Showing papers by "Mario C. Deng published in 2022"

Whole transcriptome profiling of prospective endomyocardial biopsies reveals prognostic and diagnostic signatures of cardiac allograft rejection.

Abstract: BACKGROUND Heart transplantation provides a significant improvement in survival and quality of life for patients with end-stage heart disease, however many recipients experience different levels of graft rejection that can be associated with significant morbidities and mortality. Current clinical standard-of-care for the evaluation of heart transplant acute rejection (AR) consists of routine endomyocardial biopsy (EMB) followed by visual assessment by histopathology for immune infiltration and cardiomyocyte damage. We assessed whether the sensitivity and/or specificity of this process could be improved upon by adding RNA sequencing (RNA-seq) of EMBs coupled with histopathological interpretation. METHODS Up to 6 standard-of-care, or for-cause EMBs, were collected from 26 heart transplant recipients from the prospective observational Clinical Trials of Transplantation (CTOT)-03 study, during the first 12-months post-transplant and subjected to RNA-seq (n = 125 EMBs total). Differential expression and random-forest-based machine learning were applied to develop signatures for classification and prognostication. RESULTS Leveraging the unique longitudinal nature of this study, we show that transcriptional hallmarks for significant rejection events occur months before the actual event and are not visible using traditional histopathology. Using this information, we identified a prognostic signature for 0R/1R biopsies that with 90% accuracy can predict whether the next biopsy will be 2R/3R. CONCLUSIONS RNA-seq-based molecular characterization of EMBs shows significant promise for the early detection of cardiac allograft rejection. Heart transplantation provides a significant improvement in survival and quality of life for patients with end-stage heart disease, however many recipients experience different levels of graft rejection that can be associated with significant morbidities and mortality. Current clinical standard-of-care for the evaluation of heart transplant acute rejection (AR) consists of routine endomyocardial biopsy (EMB) followed by visual assessment by histopathology for immune infiltration and cardiomyocyte damage. We assessed whether the sensitivity and/or specificity of this process could be improved upon by adding RNA sequencing (RNA-seq) of EMBs coupled with histopathological interpretation. Up to 6 standard-of-care, or for-cause EMBs, were collected from 26 heart transplant recipients from the prospective observational Clinical Trials of Transplantation (CTOT)-03 study, during the first 12-months post-transplant and subjected to RNA-seq (n = 125 EMBs total). Differential expression and random-forest-based machine learning were applied to develop signatures for classification and prognostication. Leveraging the unique longitudinal nature of this study, we show that transcriptional hallmarks for significant rejection events occur months before the actual event and are not visible using traditional histopathology. Using this information, we identified a prognostic signature for 0R/1R biopsies that with 90% accuracy can predict whether the next biopsy will be 2R/3R. RNA-seq-based molecular characterization of EMBs shows significant promise for the early detection of cardiac allograft rejection.

Variability in Donor-Derived Cell-Free DNA Scores to Predict Mortality in Heart Transplant Recipients – A Proof-of-Concept Study

Abstract: Background Over the last decade, expanding use of molecular diagnostics in heart transplantation has allowed implementation of non-invasive surveillance strategies for monitoring allograft health. The commercially available HeartCare platform combines the AlloMap gene expression profiling assay and the AlloSure donor-derived cell-free DNA test (dd-cfDNA). Beyond their established use for assessment of rejection, evidence is building for predictive utility, with the longitudinal AlloMap Variability score previously shown to correlate with the risk of future rejection, graft dysfunction, re-transplantation, or death. In this single-center, retrospective pilot study, we evaluated the performance of a novel AlloSure Variability metric in predicting mortality in a cohort of heart transplant recipients. Methods Seventy-two adult heart transplant recipients with at least 3 concurrent AlloMap/AlloSure results were included. Demographic, clinical, imaging, and laboratory parameters were captured. Variability was defined as the standard deviation of longitudinal AlloMap/AlloSure results. A Cox multivariable adjusted proportional hazards model was used to evaluate the variability metrics as predictors of mortality. Associations between AlloMap/AlloSure variability and donor specific antibody (DSA) status were also assessed. Results A total of 5 patients (6.9%) died during a median follow-up of 480 days. In a univariate Cox proportional hazards model, higher AlloSure variability (HR 1.66, 95%CI 1.14 – 2.41), but not AlloMap variability or the cross-sectional AlloSure/AlloMap results was associated with increased mortality risk. Longitudinal AlloSure variability was also higher among patients with both preformed DSA and those developing de novo DSA. Conclusion Our results suggest that increased variability of dd-cfDNA in heart transplant patients is associated with both mortality risk and the presence of donor specific antibodies. These findings highlight the added value of longitudinal data in the interpretation of AlloMap/AlloSure scores in this population and open the door to larger studies investigating the utility of these metrics in shaping post-transplant clinical care paradigms.

Assessing the Relationship Between Molecular Rejection and Parenchymal Injury in Heart Transplant Biopsies

Abstract: Background. The INTERHEART study (ClinicalTrials.gov #NCT02670408) used genome-wide microarrays to detect rejection in endomyocardial biopsies; however, many heart transplants with no rejection have late dysfunction and impaired survival. We used the microarray measurements to develop a molecular classification of parenchymal injury. Methods. In 1320 endomyocardial biopsies from 645 patients previously studied for rejection-associated transcripts, we measured the expression of 10 injury-induced transcript sets: 5 induced by recent injury; 2 reflecting macrophage infiltration; 2 normal heart transcript sets; and immunoglobulin transcripts, which correlate with time. We used archetypal clustering to assign injury groups. Results. Injury transcript sets correlated with impaired function. Archetypal clustering based on the expression of injury transcript sets assigned each biopsy to 1 of 5 injury groups: 87 Severe-injury, 221 Late-injury, and 3 with lesser degrees of injury, 376 No-injury, 526 Mild-injury, and 110 Moderate-injury. Severe-injury had extensive loss of normal transcripts (dedifferentiation) and increase in macrophage and injury-induced transcripts. Late-injury was characterized by high immunoglobulin transcript expression. In Severe- and Late-injury, function was depressed, and short-term graft failure was increased, even in hearts with no rejection. T cell–mediated rejection almost always had parenchymal injury, and 85% had Severe- or Late-injury. In contrast, early antibody-mediated rejection (AMR) had little injury, but late AMR often had the Late-injury state. Conclusions. Characterizing heart transplants for their injury state provides new understanding of dysfunction and outcomes and demonstrates the differential impact of T cell–mediated rejection versus AMR on the parenchyma. Slow deterioration from AMR emerges as a major contributor to late dysfunction.

Variability in Donor-Derived Cell-Free DNA Levels Predicts Mortality Risk in Heart Transplant Recipients

Abstract: Purpose Longitudinal surveillance of heart transplant (HTx) recipients using donor-derived cell-free DNA (dd-cfDNA) allows non-invasive detection of molecular injury patterns. We hypothesized that variability in dd-cfDNA levels over time identifies patients at risk of adverse outcomes. Methods 72 adult HTx patients monitored with gene expression profiling (GEP) and dd-cfDNA (AlloMap, AlloSure; CareDx Inc., Brisbane, CA) at our center were included. Variability (ASV) was defined as standard deviation of the 3 most recent (3-value) or all (all-value) sequential dd-cfDNA results and scaled x 10 for analysis (ASV*10). We used a Cox proportional hazards model to assess the association between ASV and mortality and a survival classification and regression tree (CART) algorithm to define a threshold for increased mortality risk. Results Patients had a median of 6 (IQR 4-9) paired GEP/dd-cfDNA results spaced 40.8 (IQR 31.5 - 66.1) days apart. 48.6% of patients had dd-cfDNA below the assay limit of detection (<0.12%); the remainder had a median dd-cfDNA of 0.17% (IQR: 0.12 - 0.45%) and a mean all-value ASV of 0.24 (SD: 0.40). ASV*10 of patients who died (2.62, SD 2.66) trended higher than of those who survived (0.54, SD 1.10) over a median follow-up of 480 days (IQR 244 - 859). In a univariate Cox model, the 3-value ASV*10 was associated with an increased risk of mortality (HR 1.66, 95% CI 1.14 - 2.41, p<0.009). Only a trend was evident after multivariate adjustment [Figure 1a]. Using a CART algorithm, a 3-value ASV*10 of 2.97 discriminated between patients at high and low risk of mortality after HTx [Figure 1b]. Conclusion In this proof-of-concept study evaluating the clinical utility of longitudinal dd-cfDNA results, we found that higher variability in dd-cfDNA over time was associated with an increased risk of mortality after HTx. Additional studies utilizing larger datasets are needed to validate and expand on our findings. Longitudinal surveillance of heart transplant (HTx) recipients using donor-derived cell-free DNA (dd-cfDNA) allows non-invasive detection of molecular injury patterns. We hypothesized that variability in dd-cfDNA levels over time identifies patients at risk of adverse outcomes. 72 adult HTx patients monitored with gene expression profiling (GEP) and dd-cfDNA (AlloMap, AlloSure; CareDx Inc., Brisbane, CA) at our center were included. Variability (ASV) was defined as standard deviation of the 3 most recent (3-value) or all (all-value) sequential dd-cfDNA results and scaled x 10 for analysis (ASV*10). We used a Cox proportional hazards model to assess the association between ASV and mortality and a survival classification and regression tree (CART) algorithm to define a threshold for increased mortality risk. Patients had a median of 6 (IQR 4-9) paired GEP/dd-cfDNA results spaced 40.8 (IQR 31.5 - 66.1) days apart. 48.6% of patients had dd-cfDNA below the assay limit of detection (<0.12%); the remainder had a median dd-cfDNA of 0.17% (IQR: 0.12 - 0.45%) and a mean all-value ASV of 0.24 (SD: 0.40). ASV*10 of patients who died (2.62, SD 2.66) trended higher than of those who survived (0.54, SD 1.10) over a median follow-up of 480 days (IQR 244 - 859). In a univariate Cox model, the 3-value ASV*10 was associated with an increased risk of mortality (HR 1.66, 95% CI 1.14 - 2.41, p<0.009). Only a trend was evident after multivariate adjustment [Figure 1a]. Using a CART algorithm, a 3-value ASV*10 of 2.97 discriminated between patients at high and low risk of mortality after HTx [Figure 1b]. In this proof-of-concept study evaluating the clinical utility of longitudinal dd-cfDNA results, we found that higher variability in dd-cfDNA over time was associated with an increased risk of mortality after HTx. Additional studies utilizing larger datasets are needed to validate and expand on our findings.

Donor Specific Allo-Antibody is Significantly Associated with Variability in Donor-Derived Cell-Free DNA Scores in Adult Heart Transplant Recipients

Abstract: PurposeIn this single-center retrospective pilot study heart transplant (HTx) patients were non-invasively monitored by donor-derived cell-free DNA (dd-cfDNA). We hypothesized that longitudinal dd-cfDNA variability scores correlate with preformed and de novo (dn) donor specific HLA allo-antibody (DSA).Methods72 adult HTx patients with at least 3 dd-cfDNA results were included (AlloSure; CareDx Inc., Brisbane, CA). We determined dd-cfDNA variability (ASV) by standard deviation (SD) of longitudinal dd-cfDNA results, scaled x10 (ASV*10), and DSA by single antigen class I/II and MFI >1000 for HLA-A, B, DR, DQB/A and >2000 for HLA-C and DPB/A.ResultsAge at HTx was 49.1 years (SD 14.3). Study enrollment occurred at 270 days +/-SD 420.2 post-HTx. Biopsy-proven rejection (ACR ≥ 2R and/or pAMR ≥ 1) was detected in 16 (22 %), preformed DSA in 9 (12.5%), dnDSA in 13 (18%) and preformed + dn in 2 (2.7%) patients while 48 (66.7%) were DSA negative. Median time from HTx to dnDSA was 329d (range: 39-827). Including all values, mean ASV*10 was highest in patients with dnDSA (3.03 ± 4.74) compared to those with no DSA (0.65 ± 2.04) or preformed DSA (1.36 ± 3.40, p=0.001; Table 1). Patients who developed dnDSA showed a significant increase of ASV*10 after dnDSA detection (1.50 ± 2.06, p = 0.014), but not before dnDSA detection (1.24 ± 1.49, p=0.380) in comparison to patients with no DSA (0.65 ± 2.04, Table 1).ConclusionThese findings highlight the value of longitudinal assessment of dd-cfDNA and DSA in HTx recipients. Our results show that increased dd-cfDNA variability is associated with the presence of preformed and dnDSA. Further, ASV*10 is significantly increased after identification of dnDSA, further supporting the mechanistic role of dnDSA in mediating poor outcomes after HTx. Confirmation in larger data sets may further advance the utility of longitudinal non-invasive surveillance using dd-cfDNA and dnDSA in transplant recipients. In this single-center retrospective pilot study heart transplant (HTx) patients were non-invasively monitored by donor-derived cell-free DNA (dd-cfDNA). We hypothesized that longitudinal dd-cfDNA variability scores correlate with preformed and de novo (dn) donor specific HLA allo-antibody (DSA). 72 adult HTx patients with at least 3 dd-cfDNA results were included (AlloSure; CareDx Inc., Brisbane, CA). We determined dd-cfDNA variability (ASV) by standard deviation (SD) of longitudinal dd-cfDNA results, scaled x10 (ASV*10), and DSA by single antigen class I/II and MFI >1000 for HLA-A, B, DR, DQB/A and >2000 for HLA-C and DPB/A. Age at HTx was 49.1 years (SD 14.3). Study enrollment occurred at 270 days +/-SD 420.2 post-HTx. Biopsy-proven rejection (ACR ≥ 2R and/or pAMR ≥ 1) was detected in 16 (22 %), preformed DSA in 9 (12.5%), dnDSA in 13 (18%) and preformed + dn in 2 (2.7%) patients while 48 (66.7%) were DSA negative. Median time from HTx to dnDSA was 329d (range: 39-827). Including all values, mean ASV*10 was highest in patients with dnDSA (3.03 ± 4.74) compared to those with no DSA (0.65 ± 2.04) or preformed DSA (1.36 ± 3.40, p=0.001; Table 1). Patients who developed dnDSA showed a significant increase of ASV*10 after dnDSA detection (1.50 ± 2.06, p = 0.014), but not before dnDSA detection (1.24 ± 1.49, p=0.380) in comparison to patients with no DSA (0.65 ± 2.04, Table 1). These findings highlight the value of longitudinal assessment of dd-cfDNA and DSA in HTx recipients. Our results show that increased dd-cfDNA variability is associated with the presence of preformed and dnDSA. Further, ASV*10 is significantly increased after identification of dnDSA, further supporting the mechanistic role of dnDSA in mediating poor outcomes after HTx. Confirmation in larger data sets may further advance the utility of longitudinal non-invasive surveillance using dd-cfDNA and dnDSA in transplant recipients.