ColoScape™ Colorectal Cancer Mutation Detection Test | DiaCarta, Inc.

ColoScape™ Colorectal Cancer Mutation Detection Test

Improved Sensitivity for Early Colorectal Cancer Screening using FFPE and Plasma Samples 

ColoScape™ is a highly sensitive novel multigene mutation biomarker qPCR-based in vitro diagnostic assay for qualitative detection of colorectal cancer associated biomarkers in liquid biopsy and FFPE tissue samples. The kit utilizes our XNA technology which leverages a sequence-specific clamp made by xeno-nucleic acid (XNA) to suppress PCR amplification of wild-type DNA template and selectively amplify only mutant DNA template, reaching sensitivity at 0.1% to 0.5% with 5 ng of blood DNA input. The detection kit identifies the presence or absence of mutations in the targeted regions of four colon cancer associated genes including KRAS, APC, CTNNB and BRAF.

The assay can be performed on readily available qPCR instrumentation that is already present in hospital pathology laboratories. Unlike all of the currently available FDA approved colorectal cancer tests on the market, ColoScape™ provides a comprehensive profile of the key colorectal cancer ‘driver’ and ‘resistance’ mutations (gene variation landscape) and directly informs oncologists of targeted therapy options without the need to reflex to another assay. ColoScape™ could also be used to improve existing colorectal cancer detection tests.

ColoScape™ Colorectal Cancer Mutation Detection Test is CE/IVD-certified. 

Advantages of ColoScape™ Colorectal Cancer Mutation Detection Test

NON-INVASIVE TESTING

A blood or FFPE samples test intended to diagnose colorectal cancer and occurrence at the early and treatable stage

ultra-sensitivity

Detect reliably 0.1% to 0.5% VAF mutant DNA out of wild-type DNA for targeted mutations

Clinical Sensitivity

>60% for advanced adenomas (pre-cancer, stage 0)  (compared to 42% for Cologuard®); 95.5% for colorectal  cancer (stage I to IV)

low input dna

Minimum 2pg input DNA per reaction. Less than 2 tubes of blood (10mL each) needed for cell-free DNA (cfDNA)

Diagnostic Aid

Aids colonoscopists in the diagnosis of serrated advanced adenomas

Prediction of Therapy Response

Proprietary XNA technology accurately identifies wild-type and mutant status of relevant genes

Fast Result

Less than 4 hours of assay run time

Comprehensive Coverage

Patented gene panel covering 4 genes and 20 mutations

Great Versatility

Only requires routine qPCR instruments tat are available in common research discovery and pathology labs

What Is Colorectal Cancer?

Colorectal cancer is cancer that starts in the colon or the rectum. These cancers can also be named colon cancer or rectal cancer, depending on where they start. Colon cancer and rectal cancer are often grouped together because they have many features in common. Colorectal cancer is the third most common cancer in the world and in the U.S.

People died of colorectal cancer in 2018

New cases a year for rectal cancer

New cases a year for colon cancer

New cases a year for colorectal cancer

%

Stage I colon cancer 5-year survival rate

%

Stage I rectal cancer 5-year survival rate

%

Stage IV colon cancer 5-year survival rate

%

Stage IV rectal cancer 5-year survival rate

Death from Colorectal Cancer is Preventable

The trend for the death rate of colorectal cancer has dropped steadily over the years. The development of colorectal cancer takes about 15 years and the death from colorectal cancer is preventable with early diagnosis. Colorectal cancer screening is strongly suggested to get diagnosed early to prevent death.

Colorectal cancer screening is strongly suggested to get diagnosed early to prevent death. According to the American Cancer Society, the guideline for colorectal screening is to start the colorectal cancer screening at age 50 (most recent changes to age 45). However, among the 98 million Americans aged 50 to 84, one-third of them are not screened for colorectal cancer for different reasons.

