
|Diagnostic Test: miRNA|15 ml of whole blood samples are collected in Vacutainer tubes with spray-coated K2EDTA and stored at room temperature. To minimize the hemolysis and nucleic acids degradation, plasma separation undergoes within 2 h. Within 1 h, tubes are subjected to a first centrifugation step at 2200 x g and room temperature for 15 min. Plasma supernatants are transferred to 15 mL tubes, carefully avoiding contact with the lymphocytic ring, and tubes are centrifuged a second time at 3000 x g and RT for 10 min to remove cellular debris. Plasma samples are then collected into 1.5 mL cryovials and all the aliquots are stored at -80 °C.|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in expression levels of plasma miRNA | the association of variation between preneoadjuvant and postneoadjuvant expression levels of miRNA with pCR | 5 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| miRNA expression and surgery | changes in expression levels of miRNA following complete surgical resection with disease-free survival | 5 years | 
| miRNA expression and surveillance | the relation between changes in miRNA during surveillance and tumor relapse | 5 years |