Aylin Sar

Histopathologic features aid in predicting risk for progression of IgA nephropathy.

BACKGROUND AND OBJECTIVES IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide. Accurately identifying patients who are at risk for progressive disease is challenging. The extent to which histopathologic features improves prognostication is uncertain. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We studied a retrospective cohort with biopsy-proven IgAN in Calgary, Canada. Renal biopsies were reviewed by a nephropathologist with histopathologic data abstracted using a standardized form. The primary outcome was the composite of doubling of serum creatinine, ESRD, or death. Spline models defined significant levels of interstitial fibrosis, glomerulosclerosis, hypertension, proteinuria, and creatinine. The prognostic significances of clinical and histopathologic parameters were determined using Cox proportional hazards models. RESULTS Data from 146 cases were available for analysis with a median follow-up of 5.8 years. Greater than 25% interstitial fibrosis, >40% glomerular sclerosis, and a systolic BP >150 mmHg were risk thresholds. In univariable analyses, baseline creatinine, proteinuria, systolic BP, glomerular sclerosis, interstitial fibrosis, and crescentic disease were predictors of the primary outcome. In multivariable models adjusted for clinical characteristics, interstitial fibrosis (hazard ratio [HR]2.7; 95% confidence interval [CI] 1.2 to 6.0), glomerular sclerosis (HR 2.6; 95% CI 1.2 to 4.5), and crescents (HR 2.4; 95% CI 1.2 to 5.1) remained independent predictors of the primary outcome and significantly improved model fit compared with clinical characteristics alone. CONCLUSIONS Baseline histopathologic parameters are independent predictors of adverse outcomes in IgAN even after taking into consideration clinical characteristics. Relatively small degrees of interstitial fibrosis confer an increased risk for progressive IgAN.

Interobserver agreement for Polyomavirus nephropathy grading in renal allografts using the working proposal from the 10th Banff Conference on Allograft Pathology

A classification schema for grading Polyomavirus nephropathy was proposed at the 2009 Banff allograft meeting. The schema included 3 stages of Polyomavirus nephropathy: early (stage A), florid (stage B), and late sclerosing (stage C). Grading categories for histologic viral load levels were also proposed. To examine the applicability and the interobserver agreement of the proposed Polyomavirus nephropathy grading schema, we evaluated 24 renal allograft biopsies with confirmed Polyomavirus nephropathy by histology and SV40. Four renal pathologists independently scored the Polyomavirus nephropathy stage (A, B, or C), without knowledge of the clinical history. Viral load was scored as a percent of tubules exhibiting viral replication, using either a 3-tier viral load score (1: ≤1%; 2: >1%-10%; 3: >10%) or a 4-tier score (1: ≤1%; 2: >1%-≤5%; 3: >5%-15%; 4: >15%). The κ score for the Polyomavirus nephropathy stage was 0.47 (95% confidence interval, 0.35-0.60; P < .001). There was a substantial agreement using both the 3-tier and the 4-tier scoring for the viral load (Kendall concordance coefficients, 0.72 and 0.76, respectively; P < .001 for both). A better complete agreement was found using the 3-tier viral load score. In this first attempt to evaluate the interobserver reproducibility of the proposed Polyomavirus nephropathy classifying schema, we demonstrated moderate κ agreement in assessing the Polyomavirus nephropathy stage and a substantial agreement in scoring the viral load level. The proposed grading schema can be applied in routine allograft biopsy practice for grading the Polyomavirus nephropathy stage and the viral load level.

Identification and characterization of demethylase JMJD1A as a gene upregulated in the human cellular response to hypoxia

Hypoxia is commonly found in human solid cancers and serves as a selective environment for the survival of aggressive cancer cells and as protection from anti-cancer therapies. In addition to a shift to anaerobic metabolism, the cellular response to hypoxia includes cessation of cell division and/or cell death. These mechanisms have still not been defined. Identification of the members of hypoxia-induced growth arrest pathways remain incomplete. We have undertaken an expression microarray analysis of the cellular response to hypoxia in diverse cell lines. An identified cohort of genes is reliably upregulated in various cells in response to hypoxia, as validated by reverse-transcriptase polymerase chain reaction (RT-PCR). One of the upregulated targets corresponds to an expressed sequence tag encoded by JMJD1A (a gene also known as JHDM2A), which has been identified as a histone demethylase that regulates the transcription of androgen receptor targets. We confirm, by RT-PCR, the upregulation of JMJD1A after hypoxia and desferroxamine treatment in multiple cell lines. We also show that JMJD1A is predominantly, but not exclusively, a nuclear protein. Immunofluorescent staining of HeLa cells shows a shift of cytoplasmic JMJD1A into the nucleus on hypoxia treatment. Immunohistochemical staining has revealed that JMJD1A is widely expressed in tissues, even in cells that are not known to express the androgen receptor, and is significantly increased in smooth muscle cells upon hypoxia treatment.

Evaluating the accuracy of functional biomarkers for detecting histological changes in chronic allograft nephropathy

The most common cause of late kidney transplant failure is chronic allograft nephropathy (CAN). Much research has focused on identifying biomarkers (or correlates) that would predict subsequent CAN and allow timely intervention. Functional biomarkers such as serum creatinine and estimated glomerular filtration rate (eGFR) have been widely adopted, even though they have not been rigorously evaluated as surrogate markers. This study evaluated serum creatinine and eGFR for predicting the early histopathological changes seen in transplant protocol biopsies (TPB). We prospectively followed 289 kidney transplant patients in the Southern Alberta Transplant Program who had TPB at 6–12 months post‐transplant. Tissue samples (n = 280) were independently examined by renal pathologists. The ability of serum creatinine or eGFR to predict the threshold level for abnormal histopathology was evaluated by calculating the area under the receiver operator characteristic curve. Serum creatinine and eGFR had poor predictive value (most confidence intervals included 0.5, indicating no predictive ability) for ten individual histological measurements (Banff 97 scores), and the Chronic Allograft Damage Index. We conclude that serum creatinine and eGFR have a limited clinical role in predicting the early histopathological changes that precede CAN and should not be used for this purpose.

Mutation or polymorphism of the CD20 gene is not associated with the response to R-CHOP in diffuse large B cell lymphoma patients.

BACKGROUND Rituximab, a chimeric antibody targeted to the human CD20 molecule, is a major component of the R-CHOP protocol used to treat patients with diffuse large B cell lymphoma (DLBCL). It is also a very expensive drug. Though the response rate of R-CHOP is higher than that of CHOP alone, patients may still experience a poor response. One possible mechanism that may mediate the poor response to R-CHOP is poor binding of the drug to the CD20 molecule, perhaps due to mutations or polymorphisms of the CD20 gene that affect its structure. To test this hypothesis and perhaps define a new predictor of R-CHOP response, our pilot study evaluated the CD20 gene sequence from patients exhibiting a poor outcome to R-CHOP. METHODS Eleven patients with DLBCL who exhibited a recurrence of their disease and/or died within 24 months of R-CHOP treatment were categorized as showing poor progression-free survival (poor outcomes). Paraffin-embedded tissue specimens from the original tumors were evaluated for mutations or polymorphisms of the CD20 gene. Furthermore, sections from these patients and as well as from matched control patients who were categorized as good outcome patients (no progression of their disease within 36 months) were stained with anti-CD20 antibody and compared for CD20 protein expression. RESULTS DNA sequence analyses revealed that no mutations were observed in DNA from the coding region of the CD20 gene in any of the initial tumors from 11 patients who showed poor outcomes with R-CHOP therapy. One case showed a synonymous single nucleotide polymorphism in exon 2 (C216T; rs2070770; Ile>>Ile). No significant differences in CD20 expression was observed between good and poor outcome patients with R-CHOP. CONCLUSIONS Our results do not support association of CD20 mutations in specimens of the initial tumor or structural polymorphisms of the CD20 gene with patients who exhibited poor outcomes to R-CHOP.

Graft Inflammation and Histologic Indicators of Kidney Chronic Allograft Failure: Low-Expressing Interleukin-10 Genotypes Cannot Be Ignored

Background. Infiltration of inflammatory cells into the renal allograft interstitium is the biologic hallmark of alloimmune responses that leads to tubulointerstitial injury and subsequent interstitial fibrosis and chronic allograft failure. The proliferation, stimulation, and infiltration of these inflammatory cells are governed by various proinflammatory and regulatory cytokines. We assessed whether the differences in the genes encoding cytokines (producing low, moderate, or high expression profiles) may affect the infiltration of inflammatory cells into the renal allograft and the histologic changes characteristics of chronic allograft failure. Methods. A total of 218 kidney transplant recipients were genotyped for 15 single-nucleotide polymorphisms located in the gene promoter or exonic regions of 10 different cytokines or their receptors. Six- to 12-month posttransplant surveillance biopsies were scored using 11 individual histologic parameters and the combined grade of interstitial fibrosis and tubular atrophy (IF/TA). B-cell, T-cell, and macrophage infiltrates were quantified by immunostaining. Results. The low-expressing interleukin (IL)-10 gene promoter genotypes were found significantly associated with high IF/TA grade (IL-10 −819 TT; P=0.035; odds ratio=3.27; 95% confidence interval 1.1–9.8). At individual histologic indices, recipients carrying low-expressing IL-10 genotypes showed 2.5-fold higher scores for interstitial fibrosis, inflammation, and tubular atrophy. High infiltration of T cells and macrophages but not B cells into the renal allograft interstitium was found strongly associated with the carriage of low-expressing IL-10 genotypes. Conclusions. The results suggest that renal transplant recipients genetically predisposed to low expression of the regulatory cytokine IL-10 are more susceptible to high grades of IF/TA scores, graft inflammation, and high influx of inflammatory cells into the graft interstitium.

Pathogenesis and management of chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) is a common cause of late kidney transplant failure, characterized by progressive histological damage in the allograft. Although functional biomarkers such as creatinine are typically used to predict CAN, recent evidence suggests that composite, quantitative histological indices may be better predictors of long-term graft outcomes. Calcineurin inhibitors (CNIs) have been associated with major improvements in early rejection outcomes, but appear to cause both acute and chronic nephrotoxicity. The acute phase is associated with functional nephrotoxicity and is reversible with a reduction in CNI dosage, whereas the chronic phase is characterized by persistent histological lesions that are typically irreversible. Results from recent clinical trials suggest that converting from a CNI to sirolimus, withdrawing a CNI from a sirolimus-based regimen or using a CNI-free strategy may improve long-term outcomes by reducing CNI-related nephrotoxicity. However, in the de novo transplant setting, triple therapy with sirolimus, mycophenolate mofetil and corticosteroids is not recommended in combination with basiliximab induction. A treatment algorithm, based on the patient’s histological score obtained on an allograft biopsy taken at approximately 6–12 months post-transplant, has been developed by our group and is described here.

Segmentation of the effective area of images of renal biopsy samples

Diagnosis and monitoring of kidney diseases and transplants is supported by microscopic analysis of needle-core biopsy samples. The current methods of analysis allow for inconsistencies, bias, and inaccuracies. We propose image processing methods for automatic segmentation of the effective biopsy area (cortex and medulla) from digital images of renal biopsy samples. The methods include opening-by-reconstruction, a morphological closing operation, and morphological erosion. The results are compared to 100 randomly selected images manually marked by an experienced renal pathologist. Comparative measures indicate that the automatically detected region of interest closely matches the ground truth; the mean distance to the closest point was 5.46 ± 3.92 µm (6 ± 4.31 pixels) and the true-positive fraction was 98.25 ± 1.77%.

Segmentation of cell nuclei in images of renal biopsy samples

Diagnosis and monitoring of kidney diseases and transplants is supported by microscopic analysis of needle-core biopsy samples. The current methods of analysis are affected by inconsistencies, bias, and inaccuracies. We propose and evaluate image processing methods for automatic segmentation of cell nuclei in digital images of renal biopsy samples. The methods evaluated include automatic thresholding, adaptive thresholding, and morphological granulometry. The results are compared to annotations made by an expert pathologist of more than 1500 cells in 18 images from different patients. The three methods provided true-positive ratios in the range 0.80 to 0.93.

GRAFT INFLAMMATION AND HISTOLOGICAL INDICATORS OF KIDNEY CHRONIC ALLOGRAFT FAILURE: GENETIC PREDISPOSITION TO LOW EXPRESSING INTERLEUKIN-10 GENOTYPES IS SIGNIFICANTLY CORRELATED: 2463

Introduction: Infiltration of inflammatory cells into the renal allograft interstitium is the biological hallmark of allo-immune responses that lead to tubulointerstitial injury and subsequent interstitial fibrosis and chronic allograft failure (CAF). The proliferation, stimulation, and infiltration of these inflammatory cells are governed by various pro-inflammatory and regulatory cytokines. We assessed whether differences in the genes encoding cytokines (producing low, moderate, or high expression profiles) may affect the infiltration of inflammatory cells into the renal allograft and the histological changes characteristic of CAF. Methodology: A total of 218 kidney transplant recipients were genotyped for 15 SNPs located in gene promoter/exonic regions of 10 different cytokines or their receptors. 6-12-months post-transplant surveillance biopsies were scored using 11 individual histological parameters and the combined grade of interstitial fibrosis/tubular atrophy (IF/TA). B cell, T cell, and macrophage infiltrates were quantified by immunostaining. A multivariate analysis based Multiple logistic regression analysis was performed with IF/TA or inflammation as dependent variables and all variables (genotypes and clinical and demographic parameters) that were significantly associated with IF/ TA or inflammation (p<0.05) in the univariate analysis as independent variables. Results: The low-expressing IL-10 gene promoter genotypes were found significantly associated with high interstitial fibrosis/tubular atrophy grade (p=0.035; Hazard’s Ratio=3.27; 95% CI=1.1–9.8). At individual histological indices, recipients carrying low expressing IL-10 genotypes showed significantly high scores for interstitial inflammation (p=0.04; Hazard’s Ratio=2.8; 95% CI=1.0-7.4). High infiltration of T cells (p=0.01) and macrophages (p=0.006) but not B cells into the renal allograft interstitium was found strongly associated with the carriage of low expressing IL-10 genotypes Conclusion: We conclude that kidney allograft recipients genetically predisposed to low expression of the regulatory cytokine IL-10 are more susceptible to high grades of IF/TA scores, graft inflammation and high influx of inflammatory cells into the graft interstitium. This suggests that a deficient regulatory milieu may represent a crucial predictor of chronic renal allograft damage and provides an important piece for a more comprehensive mathematical model predicting longterm renal transplant outcomes.