The Challenges and Drawbacks for the Current Colorectal Cancer Detection Methods

As a gold standard for colon cancer screening, invasive colonoscopy not only identifies the polyps on the surface of the colon but removes them as well. However, part of the reasons that one-third of the population aged 50 to 84 is not using this screening tool is due to either the unpleasant experience for the preparation of colonoscopy or worries of medical complications caused by bleeding and infection during the process of colonoscopy.

The traditional non-invasive assays such as FIT assays are based on blood test using immunochemistry and their sensitivity is much lower (and therefore inaccurate) compared to the invasive tests.

Molecular diagnostics as a powerful non-invasive test has only recently been approved for colorectal screening. However, the current ColoGuard test uses stool samples making it less inconvenient. In addition, the stool samples have been shipped to a central laboratory for processing and testing, eliminating the test available through the local clinical testing labs where patients often seek for these tests. In addition, the test also uses multiple instruments such as liquid handling instruments, qPCR instruments and ELISA readers, making the costly investment as a barrier for localization of the test.

Source:
Zauber, et al., Agency for Healthcare Research and Quality (2009)
Pickhardt et. al., Radiology (2011)
Pickhardt et. al.,New England Journal of Medicine (2003)
Johnson, et. al., New England Journal of Medicine (2008)
RadiologyInfo for Patients

Colonoscopy (Invasive)

Colonoscopy is the gold standard for colorectal cancer screening. It allows a doctor closely to see the inside of the entire colon and rectum for polyps which could be an early sign of cancer and grow over time to develop cancer. 

Sensitivity – Colorectal Cancer: 95% 
Sensitivity – Advanced Adenomas: 95%
Specificity: 90%

Sigmoidoscopy (Invasive)

Examination of sigmoid colon (most distant part of colon) by means of a flexible tube inserted through the anus.

Sensitivity – Colorectal Cancer: ~50% 
Sensitivity – Advanced Adenomas: ~50% 
Specificity: 92%

CT Colonography (Invasive)

Computed tomography (CT) colonography or virtual colonoscopy uses special x-ray equipment to examine the large intestine for cancer and growths called polyps.

Sensitivity – Colorectal Cancer: 96% 
Sensitivity – Advanced Adenomas: 94% 
Specificity: 86% to 96%

gFOBT (Non-Invasive)

gFOBT (guaiac fecal occult blood test) is a test that checks for occult (hidden) blood in the stool.

Sensitivity – Colorectal Cancer
Hemoccult SENSA: 70%; Hemoccult II: 40%
Sensitivity – Advanced Adenomas
Hemoccult SENSA: 24; Hemoccult II: 12%
Specificity
Hemoccult SENSA:95%; Hemoccut II: 98%

FIT (Non-Invasive)

FIT (fecal immunochemical test) is a test that checks for occult (hidden) blood in the stool.
 

Sensitivity – Colorectal Cancer: 70% 
Sensitivity – Advanced Adenomas: 22%
Specificity: 95%

ColoGuard® (Non-Invasive)

ColoGuard® is an FDA approved test (2016) for colorectal cancer by checking gene mutations and methylations from stool DNA.

Sensitivity – Colorectal Cancer: 92.3%
Sensitivity – Advanced Adenomas: 92.3% (42.3% pre-cancer)
Specificity: 100%

ColoScape™ (Non-Invasive, suitable for both liquid biopsy and FFPE samples)

ColoScape™ is a novel multigene mutation biomarker qPCR-based assay for qualitative detection of colorectal cancer (CRC) associated somatic mutations in the genes that are frequently mutated in colon cancer patients and are responsible for aberrant colonic epithelial cell proliferation.

Sensitivity – Colorectal Cancer (Stage I – IV): 95.5% (compared to 92.3% for ColoGuard®)
Sensitivity – Advanced Adenomas (Pre-Cancer): >60% (compared to 42.3% for ColoGuard®)
Specificity: >96%

ColoScape™ is Optimal for Liquid Biopsy

Recently, with liquid biopsy gaining more traction, circulating cell-free DNA in blood samples can be used for testing gene mutations for powerful cancer diagnostics tools. ColoScape™ colorectal cancer mutation detection kit is developed and used after FIT-positive test and before the colonoscopy examination. Preliminary testing data shows ColoScape™ colorectal cancer mutation detection kit has 95.5% sensitivity and specificity of >96% for stage I to IV colorectal cancer and >60% sensitivity for pre-cancer for EEPE and Plasma samples. 

Patended Colorectal Cancer Detection Panel

Based on the colorectal cancer gene mutation panels licensed from the University of Potsdam, 20 mutations in four genes associated with colorectal cancers, APC, BRAF, CTNNB1, and KRAS, are detected in three multiplex qPCR reactions. With the negative, positive, and reference gene controls, the qPCR-amplified mutation target can be used to call out for positive or negative results based on the difference in Cq value between the Cq for mutation target and the Cq for reference gene control.

The APC 1367 Amplification Plot on ABI 7500 Fast Dx showing positive control, negative control and no template control

A run file on ABI 7500 Fast Dx showing a KRAS-positive clinical sample

Colorectal Cancer Mutation Detection Powered by XNA Technology

ULTRA-SENSITIVE

XNA is the Optimal Choice for Colorectal Cancer Mutation Detection Compared to other Technologies

XNA, xenonucleic acids, are innovative new nucleic acid molecular oligomers that hybridize by Watson-Crick base pairing to target DNA sequences yet have a modified chemical backbone. XNA oligomers are highly effective at hybridizing to targeted normal DNA sequences and can be employed as molecular clamps in quantitative real-time polymerase chain reactions (PCR) or as highly specific molecular probes for detection of nucleic acid target sequences. The XNA tightly binds to the wildtype sequence that is 100% complementary in sequence and blocks DNA polymerase from DNA elongation; only the mutant target sequence gets amplified because the XNA:mutant DNA duplex is not stable due to mismatch and fall off from the template in PCR reactions.

Sanger Sequencing

Advantages: accurate result and is, therefore, the gold standard
Disadvantages: low sensitivity (20% to 25% VAF)

Pyrosequencing Assays

Advantages: better sensitivity and throughput than Sanger sequencing,  the early form of NGS
Disadvantages: low sensitivity (5% to 8% VAF)

NGS Sequencing

Advantages: high-throughput and good sensitivity – 1% to 5% VAF, or even better
Disadvantages: costly and time consuming (7 to 10 days)

Digital Droplet PCR (ddPCR)

Advantages: high sensitivity and claimed to be 0.001% VAF
Disadvantages: much less sensitivity observed in testing than claimed  and suffers false-positive results

qPCR Analysis

Sensitivity can reach 1% VAF for some targets. Rapid and little hands-on work. Multiple methods for qPCR and a lot of variations in sensitivity. Some of them are only 10% VAF

ColoScape™ Colorectal Cancer Mutation Detection Analytical Performance

Analytical Performance - Accuracy

The analytical accuracy is verified and validated through testing of well-characterized samples with known mutations verified by NGS, Sanger sequencing or digital droplet PCR. Studies are conducted to demonstrate concordance in mutation status of FFPE and plasma samples. The results demonstrate a 100% match between reference methods and the ColoScape™ kit. 

Analytical Performance - Precision

The precision of the ColoScape™ kit was determined with defined analytical levels of genomic DNA with known mutational status and allelic frequencies. Below table shows the summary of reproducibility results of ColoScape™ Colorectal Cancer Mutation Detection Test.

