Technical considerations when designing a gene expression panel for renal transplant diagnosis

Toulza, Frédéric; Dominy, Kathy; Cook, Terry; Galliford, Jack; Beadle, Jack; McLean, A.G.; Roufosse, Candice

doi:10.1038/s41598-020-74794-3

Cited by 10 publications

(11 citation statements)

References 30 publications

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“…Although the NanoString platform offers the most affordable and technically robust method of performing multiplexed expression analysis on the limited, low-quality RNA isolated from FFPE tissue, a subset of transcripts do not correlate between NanoString and quantitative real-time polymerase chain reaction (qRT-PCR) or are affected by tissue handling (FFPE tissue vs. tissue that is fresh frozen or placed in RNAlater). 35,36 Upcoming validation studies with an increased sample size will aim to determine if histologic parameters, such as severity of tubulitis and mast cell numbers, and transcriptionally detected alterations in allograft lipid metabolism are predictive of CA TCMR treatment response.…”

Section: Discussionmentioning

confidence: 99%

Chronic active T cell–mediated rejection is variably responsive to immunosuppressive therapy

Kung

Sandhu

Haas

et al. 2021

Kidney International

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Chronic active T cell-mediated rejection (CA TCMR) is a newly described variant of kidney allograft rejection associated with long-term graft loss. Whether this form of rejection is related to under immunosuppression is debated and the benefit of immunosuppressive therapy in CA TCMR is unknown. Here we investigate the amenability of CA TCMR to treatment and examine the impact of clinical, histologic, and molecular parameters on outcomes. In a retrospective single institution review, we identified 48 cases of isolated CA TCMR, of which 44 were treated with pulse steroids or anti-thymocyte globulin, or both. Defining treatment response as an at least 50% estimated glomerular filtration rate recovery, a response was achieved in 20% of cases at four weeks post initiation of immunosuppressive therapy. Treatment responsiveness did not reflect the presence of concomitant acute T cellmediated rejection, and was not significantly different between cases with mild, moderate, and severe parenchymal scarring. Although not statistically significant, there was a trend toward greater treatment responsiveness in cases with moderate as opposed to severe tubulitis. By targeted transcriptional profiling, increased allograft mast cells and alterations in lipid metabolism were identified as possible features of treatment resistant CA TCMR. Thus, our study shows that although its prognosis is generally poor, CA TCMR is not a homogenous entity and in a subset of cases, improvement in kidney function can be achieved with immunosuppressive therapy.

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Section: Discussionmentioning

confidence: 99%

Chronic active T cell–mediated rejection is variably responsive to immunosuppressive therapy

Kung

Sandhu

Haas

et al. 2021

Kidney International

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“…We did not test in this study whether the AMR 10-gene score is applicable using other techniques, or other sample types, such as formalin-fixed paraffin-embedded tissue. However, in another manuscript where we investigated whether results of gene expression analysis performed using qRT-PCR on samples preserved in RNAlater correlated with results using Nanostring nCounter analysis on formalin-fixed paraffin-embedded tissue, these 10 genes correlated well [ 15 ]. We previously observed that there was good correlation between measurements of AMR-associated genes, when comparing two different cores from the same kidney transplant, using two different techniques (Nanostring and qRT-PCR) [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

“…qRT-PCR was carried out using an Applied Biosystems Vii7 real-time qPCR machine [ 8 ]. Gene-specific primers spanning an intron were designed for 18 genes from a list of ‘top hits’ for AMR in the literature ( Supplementary data, Table S1 ) [ 1–3 , 5 , 8 , 10 , 14 , 15 ]. Gene expression levels were normalized to housekeeping gene HPRT1 and results were measured relative to Stratagene qRT-PCR Reference RNA (Agilent Technologies using the ΔΔCt method) [ 16 ].…”

Section: Methodsmentioning

confidence: 99%

Diagnostic application of transcripts associated with antibody-mediated rejection in kidney transplant biopsies

