ColoNode®
The ColoNode® kit is for research use only. 

The intended use is for detection of tumor cells in colorectal cancer (CRC) lymph node (LN) samples and determination of CRC tumor cell aggressiveness. 

The ColoNode® kit is a test intended for measurement of mRNA expression levels of six genes: CEACAM5 (carcinoembryonic antigen), KLK6 (kallikrein-related peptidases 6), MUC2 (mucin 2), SLC35D3 (solute carrier family 35 member D3), POSTN (periostin) and 18S rRNA (18S ribosomal RNA), in total RNA extracted from CRC LN samples. 

Background
Biomarker mRNAs have great potential as tools for LN analysis in CRC. Analysis of biomarker expression at the mRNA level has several advantages over protein level or cellular level expression: several biomarkers can easily be analyzed in the same RNA extract and a large volume of the LN (potentially the entire LN) can be available for analysis. 

In the ColoNode® kit six target genes are analyzed. Four of the genes are expressed in CRC epithelial cells, namely CEACAM5, KLK6, SLC35D3 and MUC2, and one biomarker, POSTN, is expressed in tumor associated fibroblasts. None of the genes are expressed in immune cells making them suitable for analysis of LN samples. 18S rRNA is analyzed for normalization and as positive control.

 

Information about the target genes: 

  • CEACAM5 protein is a wellestablished tumor marker in adenocarcinoma and is used primarily for postoperative followup. CEACAM5 is expressed in epithelial cells of the large bowel and their expression is retained in CRC. CEACAM5 mRNA has been shown to be superior in detection of disseminated tumor cells in CRC LNs compared to the routine method (hematoxylin and eosin staining of LN tissue). Moreover, CEACAM5 mRNA level have been shown to correlate with numbers of tumor cells.

  • KLK6 belongs to the human kallikrein-related gene family of serine proteases. Many of these are dysregulated in human malignancies and have a role in cell growth regulation, angiogenesis, invasion and metastasis. KLK6 mRNA has been identified as a promising progression biomarker for CRC. High levels of KLK6 mRNA correlates to poor prognosis.

  • Mucin is the major mucin in the mucous layer covering the colon and rectum epithelium. Ten to 20% of all CRC tumors are mucinous, which involves copious production of intestinal mucins, mainly MUC2. Mucinous primary tumors have been shown to be associated with better prognosis than adenocarcinoma in general.

  • POSTN is expressed in tumoral stromal fibroblasts in CRC. The level of stromal POSTN is a prognostic biomarker for CRC. 
  • SLC35D3 is analyzed because it is associated with poor prognosis and appears to represent another epithelial cell expression pattern than CEACAM5, MUC2 and KLK6 perhaps related to cellular immaturity.

  • 18S rRNA is used for normalization of mRNA expression levels in analysis of different genes and samples. 18S rRNA has been proven to be expressed at relatively stable levels in lymphocytes and colonic epithelial cells. 

The biomarker combination of ColoNode® was able to detect tumor cells and an estimated grade tumor cell aggressiveness in CRC from fresh frozen LN samples. Moreover, the ColoNode® kit could be used successfully for analysis of formalin-fixed LN sections. 

Assay principle
The ColoNode® kit is a dual system 3-plex, one-step, real-time reverse transcription-quantitative polymerase chain reaction (qRT-PCR) test for analysis of target gene expression levels. It is designed and developed for the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems™). 

In a one-step qRT-PCR reaction both reverse transcription of RNA into cDNA and PCR amplification takes place in the same reaction mixture with all required reagents added initially, thereby avoiding contaminating agents and carry over effects. This is possible because the enzyme, Tth DNA polymerase, functions both as a reverse transcriptase and a DNA polymerase. 

Starting material is total RNA, which is reverse transcribed to cDNA with a 5’-gene specific (reverse) primer that binds to the mRNA of the respective gene of interest and produces the specific cDNA. The amplification step utilizes a pair of primers (forward and reverse) complementary to one strand each and a gene specific probe that targets one of the strands in the cDNA located between the primers. The probe is labeled with a fluorescent reporter dye at the 5’ end and a quencher dye at the 3’ end. As long as the probe is intact, it will not emit fluorescence.  As the elongation of the strand proceeds the probe will be cleaved by the Tth DNA polymerase releasing the reporter dye, and as the amplicons increase exponentially with each PCR cycle, fluorescence signals increase and are detected in real-time. When the intensity of the particular fluorescence signal reaches a pre-defined threshold, the corresponding cycle is referred as the cycle threshold (ct). 

The ColoNode® kit raw data results from the qRT-PCR analysis is transferred from the QuantStudio™ 5 Real-Time PCR System and analyzed by the ColoNode® Software yielding tumor cell detection and estimation of CRC tumor cell aggressiveness. In addition, expression levels of each target gene mRNA for every analyzed sample is given.

Figure – Overview of ColoNode® analysis

Assay design
The ColoNode® kit is designed as two 3-plex, one step, qRT-PCR systems.

The kit performs the reverse transcription (RT), amplification and detection of mRNA by qRT-PCR with two 3-plex reaction mixes. It contains two master mixtures with primers and probes for three target genes each. It also contains standard samples for actual gene expression value determination (copies/µl) and negative control. Unknown LN samples, standards and negative control are analyzed in duplicate.

Specific reverse and forward primers have been constructed for each of the six target genes. They are placed in different exons of respective target gene. A sequence-specific dye-labeled probe is placed over the boundary between the two exons to make the assay RNA specific and thereby avoiding amplification of genomic DNA. Three different fluorescence dyes (FAM™, VIC™, NED™; Thermo Fisher Scientific Inc) are used to perform quantification of 3 target genes in a single reaction. Three genes are measured in half of the wells on the plate and the other 3 genes are measured in the other half.

In each analysis, standard samples containing specific amounts of mRNA from each of the 6 target genes are analyzed and form a standard curve. The analyzed unknown samples get the actual gene quantity per µl calculated from the respective target gene standard curve.

Both the standards and the analysis of the reference gene 18S rRNA function as a positive control for the test and the unknown samples. The 18S rRNA gene also verifies that enough amounts of total RNA have been analyzed. A negative control is included for control of contamination.