Edit
Variation %CV
Intra-assay ≤ 3%
Inter-assay ≤ 5%
Lot-to-Lot Variation ≤ 4%
Operator Variability ≤ 3%

Analytical Performance - Limit of Detection (LOD)

To determine the limit of detection (LOD) and analytical sensitivity of the kit, mutant allelic frequencies at 1%, 0.5% and 0.01% variant allele frequency (VAF) with 5 and 10ng/reaction DNA input levels are tested. The LOD of 0.5% is reached for all the mutant targets. Below table shows the LOD summary determined using genomic DNA reference standards.

Edit
Target Mutation DNA Input, 10 ng/well DNA Input, 5 ng/well
% Correct Call % Correct Call
APC 1309 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 20% 0%
 
APC 1367 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 50% 10%
 
APC 1450/876 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 60% 60%
 
CTNNB1 41 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 30% 10%
 
CTNNB1 45 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 80% 40%
 
KRAS 12 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 80% 45%
 
KRAS 13 1% mutation 100% 100%
0.5% mutation 100% 80%
0.1% mutation 45% 45%
 
BRAF V600 1% mutation 100% 100%
0.5% mutation 100% 100%
0.1% mutation 100% 15%

 

ColoScape™ Colorectal Cancer Mutation Detection Clinical Sample Testing

Clinical sensitivity and specificity are tested on the samples extracted from FFPE and plasma of patients with different stages of colorectal cancer from normal to advanced adenomas (pre-cancer), to colorectal cancer stages I to IV. A sample was considered positive if at least one of the target mutations tested positive based on the cutoffs.

Edit
Test Types of Clinical Samples Clinical Parameters
Specificity Sensitivity
I CRC (cfDNA) N/A 100%
CRC FFPE N/A 90%
Advanced Adenomas (Pre-cancer) 95% 60%
 
II FFPE 100% 93%
cfDNA 100% 90%
 
III cfDNA (Pre-cancer) 92% 70%
 
Average FFPE 100% 92%
cfDNA 96% 95%

Streamlined Workflow for ColoScape™ Colorectal Cancer Mutation Detection

Step 1: DNA Isolation & Quantification

Extract DNA from FFPE or plasma using a commercial DNA extraction kit followed by measuring the concentration using fluorometric analysis

Step 2: set up qpcr

Mix the assay reagents, load into PCR plate, add controls and extracted DNA ~ 30-60 minutes

Step 3: Amplification parameters

Enter amplification parameters on
qPCR instrument, load PCR plate
and start the run ~ 2.5 hours

Step 4: Data analysis

Determine the presence or absence
of mutations according to the Cq
value cutoffs ~ 15 minutes

 Product Specifications for ColoScape™ Colorectal Cancer Mutation Detection

Product Catalog Number

CE/IVD Catalog Number: DC-30-0024E;
Research-Use-Only (RUO) Catalog Number: DC-30-0024R

Intended Use

For in vitro diagnostic use (CE/IVD) or for research use

Sample Type

FFPE and Plasma

Input DNA

5-10ng/Reaction

Pack Size

24 Reactions

Instruments Validated

Roche LightCycler® 480, Bio-Rad CFX384. ABI QuantStudio 5 and ABI 7500 Fast Dx 

Detection Chemistry

TaqMan

Turnaround Time

Less Than 4 hours

Stability

Stable for 12 Months at -25°C  to -15°C

Webinar

Title: Colorectal Cancer Genetics & A Non-Invasive Test for Colorectal Cancer Mutation Detection Powered by XNA

Speaker: Sir Walter Bodmer (Head of Laboratory for Cancer and Immunogenetics, Weatherall Institute for Molecular Medicine, University of Oxford) and Michael J. Powell, Ph.D. (Chief Scientific Officer, DiaCarta, Inc.)

Highlight: 

  • Colorectal cancer genetics
  • Driver mutations in a limited number of genes are the ideal candidates for screening & early detection of recurrence
  • ColoScape™: Discuss future use of early identification of subclinical disease, cancer recurrence & monitoring

Need Help?

Call us: +1 (800) 878-6662

Email us: information@diacarta.com

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