Benjamin Adam

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility of Polyomavirus Immunohistochemistry in Kidney Allografts

Immunohistochemistry (IHC) is the gold standard for diagnosing (positive vs. negative) polyomavirus BK (BKV) nephropathy and has the potential for disease staging based on staining intensity and quantification of infected cells. This multicenter trial evaluated the reproducibility of BKV IHC among 81 pathologists at 60 institutions. Participants stained tissue microarray slides and scored them for staining intensity and percentage of positive nuclei. Staining protocol details and evaluation scores were collected online. Slides were returned for centralized panel re‐evaluation and kappa statistics were calculated. Individual assessment of staining intensity and percentage was more reproducible than combined scoring. Inter‐institutional reproducibility was moderate for staining intensity (κ = 0.49) and percentage (κ = 0.42), fair for combined (κ = 0.25) and best for simple positive/negative scoring (κ = 0.78). Inter‐observer reproducibility was substantial for intensity (κ = 0.74), percentage (κ = 0.66), positive/negative (κ = 0.78) and moderate for combined scoring (κ = 0.43). Inter‐laboratory reproducibility was fair for intensity (κ = 0.37), percentage (κ = 0.40) and combined (κ = 0.24), but substantial for positive/negative scoring (κ = 0.67). BKV RNA copies/cell correlated with staining intensity (r = 0.56) and percentage (r = 0.62). These results indicate that BKV IHC is reproducible between observers but scoring should be simplified to a single‐feature schema. Standardization of tissue processing and staining protocols would further improve inter‐laboratory reproducibility.

Banff Fibrosis Study: Multicenter Visual Assessment and Computerized Analysis of Interstitial Fibrosis in Kidney Biopsies

Increasing interstitial fibrosis (IF) in native and kidney transplant biopsies is associated with functional decline and serves as a clinical trial end point. A Banff 2009 Conference survey revealed a range in IF assessment practices. Observers from multiple centers were asked to assess 30 renal biopsies with a range of IF and quantitate IF using two approaches on trichrome, Periodic acid‐Schiff (PAS) and computer‐assisted quantification of collagen III immunohistochemistry (C‐IHC) slides, as well as assessing percent of cortical tubular atrophy% (TA%) and Banff total cortical inflammation score (ti‐score). C‐IHC using whole slide scans was performed. C‐IHC assessment showed a higher correlation with organ function (r = −0.48) than did visual assessments (r = −0.32–−0.42); computerized and visual C‐IHC assessment also correlated (r = 0.64–0.66). Visual assessment of trichrome and C‐IHC showed better correlations with organ function and C‐IHC, than PAS, TA% and ti‐score. However, visual assessment of IF, independent of approach, was variable among observers, and differences in correlations with organ function were not statistically significant among C‐IHC image analysis and visual assessment methods. C‐IHC image analysis correlated among three centers (r > 0.90, p < 0.0001, between all centers). Given the difficulty of visual IF assessment standardization, C‐IHC image could potentially accomplish standardized IF assessment in multicenter settings.

Multiplexed color‐coded probe‐based gene expression assessment for clinical molecular diagnostics in formalin‐fixed paraffin‐embedded human renal allograft tissue

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard‐of‐care workflows for transplant biopsies, that is, formalin‐fixed paraffin‐embedded (FFPE) processing. The NanoString® gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature‐derived antibody‐mediated rejection (ABMR) 34‐gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString® results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString® with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) with corresponding dedicated fresh‐stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString® (r = 0.354) than with qRT‐PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Students t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection

Precise diagnosis of antibody‐mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin‐fixed, paraffin‐embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor‐specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti‐HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy‐based molecular assessment of antibody‐mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes

Molecular testing represents a promising adjunct for the diagnosis of antibody‐mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin‐fixed paraffin‐embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10‐fold cross‐validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non‐AMR samples (area under the curve = 0.92). This three‐gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39–0.63, p < 0.001). Principal component analysis confirmed the association between three‐gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell–mediated rejection (TCMR). Elevated three‐gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Transplant biopsy beyond light microscopy.

