The CARGO Study and Development of AlloMap Testing
Scientific Rationale
The Human Genome Project advanced the knowledge of the role of gene expression in disease. Building on this new knowledge and advances in genomics, bioinformatics, transplant immunology and molecular diagnostics technology, scientists at XDx and clinical investigators at leading cardiac transplant centers postulated that a non-invasive testing method to monitor for acute cellular rejection using gene expression profiling could be developed for clinical use.
The CARGO study participants
| Transplant Center |
Principal Investigator(s) |
| Cleveland Clinic |
Randall Starling, MD |
| Columbia University |
Mario Deng, MD
Seema Mital, MD (pediatrics)
Linda Addonizio, MD (pediatrics) |
| Kaiser Permanente |
Dana Weisshaar, MD |
| Ochsner Clinic |
Mandeep Mehra, MD |
| Stanford University |
Sharon Hunt, MD
Daniel Bernstein, MD (pediatrics) |
| Temple University |
Howard Eisen, MD |
| University of California at Los Angeles |
Jon Kobashigawa, MD |
| University of Florida |
James Hill, MD
Dan Pauly, MD, PhD |
| University of Pittsburgh |
Srinivas Murali, MD
Adrianna Zeevi. PhD
Steven Weber, MD (pediatrics) |
| Independent Study Pathologists |
|
| Columbia University |
Charles Marboe, MD |
| Stanford University |
Gerald Berry, MD
Margaret Billingham, MD |
|
The CARGO Study:
Collecting Samples and Data to Develop AlloMap Testing
Overview
The CARGO study was initiated at eight major U.S. transplant centers representing more than 20% of the cardiac transplant volume. This observational study collected paired biopsy and peripheral blood samples and clinical data from cardiac transplant recipients at various times post-transplant for use in the development and validation of AlloMap molecular expression testing.
Methodology
XDx used genomic technologies—Microarrays and qRT-PCR—to identify the genes that strongly correlated with the presence or absence of acute cellular rejection in microarray gene expression studies. 68 differentiating gene sequences correlated significantly with the presence or absence of acute cellular rejection.
The 68 differentiating genes were distributed among several immune pathways, and in many instances included several covariant genes from the same pathway. To improve precision, covariant genes were grouped for further analysis. Using linear discriminant analysis, an algorithm was developed that combined 11 genes whose differential expression distinguishes most reproducibly between the presence and absence of acute cellular rejection. Then, 9 genes were added for quality control, of which 6 also function as normalization genes, yielding a total of 20 genes.
Results: Validation of AlloMap Testing
Once the test was developed, a prospective, blinded, and powered validation study at the XDx Reference Laboratory compared AlloMap testing results with the consensus biopsy diagnosis for the paired sample and the patient’s clinical data.
This case-controlled clinical validation study showed that AlloMap testing distinguished between the presence and absence of acute cellular rejection with high statistical significance (p = 0.0001) .
A representative population study provided clinical performance characteristics (e.g. NPV) that should apply to the general cardiac transplant population.
A subsequent CARGO sub-study showed that in heart transplant patients in the first 6 months post-transplant were free from ACR ≥3A[2R] for the next 80 days (~12 weeks).
In addition to providing clinical performance characteristics, analytical validation studies performed by the XDx Reference Laboratory verified the reliability and reproducibility of the AlloMap test procedure for routine clinical use.
Prediction Study
In a CARGO sub-study (n=74), patients in the first six months post transplant who had AlloMap scores < 20 were free from ACR (ISHLT ≥3A(2R)) for the next 80 days (~12 weeks). Of the 11 informative gene panel, there were 3 genes that most significantly discriminated future ACR: IL1R2, FLT3 and PDCD1 (p ≤ .02). IL1R2 and FLT3 are corticosteroid responsive genes which decrease in expression before ACR. PDCD1, a market of T cell activation, increased in expression before ACR.
Find out more:
