Limitations of biopsy-based transcript diagnostics to detect T cell-mediated allograft rejection.

BACKGROUND AND HYPOTHESIS: Isolated Tubulitis, Borderline Changes, and Isolated Arteritis suspicious for histologic T cell-mediated rejection (hTCMR) remain findings of uncertain significance. Although the Molecular Microscope Diagnostics System (MMDx) has not been trained on those lesions, it was suggested that MMDx might reclassify a subgroup to molecular TCMR (mTCMR). METHODS: In this single-center cohort of 326 consecutive, unselected kidney allograft biopsies assessed by histology and MMDx, we analyzed 249 cases with Isolated Tubulitis (i0, t1-3, v0; n=101), Borderline Changes (according to Banff 2022, v0; n=9), Isolated Arteritis (no borderline, v1; n=37), No Inflammation (i0, t0, v0; n=67) and a Positive Control Cohort (hTCMR, n=27; Mixed histologic Rejection, n=8; both according to Banff 2022; total n=35). The first three groups were summarized as TCMR-Suspicion (n=147). Subcategorization included the presence and absence of microvascular inflammation (MVI; g+ptc≥2). Molecular rejection rates and differentiation were investigated. RESULTS: Molecular rejection rates were 37/147 cases (25.2%; 32 with MVI) in TCMR-Suspicion, 6/67 (9%; 4 with MVI) in No Inflammation and 30/35 (85.7%; 19 with MVI) in the Positive Control Cohort. Molecular antibody-mediated rejection (mAMR) was present in 39/73 (53.4%) of cases. The presence of donor-specific antibodies (DSA) at the time of the biopsy was high (127/249, 51%). Only 3 mAMR/TCMR and no pure mTCMR cases were detected in TCMR-Suspicion and No Inflammation, compared to 12 mAMR/TCMR and 10 mTCMR cases in the Positive Control Cohort (p<0.001). Even though the TCMR-specific molecular (Classifier) score differentiated between TCMR-Suspicion and No Inflammation (p=0.005), rejection phenotype scores (R2 and R3) did not (p=0.157 and 0.121). CONCLUSIONS: MMDx did not identify pure mTCMR among Isolated Tubulitis, Borderline Changes, or Isolated Arteritis, likely due to low sensitivity for TCMR-lesions. However, it identified mAMR or mAMR/TCMR, especially in cases with MVI. Subthreshold findings remain to be further studied.

The utilization of molecular microscope in management of heart transplant recipients in the era of noninvasive monitoring.

INTRODUCTION: Monitoring for graft rejection is a fundamental tenet of post-transplant follow-up. In heart transplantation (HT) in particular, rejection has been traditionally assessed with endomyocardial biopsy (EMB). EMB has potential complications and noted limitations, including interobserver variability in interpretation. Additional tests, such as basic cardiac biomarkers, cardiac imaging, gene expression profiling (GEP) scores, donor-derived cell-free DNA (dd-cfDNA) and the novel molecular microscope diagnostic system (MMDx) have become critical tools in rejection surveillance beyond standard EMB. METHODS: This paper describes an illustrative case followed by a review of MMDx within the context of other noninvasive screening modalities for rejection. CONCLUSIONS: We suggest MMDx be used to assist with early detection of rejection in cases of discordance between EMB and other noninvasive studies.

The Molecular Microscope (MMDXR ) interpretation of thoracic and abdominal allograft biopsies: Putting things in perspective for the clinician.

