Methods
Patient Population, Specimens and Data Collection
INTERCOM (clinicaltrials.gov NCT01299168) was a prospective observational study involving Edmonton, Minneapolis, Baltimore, Barcelona, Hannover and Manchester. Local institutional review board approval was obtained in each center. Inclusion criteria specified all kidney transplant recipients ≥18 years of age undergoing a kidney biopsy for clinical indications that were able and willing to give informed consent. However, the centers did enroll two patients under age 18, and they were left in the study.
The local biopsy assessment was performed as per standard of care, without knowledge of the microarray results. The local C4d staining method was either immunofluorescence (University of Alberta and University of Minnesota at Fairview) or deparaffinized immunoperoxidase (University of Maryland, Hospital Vall d'Hebron, Hannover Medical School and Manchester Royal Infirmary). Positivity was determined using published guidelines: diffuse staining by immunofluorescence (Grade 3) or diffuse/focal by immunoperoxidase (Grades 2 and 3). One 5 mL serum sample was collected for local HLA antibody testing at the time of biopsy.
The study required one additional biopsy core beyond those obtained as standard of care. The six centers contributed 320 samples collected between October 2008 and March 2012, of which 300 were suitable for microarray processing. The 20 excluded biopsy specimens were either too small or improperly collected and stabilized. We also included control kidney samples from histologically normal areas of six nephrectomies for renal carcinoma.
Data Collection
Copies of the original pathology reports (with patient identifiers removed), along with relevant clinical and laboratory data, were sent to the Alberta Transplant Applied Genomics Center (ATAGC). This information was stored in anonymous fashion in a Scientific Data Management System (SDMS) at the University of Alberta.
Classification of the Local Center Pathology Reports
The local assessment of ABMR based on conventional features (histology, C4d staining and DSA) was often expressed using terms of uncertainty such as "suspicious for" and "rule out." To compare the local assessment to the ABMR score, a pathologist in Edmonton (Dr. M. Mengel) read all reports to ensure that the diagnoses recorded in our SDMS represented the opinion or suspicion of the local center as stated in the pathology report. For comparison with the reference set, these diagnoses were assembled into categories previously used for the reference set without knowledge of the ABMR scores, using the rules outlined in Table S1.
Biopsy Processing and Microarray Analyses
To prevent mRNA degradation, the study core was immediately stabilized in RNAlater® (Life Technologies, Carlsbad, CA), kept at −4°C for 24 h and stored at −20°C until processing. Samples from distant centers were batched and shipped to ATAGC on dry ice for processing on Affymetrix U133 2.0 microarrays (Santa Clara, CA).
We normalized microarray results using the Bioconductor "RefPlus" package. Because Affymetrix changed their labeling kit before the INT300 samples were processed, BFC403 and INT300 were normalized as separate batches. The BFC403 expression values were then adjusted for batch effects using the Ratio-G method. All analyses used "R" version 2.12.1 (64-bit), with various libraries from Bioconductor 2.8. Microarray expression files for BFC403 are posted on the Gene Expression Omnibus website (GSE36059). Files for INT300 will be posted at GEO on publication.
Assigning ABMR Scores
ABMR scores were assigned using a classifier algorithm built using the BFC403 reference set, details of which can be found in Supplementary Methods. Since INT300 can be considered an independent test set, the entire BFC403 data set was used to train the classifier, rather than using the cross-validation method from the original paper. The classifier output is a score between 0.0 and 1.0, reflecting the probability that ABMR is operating in the biopsy.
Survival Analysis
Since graft failure is uncommon in patients undergoing an indication biopsy less than 1 year after transplantation, survival analyses were restricted to late (>1 year) biopsies. Within this subpopulation, if a patient had more than one biopsy, the first that had either histologic or molecular ABMR was used. If none had ABMR, the earliest biopsy was used. In order to compare the predictive ability of the molecular and histologic diagnoses, a time cutoff was required and we chose 3 years postbiopsy. All biopsies that were functioning at 3 years were given a censor time of 3 years, and assigned a graft status of "working," regardless of any failures in the post–3-year period. Thus, all survival analyses in this study refer to 3-year survival. Cox regression and Kaplan–Meier plots were performed using the "R" package "survival." Comparison of the predictive ability of different models used the integrated discrimination improvement (IDI), net reclassification index (NRI) and median risk score (MRS) improvement methods in the "R" package "survIDINRI".