Toulza

Dominy

Willicombe

et al. 2021

Nephrology Dialysis Transplantation

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Background The diagnosis of antibody-mediated rejection (AMR) is reached using the Banff Classification for Allograft Pathology, which now includes gene expression analysis. In this study, we investigate the application of “Increased Expression Of Thoroughly Validated Gene Transcripts/Classifiers Strongly Associated With AMR” as a diagnostic criteria. Method We used qRT-PCR for 10 genes associated with AMR in a retrospective cohort of 297 transplant biopsies, including biopsies that met the full diagnostic criteria for AMR, even without molecular data (AMR, n = 27); biopsies that showed features of AMR, but that would only meet criteria for AMR with increased transcripts (AMRsusp, n = 49) and biopsies that would never meet criteria for AMR (No-AMR, n = 221). Results A 10-gene AMR score trained by a receiver-operating characteristic to identify AMR found 16 cases with a high score amongst the AMRsusp cases (AMRsusp-high) that had significantly worse graft survival than those with a low score (AMRsusp-low) (n = 33). In both univariate and multivariate Cox regression analysis, the AMR 10-gene score was significantly associated with an increased hazard ratio for graft loss in the AMRsusp group (HR 1.109, p = 0.004 and HR 1.138, p = 0.012), but not in the whole cohort. Net reclassification index and integrated discrimination improvement analyses demonstrated improved risk classification and superior discrimination respectively for graft loss when considering the gene score in addition to histological and serological data, but only in the AMRsusp group, not the whole cohort. Conclusions This study provides evidence that a gene score strongly associated with AMR helps identify cases at higher risk of graft loss in biopsies that are suspicious for AMR but don’t meet full criteria.

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“…This has enabled the ability to perform RNA analyses using the aforementioned microarray techniques or bulkSeq. Despite this advancement, which opens the door for in-depth, retrospective analyses of preserved samples, formalin fixation of the tissue may lead to inferior RNA quality ( 64 , 65 ), producing artificial associations and overrepresentation of histone transcripts ( 66 ).…”

Section: Bulk Rna Sequencingmentioning

confidence: 99%

Advanced Genomics-Based Approaches for Defining Allograft Rejection With Single Cell Resolution

et al. 2021

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Solid organ transplant recipients require long-term immunosuppression for prevention of rejection. Calcineurin inhibitor (CNI)-based immunosuppressive regimens have remained the primary means for immunosuppression for four decades now, yet little is known about their effects on graft resident and infiltrating immune cell populations. Similarly, the understanding of rejection biology under specific types of immunosuppression remains to be defined. Furthermore, development of innovative, rationally designed targeted therapeutics for mitigating or preventing rejection requires a fundamental understanding of the immunobiology that underlies the rejection process. The established use of microarray technologies in transplantation has provided great insight into gene transcripts associated with allograft rejection but does not characterize rejection on a single cell level. Therefore, the development of novel genomics tools, such as single cell sequencing techniques, combined with powerful bioinformatics approaches, has enabled characterization of immune processes at the single cell level. This can provide profound insights into the rejection process, including identification of resident and infiltrating cell transcriptomes, cell-cell interactions, and T cell receptor α/β repertoires. In this review, we discuss genomic analysis techniques, including microarray, bulk RNAseq (bulkSeq), single-cell RNAseq (scRNAseq), and spatial transcriptomic (ST) techniques, including considerations of their benefits and limitations. Further, other techniques, such as chromatin analysis via assay for transposase-accessible chromatin sequencing (ATACseq), bioinformatic regulatory network analyses, and protein-based approaches are also examined. Application of these tools will play a crucial role in redefining transplant rejection with single cell resolution and likely aid in the development of future immunomodulatory therapies in solid organ transplantation.

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Technical considerations when designing a gene expression panel for renal transplant diagnosis

Cited by 10 publications

References 30 publications

Chronic active T cell–mediated rejection is variably responsive to immunosuppressive therapy

Chronic active T cell–mediated rejection is variably responsive to immunosuppressive therapy

Diagnostic application of transcripts associated with antibody-mediated rejection in kidney transplant biopsies

Advanced Genomics-Based Approaches for Defining Allograft Rejection With Single Cell Resolution

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