Urinary-Cell mRNA Profile and Acute Cellular Rejection in Kidney Allografts

Background—The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. Methods—We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. Results—A three-gene signature of 18S ribosomal (rRNA)–normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operatingcharacteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer–Lemeshow test indicated good fit (P = 0.77). In an externalvalidation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P = 0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti–interleukin-2 receptor antibodies from those who received T-cell–depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P = 0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). Conclusions—A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts

Keratin immunohistochemistry detects clinically significant metastases in bone marrow biopsy specimens in women with lobular breast carcinoma

Some patients with breast cancer currently undergo bone marrow biopsy to make clinical decisions regarding therapy; however, lobular carcinoma can be difficult to detect in routine histologic sections. The authors reviewed retrospectively all available bone marrow biopsies from patients with lobular carcinoma diagnosed between January, 1, 1989, and September, 25, 1997, at the City of Hope National Medical Center to identify useful morphologic features and to determine the utility of pan-keratin immunohistochemical (IHC) staining. A total of 65 biopsies from 54 patients were reviewed. Thirteen of the 65 biopsies were classified initially as containing metastatic tumor based on histologic features alone. With the addition of keratin IHC, seven additional cases of metastatic disease were detected. Forty of the 54 patients received stem cell replacement or autologous bone marrow transplantation. Disease-free survival after high-dose chemotherapy with stem cell replacement or autologous bone marrow transplantation was stratified into three groups based on hematoxylin and eosin (HandE) staining and IHC results. Two-year disease-free survival was 33% for the HandE-/IHC+ group versus 90% for the HandE-/IHC- group (p = 0.005) among patients clinically free of disease at the time of stem cell replacement or autologous bone marrow transplantation. Two-year disease-free survival was 0% in the HandE+/IHC+ group (p = 0.04, compared with the HandE-/IHC+ group). The authors conclude that routine morphologic examination without the aid of keratin IHC is unreliable in detecting clinically relevant metastatic lobular carcinoma in bone marrow biopsies. These findings suggest that pan-keratin immunostaining may be indicated on bone marrow biopsy specimens from lobular carcinoma patients if the biopsy appears histologically negative for metastatic tumor on HandE sections

Five‐year histological and serological follow‐up of operationally tolerant pediatric liver transplant recipients enrolled in WISP‐R

Pediatric liver transplant recipients arguably have the most to gain and the most to lose from discontinuing immunosuppression (IS). While IS undoubtedly exerts a cumulative toll, there is concern that insufficient or no IS may contribute to allograft deterioration. Twelve pediatric recipients of parental living donor liver grafts, identified as operationally tolerant through complete IS withdrawal (WISP-R; NCT00320606) were followed for a total of five years (one year of IS withdrawal and four years off IS) with serial liver tests, auto- and allo-antibody assessments. Liver biopsies were performed two and four years off IS and, at these time points, immunoglobulin G (IgG) subclass and C1q binding activity for donor specific antibodies (DSAs) were determined. There were no cases of chronic rejection, graft loss, or death. Allografts did not exhibit progressive increase in inflammation or fibrosis. Smooth muscle actin (SMA) expression by stellate cells and CD34 expression by liver sinusoidal endothelial cells (LSECs) remained stable, consistent with the absence of progressive graft injury. Three subjects never exhibited DSA. However, three subjects showed intermittent de novo Class I DSA, four subjects showed persistent de novo Class II DSA and five subjects showed persistent pre-existing Class II DSA. Class II DSA was predominantly against donor DQ antigens, often of high mean fluorescence intensity (MFI), rarely of the IgG3 subclass, and often capable of binding C1q. CONCLUSION: Operationally tolerant pediatric liver transplant recipients maintain generally stable allograft histology in spite of apparently active humoral allo-immune responses. The absence of increased inflammation or progressive fibrosis suggests that a subset of liver allografts seem resistant to the chronic injury that is characteristic of antibody-mediated damage

Serum MicroRNA Transcriptomics and Acute Rejection or Recurrent Hepatitis C Virus in Human Liver Allograft Recipients: A Pilot Study

BackgroundAcute rejection (AR) and recurrent hepatitis C virus (R-HCV) are significant complications in liver allograft recipients. Noninvasive diagnosis of intragraft pathologies may improve their management.MethodsWe performed small RNA sequencing and microRNA (miRNA) microarray profiling of RNA from sera matched to liver allograft biopsies from patients with nonimmune, nonviral (NINV) native liver disease. Absolute levels of informative miRNAs in 91 sera matched to 91 liver allograft biopsies were quantified using customized real-time quantitative PCR (RT-qPCR) assays: 30 biopsy-matched sera from 26 unique NINV patients and 61 biopsy-matched sera from 41 unique R-HCV patients. The association between biopsy diagnosis and miRNA abundance was analyzed by logistic regression and calculating the area under the receiver operating characteristic curve.ResultsNine miRNAs-miR-22, miR-34a, miR-122, miR-148a, miR-192, miR-193b, miR-194, miR-210, and miR-885-5p-were identified by both sRNA-seq and TLDA to be associated with NINV-AR. Logistic regression analysis of absolute levels of miRNAs and goodness-of-fit of predictors identified a linear combination of miR-34a + miR-210 (P &lt; 0.0001) as the best statistical model and miR-122 + miR-210 (P &lt; 0.0001) as the best model that included miR-122. A different linear combination of miR-34a + miR-210 (P &lt; 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft inflammation, and miR-34a + miR-122 (P &lt; 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft fibrosis.ConclusionsCirculating levels of miRNAs, quantified using customized RT-qPCR assays, may offer a rapid and noninvasive means of diagnosing AR in human liver allografts and for discriminating AR from intragraft inflammation or fibrosis due to R-HCV

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants

Background &amp; aimsA substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles.MethodsWe conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; &gt; 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an&nbsp;unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data.ResultsThe mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n&nbsp;= 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n&nbsp;= 45), and cluster 3 had neither feature (n&nbsp;=&nbsp;78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or&nbsp;3.ConclusionIn an analysis of biopsies from&nbsp;an apparently homogeneous group of stable, long-term&nbsp;pediatric liver transplant recipients with consistently normal&nbsp;liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with&nbsp;TCMR

Rituximab Versus Cyclophosphamide for ANCA-Associated Vasculitis with Renal Involvement

Rituximab (RTX) is non-inferior to cyclophosphamide (CYC) followed by azathioprine (AZA) for remission-induction in severe ANCA-associated vasculitis (AAV), but renal outcomes are unknown. This is a post hoc analysis of patients enrolled in the Rituximab for ANCA-Associated Vasculitis (RAVE) Trial who had renal involvement (biopsy proven pauci-immune GN, red blood cell casts in the urine, and/or a rise in serum creatinine concentration attributed to vasculitis). Remission-induction regimens were RTX at 375 mg/m(2) x 4 or CYC at 2 mg/kg/d. CYC was replaced by AZA (2 mg/kg/d) after 3-6 months. Both groups received glucocorticoids. Complete remission (CR) was defined as Birmingham Vasculitis Activity Score/Wegener's Granulomatosis (BVAS/WG)=0 off prednisone. Fifty-two percent (102 of 197) of the patients had renal involvement at entry. Of these patients, 51 were randomized to RTX, and 51 to CYC/AZA. Mean eGFR was lower in the RTX group (41 versus 50 ml/min per 1.73 m(2); P=0.05); 61% and 75% of patients treated with RTX and 63% and 76% of patients treated with CYC/AZA achieved CR by 6 and 18 months, respectively. No differences in remission rates or increases in eGFR at 18 months were evident when analysis was stratified by ANCA type, AAV diagnosis (granulomatosis with polyangiitis versus microscopic polyangiitis), or new diagnosis (versus relapsing disease) at entry. There were no differences between treatment groups in relapses at 6, 12, or 18 months. No differences in adverse events were observed. In conclusion, patients with AAV and renal involvement respond similarly to remission induction with RTX plus glucocorticoids or CYC plus glucocorticoids