Analyzing Inflammatory Plasma Biomarkers in Samples from Cancer Patients

The Simoa CorPlex Human Cytokine Panel 1 is a multiplex immunoassay for the quantitative analysis of 10 inflammatory biomarkers (IFNγ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, and TNFα) in EDTA plasma and serum on the Quanterix SP-X™ analysis and imaging platform.

This assay has been verified in adherence to Quanterix principles of design control and fit-for-purpose guidance. The validation data is outlined in the validation report and datasheets are available to download from quanterix.com. This article describes the analysis of EDTA plasma samples from 1st line gastric and 1st line colorectal cancer samples.

Methods

The Simoa CorPlex Human Cytokine Panel 1 (Item # 85-0329) assay plate is prespotted with analyte-specific capture antibodies in each well of the 96-well microplate. The assay was run in line with the instructions on the kit. In short, each sample result utilizes 12.5 μl of EDTA plasma diluted 4x with the kit sample diluent.

Along with the diluted samples, multi-constituent calibrators are arranged and added to the plate, neat. The plate is incubated for two hours. Once the incubation period is over, unbound proteins are removed, and a biotinylated detection antibody reagent is added for 30 minutes.

Once the unbound detection antibody is removed, streptavidin-horseradish peroxidase (SA-HRP) is then added for a further 30 minutes. The plates are then washed and the substrate is added. Shortly after this, the plate is imaged on the SP-X platform.

In adjacent wells of the plate, the calibrators and diluted samples run in duplicate (n=2). Each of the plates had calibrators which were utilized to assess the sample concentrations for that plate.

EDTA plasma samples measured include 35 1st line gastric cancer samples, 45 1st line colorectal samples, and 29 seemingly healthy, random, and normal samples. The colorectal and gastric samples were collected from the Duke University School of Medicine. The apparently healthy, normal samples were supplied by BioIVT.

Results

The different assay results demonstrate the three sample populations according to the limit of detection (LOD) and the lower limit of quantification (LLOQ). The LLOQ and LOD cutoffs are the same as defined in the CorPlex Human Cytokine Panel 1 validation report.

Table 1 outlines the percentage of samples higher than the limit of detection (LOD). In brief, the LOD is calculated using 2.5 standard deviations above the background (the mean of the zero-calibration standard) acquired through several runs and kit lots.

Table 2 outlines the percentage of samples higher than the lower limit of quantification (LLOQ). The definition of LLOQ is the lowest concentration of calibrator with ≤ 20% pooled CV and 80 to 120% mean recovery overall runs, multiplied by the Minimum Required Dilution (MRD; for this assay MRD = 4).

Table 1: Percentage of samples that measure above the limit of detection.

Sample Detection (>LOD)
  IL-12 p70 IL-1b IL-4 IL-5 IFNγ IL-6 IL-8 IL-22 TNFα IL-10
% Gastric cancer samples >LOD 74.3% 100.0% 100.0% 97.1% 91.4% 100.0% 100.0% 100.0% 100.0% 100.0%
% Colorectal cancer samples >LOD 100.0% 100.0% 93.3% 95.6% 86.7% 100.0% 100.0% 100.0% 100.0% 100.0%
% Normal samples >LOD 72.4% 96.6% 93.1% 89.7% 86.2% 100.0% 100.0% 100.0% 100.0% 100.0%

 

Table 2: Percentage of samples that measure above the lower limit of quantification.

Sample Measurement (>LOQ)
  IL-12 p70 IL-1b IL-4 IL-5 IFNγ IL-6 IL-8 IL-22 TNFα IL-10
% Gastric cancer samples >LOQ 51.4% 100.0% 62.9% 74.3% 91.4% 100.0% 100.0% 100.0% 100.0% 100.0%
% Colorectal cancer samples >LOQ 73.3% 91.1% 66.7% 82.2% 84.4% 100.0% 100.0% 100.0% 100.0% 100.0%
% Normal samples >LOQ 34.5% 62.1% 72.4% 55.2% 79.3% 100.0% 100.0% 100.0% 100.0% 100.0%

 

Conclusions

The Simoa CorPlex Human Cytokine Panel 1 exhibits the ability in human EDTA plasma to analyze ten important cytokines at the same time, these are Interferon gamma (IFNγ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, and Tumor Necrosis Factor alpha (TNFα).

This multiplexed assay demonstrates that the endogenous biomarkers in most samples tested from these cohorts can be calculated as above the lower limit of quantification.

Discussion

The cytokines profiles of colorectal stromal and tumor cells manifest systems of bivalent, anti-tumor, or pro-tumor agents. As an example, IFNγ, interleukin-12 (IL-12) perform as inhibitory agents. However, IL-4, IL-6, IL-8, IL-17A, IL-22, TNFα are protumorigenic.¹

Additionally, patients with inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease) or alternative factors that promote inflammation (such as obesity, smoking, environmental pollutants, and infection) have an increased risk of developing colorectal cancer.

The serum levels of various cytokines, including TNFα, IL-8, and IL-6, are higher in colorectal cancer patients, which may be beneficial in understanding disease prognosis.²,³ Gastric cancer and long-term inflammation of the stomach are strongly correlated.

The cytokines produced in chronic inflammation can impact the epithelial and immune cells to affect the progression of the disease. Studies in human models of gastric cancer and gastritis are determining significant roles for cytokines in the regulation of metaplasia, oxyntic atrophy, hyperplasia, and the advancement to gastric cancer.⁴

The pathological roles of several cytokines continue to be undefined and as such, the characterization of the profiles of these cytokines will be crucial in understanding disease pathology and the progression of therapies.⁵

References and Further Reading

  1. Mager, Lukas F et al. “Cytokine-Induced Modulation of Colorectal Cancer” Frontiers in oncology vol. 6 96. 19 Apr. 2016, doi:10.3389/fonc.2016.00096
  2. Klampfer, Lidija. “Cytokines, inflammation and colon cancer” Current cancer drug targets vol. 11,4 (2011): 451- 64.
  3. Knüpfer, H. & Preiss R. “Serum interleukin-6 levels in colorectal cancer patients” International Journal of Colorectal Disease (2010) 25: 135. doi:10.1007/s00384-009-0818-8
  4. Bockerstett, Kevin A and Richard J DiPaolo. “Regulation of Gastric Carcinogenesis by Inflammatory Cytokines” Cellular and molecular gastroenterology and hepatology vol. 4,1 47-53. 14 Mar. 2017, doi:10.1016/j.jcmgh.2017.03.005
  5. Amedei A. et al. “The use of cytokines and chemokines in the cancer immunotherapy.” Recent Patents on AntiCancer Drug Discovery vol. 8 2. May 2013, doi: 10.2174/1574892811308020002

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Last updated: Aug 13, 2019 at 3:53 AM

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