Donor-Specific Antibody Is Associated with Increased Expression of Rejection Transcripts in Renal Transplant Biopsies Classified as No Rejection
Authors: Madill-Thomsen Katelynn S. , Böhmig Georg A. , Bromberg Jonathan , Einecke Gunilla , Eskandary Farsad , Gupta Gaurav , Hidalgo Luis G. , Myslak Marek , Viklicky Ondrej , Perkowska-Ptasinska Agnieszka , Halloran Philip F. ,   the INTERCOMEX Investigators
Published: July 2021

Significance Statement
Many kidney transplant patients in INTERCOMEX whose biopsy specimens are diagnosed molecularly or histologically as no rejection have donor-specific HLA antibodies (DSAs, 32%). Although the significance of DSA in no rejection has been unclear, we hypothesized that current diagnostic thresholds miss some DSA-positive patients who may have subtle antibody-mediated rejection (ABMR)–related stress, with potential effect on outcomes. To search for subtle ABMR-related gene expression in “no rejection” biopsy samples, we developed a “DSA-probability” classifier (trained on DSA positivity) in microarray results from 1679 biopsy samples that detected ABMR-related transcripts (e.g., NK cell and IFNG-inducible). Many no rejection biopsy samples had mildly increased expression of ABMR-related transcripts, associated with DSA positivity, and these kidneys had increased risk of failure. Thus, mild ABMR-related stress is more common than previously thought.

Background
Donor -specific HLA antibody (DSA) is present in many kidney transplant patients whose biopsies are classified as no rejection (NR). We explored whether in some NR kidneys DSA has subtle effects not currently being recognized.

Methods
We used microarrays to examine the relationship between standard-of-care DSA and rejection-related transcript increases in 1679 kidney transplant indication biopsies in the INTERCOMEX study (ClinicalTrials.gov NCT01299168), focusing on biopsies classified as NR by automatically assigned archetypal clustering. DSA testing results were available for 835 NR biopsies and were positive in 271 (32%).

Results
DSA positivity in NR biopsies was associated with mildly increased expression of antibody-mediated rejection (ABMR)–related transcripts, particularly IFNG-inducible and NK cell transcripts. We developed a machine learning DSA probability (DSAProb) classifier based on transcript expression in biopsies from DSA-positive versus DSA-negative patients, assigning scores using 10-fold cross-validation. This DSAProb classifier was very similar to a previously described “ABMR probability” classifier trained on histologic ABMR in transcript associations and prediction of molecular or histologic ABMR. Plotting the biopsies using Uniform Manifold Approximation and Projection revealed a gradient of increasing molecular ABMR-like transcript expression in NR biopsies, associated with increased DSA (P<2 × 10−16). In biopsies with no molecular or histologic rejection, increased DSAProb or ABMR probability scores were associated with increased risk of kidney failure over 3 years.

Conclusions
Many biopsies currently considered to have no molecular or histologic rejection have mild increases in expression of ABMR-related transcripts, associated with increasing frequency of DSA. Thus, mild molecular ABMR-related pathology is more common than previously realized.