Despite its long-standing status as the diagnostic “gold standard”, the renal transplant biopsy is limited by a fundamental dependence on descriptive, empirically-derived consensus classification. The recent shift towards personalized medicine has resulted in an increased demand for precise, mechanism-based diagnoses, which is not fully met by the contemporary transplantation pathology standard of care. The expectation is that molecular techniques will provide novel pathogenetic insights that will allow for the identification of more accurate diagnostic, prognostic, and therapeutic targets. Here we review the current state of molecular renal transplantation pathology. Despite significant research activity and progress within the field, routine adoption of clinical molecular testing has not yet been achieved. The recent development of novel molecular platforms suitable for use with formalin-fixed paraffin-embedded tissue will offer potential solution for the major barriers to implementation. The recent incorporation of molecular diagnostic criteria into the 2013 Banff classification is a reflection of progress made and future directions in the area of molecular transplantation pathology. Transcripts related to endothelial injury and NK cell activation have consistently been shown to be associated with antibody-mediated rejection. Prospective multicenter validation and implementation of molecular diagnostics for major entities remains an unmet clinical need in transplantation. It is expected that an integrated system of transplantation pathology diagnosis comprising molecular, morphological, serological, and clinical variables will ultimately provide the greatest diagnostic precision.

Immune Cell Infiltrates in Pituitary Adenomas: More Macrophages in Larger Adenomas and More T Cells in Growth Hormone Adenomas

Tumor immune microenvironment has been gradually recognized as a key contributor to tumor development, progression, and control. Immune cell infiltrates in brain tumors have been increasingly studied, but few have published on immune cell infiltrates in pituitary adenomas. We quantitatively assessed the infiltration of macrophages and lymphocytes in 35 pituitary adenomas, including 9 densely granulated growth hormone (DG-GH), 9 sparsely granulated growth hormone (SG-GH), 9 null cell (NC), and 8 adrenocorticotropic hormone (ACTH) adenomas. All the adenomas showed varying degrees of CD68+ macrophage infiltration. While SG-GH adenomas were significantly larger in size than DG-GH and ACTH adenomas, the infiltration of CD68+ macrophages was significantly greater in SG-GH than in DG-GH and ACTH adenomas. Similarly, NC adenomas that were significantly larger than DG-GH and ACTH adenomas had significantly greater infiltration of CD68+ macrophages than DG-GH and ACTH adenomas. The numbers of CD68+ macrophages were positively correlated with the tumor sizes and Knosp classification grades for tumor invasiveness. The infiltration of CD4+ and CD8+ T cells was relatively scant in these adenomas, but GH adenomas exhibited significantly more CD4+ and CD8+ T cells than non-GH adenomas. Both DG-GH and SG-GH adenomas had significantly more CD4+ cells than ACTH adenomas and significantly more CD8+ cells than NC adenomas. These results suggest an association of CD68+ macrophage infiltration with an increase in the pituitary adenoma size and invasiveness. Our observation contributes to understanding the growth environment of pituitary adenomas, for which adjuvant immunotherapy may help to constrain the tumor enlargement and invasiveness.

Molecular nephropathology: ready for prime time?

In the current era of precision medicine, the existing nephropathology paradigm of light microscopy, immunofluorescence, and electron microscopy will become increasingly insufficient. There will be an expectation to supplement these traditional diagnostic tools with patient-specific information related to a growing understanding of molecular pathophysiology. Next generation sequencing technologies are expected to play a key role in the future of nephropathology, but transcriptomics is poised to represent the first major foray into routine molecular testing. The introduction of molecular techniques into clinical nephropathology has been hindered in part by the reliance of existing platforms on fresh tissue samples. The NanoString gene expression system works with formalin-fixed paraffin-embedded tissue and thus represents a promising solution to this technical barrier that may finally allow for the translation of recent transcriptomics discoveries into the enhancement of patient care. Widespread adoption of this new diagnostic dimension will require ongoing multidisciplinary cooperation between pathologists and clinicians, including molecular testing consensus generation and rigorous multicenter validation.

Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.Tumour metastasis is dependent on tumour cell motility. Here, the authors investigate genes required for tumour cell motility by establishing a quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos.