Search

Supplemental Digital Content is available in the text.

In the clinical practice of pathology, trichrome stains are commonly used to highlight collagen and to help evaluate fibrosis. Such stains do delineate collagen deposits but are not molecularly specific and can suffer from staining inconsistencies. Moreover, performing histochemical stain evaluation requires the preparation of additional sections beyond the original hematoxylin- and eosin-stained slides, as well as additional staining steps, which together add cost, time, and workflow complications. We have developed a new microscopy approach, termed DUET (DUal-mode Emission and Transmission) that can be used to extract signals that would typically require special stains or advanced optical methods. Our preliminary analysis demonstrates the potential of using the resulting signals to generate virtual histochemical images that resemble trichrome-stained slides and can support clinical evaluation. We demonstrate advantages of this approach over images acquired from conventional trichrome-stained slides and compare them with images created using second harmonic generation microscopy.

Kidney complications can occur due to infective endocarditis, one of which is glomerulonephritis. Most often, an immune complex or complement-mediated glomerulonephritis is seen on kidney biopsy. In a minor subset of cases, pauci-immune glomerulonephritis may be present. Most often, such patients will demonstrate the presence of antineutrophil cytoplasmic antibodies (ANCA) on serologic testing. A growing number of cases of ANCA-associated glomerulonephritis due to Bartonella endocarditis have been reported. This type of endocarditis can present diagnostic difficulties given that these patients are often culture negative. Herein, we report a challenging case of ANCA-negative pauci-immune glomerulonephritis showing florid crescents on biopsy that was associated with Bartonella endocarditis.

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53alteration without RB1alteration; half of these harbored high-level amplifications in CCNE1and MYCor MYCNand arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1or TP53but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

The combined use of ex vivo hypothermic and normothermic perfusion may be a useful strategy to more adequately assess and preserve high-risk kidneys deemed unsuitable for transplantation.

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53alteration without RB1alteration; half of these harbored high-level amplifications in CCNE1and MYCor MYCNand arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1or TP53but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

ObjectiveThe Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum.DesignWe monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis.ResultsCC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS.ConclusionsCC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Although donor livers with <30% large droplet macrovesicular steatosis (MaS) and/or small droplet MaS (irrespective of percentage) are considered safe to use, this consensus is based on variable definitions of MaS subtypes and/or without a reproducible scoring system. We analyzed 134 donor liver biopsies from allografts transplanted at University of California at San Francisco between 2000 and 2015 to determine whether large and/or small droplet MaS is a risk factor for poor outcomes. Large droplet MaS was defined as a fat droplet occupying greater than one half of an individual hepatocyte, with nuclear displacement, and scored as the percentage of total parenchymal area replaced by large fat droplets on ×40 magnification. Small droplet MaS was defined as 1 to several discrete fat droplets, each occupying less than one half of an individual hepatocyte, and scored as the percentage of remaining hepatocytes (ie, hepatocytes not occupied by large fat droplets) containing small fat droplets on ×200 magnification (ie, small droplet MaS is the percentage of “remaining hepatocytes” with small fat droplets, and “remaining hepatocytes” is defined as 100% minus percent large droplet MaS). Thus, total MaS equals the sum of large and small droplet MaS, which cannot exceed 100%. Electronic medical records were reviewed to determine outcomes. There was an increased risk for acute cellular rejection (hazard ratio=2.5, P=0.0108) and bile duct loss suggestive of chronic ductopenic rejection (hazard ratio=2.4, P=0.0130) in donor livers with ≥30% small droplet MaS. Large droplet MaS (up to 60%) was not associated with adverse outcomes. Patient survival was not adversely affected by steatosis. Excellent agreement on the estimation of large (weighted κ=0.682) and small droplet MaS (weighted κ=0.780) was achieved. Our approach to donor steatosis scoring can identify liver allograft recipients at increased risk for rejection and highlights the importance of distinguishing between small and large droplet MaS in this evaluation.

Understanding tumor metastasis is crucial to developing more effective cancer therapies. Here McCreery et al. analyzed the mutational profile of metastases from chemically induced skin tumors in mice and found that parallel evolution of synchronously disseminated tumor cells underlies most metastasis.

Exposure to levamisole-adulterated cocaine can induce a distinct clinical syndrome characterized by retiform purpura andor agranulocytosis accompanied by an unusual constellation of serologic abnormalities including antiphospholipid antibodies, lupus anticoagulants, and very high titers of antineutrophil cytoplasmic antibodies. Two recent case reports suggest that levamisole-adulterated cocaine may also lead to renal disease in the form of pauci-immune glomerulonephritis. To explore this possibility, we reviewed cases of pauci-immune glomerulonephritis between 2010 and 2012 at an inner city safety net hospital where the prevalence of levamisole in the cocaine supply is known to be high. We identified 3 female patients and 1 male patient who had biopsy-proven pauci-immune glomerulonephritis, used cocaine, and had serologic abnormalities characteristic of levamisole-induced autoimmunity. Each also had some other form of clinical disease known to be associated with levamisole, either neutropenia or cutaneous manifestations. One patient had diffuse alveolar hemorrhage. Three of the 4 patients were treated with short courses of prednisone and cyclophosphamide, 2 of whom experienced stable long-term improvement in their renal function despite ongoing cocaine use. The remaining 2 patients developed end-stage renal disease and became dialysis-dependent. This report supports emerging concern of more wide spread organ toxicity associated with the use of levamisole-adulterated cocaine.

A spectrum of kidney diseases besides classic human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) exists in HIV-infected patients. Immune complex–mediated glomerulonephritis has emerged as a significant contributor to the burden of kidney disease in this population, particularly in patients of non-African descent. Lupus-like nephritis, a form of immune complex glomerulonephritis with histologic features identical to lupus nephritis in the absence of clinical or serologic markers of lupus, is well recognized as a cause of end-stage renal disease in HIV-infected patients. None of the HIV-associated kidney lesions, whether classic HIVAN or non-HIVAN, has been reported to recur in kidney transplants. We report here for the first time clinical and histologic recurrence of HIV-associated lupus-like nephritis after successful kidney transplantation, causing proteinuria, hematuria, and impaired kidney transplant function.

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.

T-cell acute lymphoblastic lymphomas commonly demonstrate activating Notch1 mutations as well as mutations or deletions in Fbxw7. However, because Fbxw7 targets Notch1 for degradation, genetic alterations in these genes are expected to be mutually exclusive events in lymphomagenesis. Previously, by using a radiation-induced Tp53-deficient mouse model for T-cell acute lymphoblastic lymphoma, we reported that loss of heterozygosity at the Fbxw7 locus occurs frequently in a Tp53-dependent manner. In the current study, we show that these thymic lymphomas also commonly exhibit activating Notch1 mutations in the proline-glutamic acid-serine-threonine (PEST) domain. Moreover, concurrent activating Notch1 PEST domain mutations and single-copy deletions at the Fbxw7 locus occur with high frequency in the same individual tumors, indicating that these changes are not mutually exclusive events. We further demonstrate that although Notch1 PEST domain mutations are independent of Tp53 status, they are completely abolished in mice with germline Fbxw7 haploinsufficiency. Therefore, Notch1 PEST domain mutations only occur when Fbxw7 expression levels are intact. These data suggest a temporal sequence of mutational events involving these important cancer-related genes, with Notch1 PEST domain mutations occurring first, followed by Fbxw7 deletion, and eventually by complete loss of Tp53.

T-cell acute lymphoblastic lymphomas commonly demonstrate activating Notch1mutations as well as mutations or deletions in Fbxw7. However, because Fbxw7 targets Notch1 for degradation, genetic alterations in these genes are expected to be mutually exclusive events in lymphomagenesis. Previously, by using a radiation-induced Tp53-deficient mouse model for T-cell acute lymphoblastic lymphoma, we reported that loss of heterozygosity at the Fbxw7locus occurs frequently in a Tp53-dependent manner. In the current study, we show that these thymic lymphomas also commonly exhibit activating Notch1mutations in the proline-glutamic acid-serine-threonine (PEST) domain. Moreover, concurrent activating Notch1PEST domain mutations and single-copy deletions at the Fbxw7locus occur with high frequency in the same individual tumors, indicating that these changes are not mutually exclusive events. We further demonstrate that although Notch1PEST domain mutations are independent of Tp53status, they are completely abolished in mice with germline Fbxw7haploinsufficiency. Therefore, Notch1PEST domain mutations only occur when Fbxw7expression levels are intact. These data suggest a temporal sequence of mutational events involving these important cancer-related genes, with Notch1PEST domain mutations occurring first, followed by Fbxw7deletion, and eventually by complete loss of Tp53.

Classic antiglomerular basement membrane (anti-GBM) disease is an exceedingly rare but extremely aggressive form of glomerulonephritis, typically caused by autoantibodies directed against cryptic, conformational epitopes within the noncollagenous domain of the type IV collagen alpha-3 subunit. Pathologic diagnosis is established by the presence of strong, diffuse, linear staining for immunoglobulin on immunofluorescence microscopy. Recently, patients with atypical clinical and pathologic findings of anti-GBM disease have been described. These patients tend to have an indolent clinical course, without pulmonary involvement, and laboratory testing rarely reveals the presence of anti-GBM antibodies. Specific guidelines for the treatment and management of these patients are unclear. Here, we describe a case of atypical anti-GBM disease in a young child who presented with hematuria and prominent proteinuria. Throughout the course of his illness, creatinine remained normal. He was conservatively treated with steroids and rituximab, resulting in resolution of his clinical symptoms and normalization of laboratory findings.