Development and Validation of HistoMX, a Gene-Expression Platform for Molecular Interpretation of Kidney Allograft Biopsies (HistoMX)

An Automated Gene Expression-Based Platform for Multidimensional Assessment and Interpretation of Kidney Allograft Biopsies: Development and Multicenter External Validation (HistoMX)

This retrospective multicentre observational study developed and validated HistoMX, an automated gene-expression-based platform for multidimensional molecular interpretation of kidney allograft biopsies.

The study included 2,410 archived post-transplant kidney allograft biopsy samples from adult kidney transplant recipients. Formalin-fixed paraffin-embedded biopsy tissue was profiled using the Banff Human Organ Transplant panel on the NanoString nCounter platform. HistoMX integrates locked preprocessing, quality control, single-sample normalization, diagnostic classification, Banff lesion-level modelling, molecular lesion-severity scores, composite injury indices, immune-cell estimation, pathway-enrichment analysis, and automated clinician-facing reporting.

The platform was evaluated against reference histology based on the Banff classification. HistoMX was designed to complement, not replace, conventional histopathology by providing standardized molecular evidence across diagnostic, lesion-level, continuous injury, and biological dimensions of kidney allograft injury.

Study Overview

• Status: Completed
• Conditions: Kidney Transplant; BK Virus Nephropathy; Antibody Mediated Rejection; Rejection of Renal Transplant; Chronic Kidney Disease (CKD) Stage 5; T-Cell Mediated Rejection; Kidney Allograft Injury

Detailed Description

Kidney allograft biopsy interpretation remains central to post-transplant care, but conventional histology may not fully capture the biological continuum of alloimmune and non-alloimmune injury. Targeted transcriptomic profiling of formalin-fixed paraffin-embedded biopsy tissue using the Banff Human Organ Transplant panel provides a practical molecular layer aligned with routine pathology workflows.

HistoMX was developed as an automated molecular interpretation platform intended to transform B-HOT nCounter expression data into standardized diagnostic probabilities, molecular lesion scores, composite injury indices, immune-cell estimates, pathway-enrichment outputs, and a clinician-facing report.

HistoMX was developed and validated using 2,410 post-transplant kidney allograft biopsies from adult recipients across eleven transplant centers in Europe and North America. The population comprised a development cohort (including 7 centers: PITOR [Saint-Louis, Necker], Montpellier, Lille, Bordeaux, Toulouse, Barcelona, and Cedars-Sinai Medical Center) and three independent external validation cohorts (Arkana Laboratories, USA, Massachusetts General Hospital, USA, and University of Alberta, Canada).

The development cohort included 693 adult kidney transplant recipients contributing to 764 biopsies collected between 2004 and 2021 across eight centres in Europe and North America. This cohort was split, stratified by main histological diagnosis, into a derivation set of 571 biopsies and an internal validation set of 193 biopsies. The derivation set was used for feature selection, model development, hyperparameter tuning, and estimation of preprocessing and normalization parameters. The internal validation set was kept independent from all model-development steps.

External validation was performed in three independent cohorts: Arkana Laboratories, USA, with 1,309 biopsies; Massachusetts General Hospital, USA, with 241 biopsies; and the University of Alberta / Alberta University Hospital, Canada, with 96 biopsies. The same eligibility and assay-quality criteria were applied to the external validation cohorts.

Repeated biopsies from the same recipient were retained where applicable because the analytical unit was the biopsy, consistent with the diagnostic objective of the study.

Eligible samples were post-transplant kidney allograft biopsies from adult recipients submitted for transcriptomic profiling. Exclusion criteria were recipient age younger than 18 years at transplantation, multi-organ transplantation, pre-transplant biopsy, missing Banff reference pathology diagnosis, or failure of prespecified nCounter quality-control criteria.

Clinical, histological, immunological, and virological data were extracted from local databases and transferred in anonymized or de-identified form. Biopsies were evaluated according to the Banff classification. For model development and validation, a single reference allograft diagnosis was assigned to each biopsy using the Banff Automation System adapted to the Banff 2022 kidney allograft classification, based on available Banff lesion scores, C4d results, donor-specific antibody status, and virological information.

RNA was extracted from formalin-fixed paraffin-embedded biopsy tissue and profiled using the B-HOT panel on the NanoString nCounter platform. The panel includes 770 genes, comprising 758 transplant-relevant endogenous genes and 12 housekeeping genes. After gene-level filtering, 702 endogenous genes and six housekeeping genes were retained for downstream analysis.

Validation samples were processed using locked derivation-cohort preprocessing and single-sample normalization parameters. This allowed each biopsy to be analysed independently and limited information leakage between derivation and validation datasets.

Three molecular model domains were evaluated: binary classifiers for diagnostic endpoints and binary Banff lesion presence; ordinal classifiers for Banff lesion severity and simplified histological indices; and regression models for weighted continuous histological indices.

Diagnostic endpoints included antibody-mediated rejection, T cell-mediated rejection, any rejection, BK virus nephropathy, acute tubular injury without rejection, and no active inflammatory complication. Banff lesion endpoints included glomerulitis, peritubular capillaritis, interstitial inflammation, tubulitis, intimal arteritis, transplant glomerulopathy, interstitial fibrosis, tubular atrophy, vascular fibrous intimal thickening, microvascular inflammation, total inflammation, and interstitial fibrosis/tubular atrophy.

Feature selection was performed in the derivation cohort using Boruta. Multiple machine-learning algorithms were trained for each endpoint and combined into equally weighted ensembles. Model performance was evaluated in the internal validation set, the pooled external validation cohort, and cohort-specific external validation analyses.

This study did not test a single confirmatory two-group hypothesis with a prespecified effect size. The sample size rationale was based on diagnostic prediction-model development and validation. All eligible B-HOT-profiled biopsies meeting quality-control and reference-diagnosis requirements were included to maximize precision, diagnostic representation, and generalisability.

For prevalent diagnostic endpoints, the derivation dataset supported model estimation and cross-validation across major diagnostic categories. For rarer endpoints, including BK virus nephropathy, acute tubular injury without rejection, transplant glomerulopathy, and intimal arteritis, model training and interpretation explicitly accounted for lower event counts through adapted cross-validation and class-imbalance approaches.

External validation used all available independent validation cohorts to assess performance, calibration, and transportability across settings. The immunophenotyping substudy included 32 paired biopsies and was used as exploratory supportive biological validation of transcriptomic cell-deconvolution estimates.

• Study Type: Observational
• Enrollment (Actual): 2410

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Post-transplant kidney allograft biopsies from adult recipients submitted for B-HOT transcriptomic profiling across eleven European and North American centers.

Description

Inclusion Criteria:

• Adult kidney transplant recipient, defined as age 18 years or older at transplantation
• Post-transplant kidney allograft biopsy
• Archived FFPE kidney allograft biopsy tissue is available and suitable for transcriptomic profiling
• Biopsy submitted for B-HOT nCounter transcriptomic profiling
• Available Banff reference pathology diagnosis
• Available Banff lesion information required for endpoint definition
• Sample meeting prespecified nCounter assay quality-control criteria

Exclusion Criteria:

• Recipient age <18 years at transplantation
• Multi-organ transplantation
• Pre-transplant biopsy
• Missing Banff reference pathology diagnosis
• Failure of prespecified nCounter quality-control criteria
• Missing or insufficient data required to define the relevant reference endpoint

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort
Development cohort; Multicenter Development cohort. Retrospective. No intervention.
Alberta cohort; Monocenter External validation cohort. Retrospective. No intervention.
Massachussets General Hospital cohort; Monocenter External validation cohort. Retrospective. No intervention.
Arkana Laboratories cohort; External validation cohort. Retrospective available on NCBI. No extervention.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Discrimination of molecular diagnostic classifiers (AUROC)	Area under the ROC curve for prediction of each main diagnostic category (antibody-mediated rejection, T cell-mediated rejection, any rejection, BK virus nephropathy, acute tubular injury without rejection, no-active-inflammatory complication) versus the Banff reference histological diagnosis, in internal and external validation cohorts.	At baseline defined by kidney allograft biopsy (single time-point assessment).
Calibration of molecular diagnostic classifiers (Brier score, log loss)	Calibration of predicted diagnostic probabilities for each main diagnostic endpoint, assessed using calibration plots, calibration intercept and slope, Brier score, and log loss, compared with the Banff reference histological diagnosis.	At baseline defined by kidney allograft biopsy (single time-point assessment).