The Molecular Microscope System (MMDXR ) has significant potential to enhance biopsy interpretation. However, discussions of MMDx do not acknowledge the basic accuracy of histology readings, and the ability of pathology as a stand-alone tool to guide patient management. MMDx overstates its ability to automatically correct for problems in biopsy readings. Assertions of accuracy approaching 99% are not supported by "real world" data. The high rate of discrepancies between MMDx® and standard biopsy readings can be attributed to the summation of many factors other than histology interpretation, including molecular noise, assay thresholding, limited sensitivity of microarray technology for low expression genes, errors in classifier development, narrow data interpretation, and lack of spatial context. It is not widely recognized that molecular signatures are not disease-specific and are affected by the stage of disease as well as the extent of tissue injury. The effect of sampling error on MMDx performance is significantly under-estimated, particularly in heart and lung biopsies. Therefore, MMDx reports should always be interpreted in the context of conventional biopsy readings. The clinical picture and conventional biopsy reading should be allowed to over-ride the molecular interpretation when there is concern that confounding factors are at play.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

Rejection! Or is it? Correlation among molecular microscope diagnostic system, histopathology and clinical judgement following heart transplantation.

PURPOSE: Little is known about clinical decision making among discordant findings concerning for rejection with endomyocardial biopsy (EMBx) and Molecular Microscope Diagnostic System (MMDx) in patients following heart transplantation. METHODS: Two hundred and twenty-eight corresponding EMBx and MMDx specimens from 135 adult heart transplant patients were retrospectively reviewed. Rejection was classified as t-cell mediated rejection ≥2R and/or antibody mediated rejection ≥1. Clinical decision making among concordant and discordant cases of EMBx and MMDx results were reviewed. RESULTS: Patient characteristics were comparable between concordant and discordant patient groups (median age 60 yrs., 76% male, and 71% White). A total of 167/228 specimens (73%) were concordant for no rejection with 98% agreement in clinical decision making and 25/228 (11%) concordant for rejection with 64% agreement in clinical decision making. Among the 36/228 (16%) discordant samples, clinical decision-making agreed on treatment for rejection in five of the MMDx samples and three of the EMBx samples. CONCLUSIONS: MMDx can be an additional tool to diagnose rejection not detected by the traditional EMBx and influence clinical decision making in guiding appropriate treatment. Ongoing investigation into the clinical utility of MMDx is warranted to determine the significance of discordant findings among diagnostic modalities when assessing for rejection.

Kidney Allograft Monitoring by Combining Donor-Derived Cell-Free DNA and Molecular Gene Expression: A Clinical Management Perspective.

Donor-derived cell-free DNA (dd-cfDNA) may safely assess kidney allograft rejection. Molecular Microscope (MMDx®) gene expression may offer increased precision to histology. This single-center retrospective study monitored kidney transplant recipients for rejection at specified time intervals by utilizing creatinine (SCr), proteinuria, donor-specific antibodies (DSAs), and dd-cfDNA. A clinically indicated biopsy sample was sent for histopathology and MMDx®. Patients were categorized into rejection (Rej) and non-rejection (NRej) groups, and further grouped according to antibody-mediated rejection (ABMR) subtypes. Rej and NRej groups included 52 and 37 biopsies, respectively. Median follow-up duration was 506 days. DSAs were positive in 53% and 22% of patients in both groups, respectively (p = 0.01). Among these groups, pre- and post-intervention median SCr, proteinuria, and dd-cfDNA at 1 month, 2 months, and at the last follow-up revealed significant difference for dd-cfDNA (all p = 0.01), however, no difference was found for SCr and proteinuria (p > 0.05). The AUC was 0.80 (95% CI: 0.69-0.91), with an optimal dd-cfDNA criterion of 2.2%. Compared to histology, MMDx® was more likely to diagnose ABMR (79% vs. 100%) with either C4d positivity or negativity and/or DSA positivity or negativity. Hence, a pre- and post-intervention allograft monitoring protocol in combination with dd-cfDNA, MMDx®, and histology has aided in early diagnosis and timely individualized intervention.

Current Status of Biomarkers and Molecular Diagnostic Tools for Rejection in Liver Transplantation: Light at the End of the Tunnel?

Strategies to minimize immune-suppressive medications after liver transplantation are limited by allograft rejection. Biopsy of liver is the current standard of care in diagnosing rejection. However, it adds to physical and economic burden to the patient and has diagnostic limitations. In this review, we aim to highlight the different biomarkers to predict and diagnose acute rejection. We also aim to explore recent advances in molecular diagnostics to improve the diagnostic yield of liver biopsies.