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Precision-recall performance (AUPRC) of molecular diagnostic classifiers	Area under Precision-recall curve (AUPRC) for each diagnostic endpoint, including antibody-mediated rejection, T cell-mediated rejection, any rejection, BK virus nephropathy, acute tubular injury without rejection, and no active inflammatory complication.	At baseline defined by kidney allograft biopsy (single time-point assessment).
Discrimination of Banff lesion classifiers (AUROC) for binary lesion.	Discrimination of Banff lesion classifiers (AUROC) for binary lesion presence including glomerulitis (g), peritubular capillaritis (ptc), microvascular inflammation (mvi), interstitial inflammation (i), tubulitis (t), intimal arteritis (v), transplant glomerulopathy (cg), interstitial fibrosis (ci), tubular atrophy (ct), vascular fibrous intimal thickening (cv), and interstitial fibrosis/tubular atrophy. (IFTA).	At baseline defined by kidney allograft biopsy (single time-point assessment).
Correlation of continuous molecular lesion-severity scores with histological Banff grades.	Correlation and concordance between continuous molecular lesion-severity scores derived from ordinal classifiers and histological Banff lesion grades, assessed using Spearman correlation, Pearson correlation, concordance index, and related calibration approaches.	At baseline defined by kidney allograft biopsy (single time-point assessment).
Performance of composite molecular injury indices (AMR/MVI, TCMR/TI, activity, chronicity).	Performance of composite molecular injury indices (AMR/MVI, TCMR/TI, activity, chronicity) assessed by correlation with corresponding histological indices and, where appropriate, binary discrimination using area under the receiver-operating characteristic curve.	At baseline defined by kidney allograft biopsy (single time-point assessment).
Validation of cell-deconvolution estimates	Correlation between HistoMX transcriptomic immune-cell estimates and multiplex immunofluorescence-derived tissue cell densities in an independent paired immunophenotyping subset of 32 patients contributing to 32 biopsies, focusing on T cells and macrophages.	At baseline defined by kidney allograft biopsy (single time-point assessment).
Concordance between molecular and histological diagnoses	Proportion of histology-defined antibody-mediated rejection, T cell-mediated rejection, and any-rejection biopsies classified concordantly by HistoMX, with characterization of molecular-histological discordant biopsies.	At baseline defined by kidney allograft biopsy (single time-point assessment).

Collaborators and Investigators

• Sponsor: Paris Translational Research Center for Organ Transplantation
• Collaborators: Massachusetts General Hospital; University of Alberta; Cedars-Sinai Medical Center; CareDx; Université Paris Cité; Arkana Labs; OrganX

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2004
• Primary Completion (Actual): May 15, 2026
• Study Completion (Actual): May 15, 2026

Study Registration Dates

• First Submitted: June 24, 2026
• First Submitted That Met QC Criteria: July 7, 2026
• First Posted (Actual): July 14, 2026

Study Record Updates

• Last Update Posted (Actual): July 14, 2026
• Last Update Submitted That Met QC Criteria: July 7, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Keywords: machine learning; kidney transplantation; gene expression; precision medicine; allograft rejection; antibody-mediated rejection; Molecular diagnostics; transcriptomic; FFPE tissue; kidney allograft biopsy; T-cell mediated rejection; BK virus Nephropathy; Acute tubular injury; Banff Classification; B-HOT panel; nCounter; HistoMX
• Additional Relevant MeSH Terms: Urogenital Diseases; Pathologic Processes; Male Urogenital Diseases; Kidney Diseases; Urologic Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Chronic Disease; Disease Attributes; Renal Insufficiency; Pathological Conditions, Signs and Symptoms; Renal Insufficiency, Chronic
• Other Study ID Numbers: HistoMXv3

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No