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Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC) is an aggressive clinical entity. Current diagnostic guidelines for premalignant lesions are ambiguous, and their molecular profile and progression events are still unclear. We selected 75 samples, from 40 patients, including 33 VSCC, 8 verrucous carcinomas (VC), 13 differentiated-type vulvar intraepithelial neoplasia (dVIN), 11 suspicious for dVIN (?dVIN), 6 differentiated exophytic vulvar intraepithelial lesions (DE-VIL), 2 vulvar acanthosis with altered differentiation (VAAD), and 2 usual-type vulvar intraepithelial neoplasia (uVIN/HSIL). Invasive and precursor lesions were matched in 29 cases. Clinical information, p16 immunohistochemistry, and mutation analysis were performed on all lesions. All dVIN, ?dVIN, DE-VIL, and VAAD were p16 negative, all uVIN/HSIL were p16 positive. In the HPV-independent group, mutations were identified in 6 genes: TP53 (n = 40), PIK3CA (n = 20), HRAS (n = 12), MET (n = 5), PTEN (n = 4), and BRAF (n = 1). TP53 mutations occurred in 73% (22/30) VSCC, 85% (11/13) dVIN, 70% (7/10) ?dVIN and no VC (0/8), DE-VIL (0/6) nor VAAD (0/2). Basal atypia was the only reliable feature of TP53 mutations. ?dVIN lesions that were non-acanthotic and atypical but obscured by inflammation, all harbored TP53 mutations. In lesions without TP53 mutations, PIK3CA (50% VC, 33% DE-VIL, 100% VAAD, 40% VSCC) and HRAS (63% VC, 33% DE-VIL, 0% VAAD, 20% VSCC) mutations were found. Mutational progression from in situ to invasive was seen (7/26, 27%) and usually involved TP53 (4/26, 15%). Cases with TP53 and PIK3CA co-mutations had the worse clinical outcomes (p

Objectives All patients who undergo cardiopulmonary bypass (CPB) experience some degree of ischemia reperfusion injury (IRI). Severe IRI-induced acute kidney injury (AKI) occurs in approximately 1–2% of patients undergoing CPB. Previous studies using activity-based protein profiling of urine identified group XV phospholipase A2, PLA2G15/LPLA2, as potentially associated with patients who develop AKI post CPB. The present study examined urinary PLA2G15/LPLA2 activity during CPB and in the near postoperative period for associations with subsequent AKI development. Design & methods Samples were collected in a nested case controlled cohort of 21 patients per group who either did (AKI) or did not (non-AKI) develop AKI post-operatively. Serum and urine samples from each patient before, during and after CPB were assayed for PLA2G15/LPLA2 activity. Results Urine activity significantly increased during the intra operative period. In contrast the activities in paired sera were markedly decreased during CPB. There was no correlation between the serum and urine activity levels of patients. There were no significant differences in activity levels of PLA2G15/LPLA2 in the urine or sera from patients that did and did not develop AKI. Conclusions The lack of correlation between serum and urine activity levels suggests that the rapid intraoperative increases in PLA2G15/LPLA2 activity may originate from the kidney and as such offer an intraoperative indicator of early renal response to CPB associated stressors.

Improving long‐term kidney transplant outcomes requires novel treatment strategies, including delayed calcineurin inhibitor (CNI) substitution, tested using informative trial designs. An alternative approach to the usual superiority‐based trial is a noninferiority trial design that tests whether an investigational agent is not unacceptably worse than standard of care. An informative noninferiority design, with biopsy‐proven acute rejection (BPAR) as the endpoint, requires determination of a prespecified, evidence‐based noninferiority margin for BPAR. No such information is available for delayed CNI substitution in kidney transplantation. Herein we analyzed data from recent kidney transplant trials of CNI withdrawal and “real world” CNI‐ based standard of care, containing subjects with well‐documented evidence of immune quiescence at 6 months posttransplant—ideal candidates for delayed CNI substitution. Our analysis indicates an evidence‐based noninferiority margin of 13.8% for the United States Food and Drug Administration's composite definition of BPAR between 6 and 24 months posttransplant. Sample size estimation determined that ~225 randomized subjects would be required to evaluate noninferiority for this primary clinical efficacy endpoint, and superiority for a renal function safety endpoint. Our findings provide the basis for future delayed CNI substitution noninferiority trials, thereby increasing the likelihood they will provide clinically implementable results and achieve regulatory approval., This meta‐analysis defines a noninferiority margin to use in the design of a phase II/III delayed calcineurin inhibitor substitution trial in kidney transplantation.

AIMS The role of mismatch repair (MMR) testing has evolved from identifying Lynch syndrome patients to predicting response to immune checkpoint inhibitors. This has led to requests from clinicians to retest recurrences of MMR-proficient primary tumours in the hope that the recurrence may show a different MMR status and qualify the patient for treatment. We aimed to determine whether repeat testing is warranted. METHODS AND RESULTS We evaluated recurrent tumours (local recurrences or metastases) from 137 patients with MMR-proficient primary tumours of the gastrointestinal and gynaecological tracts. The local recurrences and metastases all occurred at least 30 days after resection of the primary tumour. We used a combination of a tissue microarray and whole slide staining to perform immunohistochemistry (IHC) for PMS2, MLH1, MSH2, and MSH6, and compared the results with the MMR status of the primary tumour. Three of 137 (2%) initially showed a discordant staining pattern. However, further investigation showed that these discordances were attributable to some of the known pitfalls associated with MMR IHC interpretation - post-radiotherapy loss of MSH6 expression and subclonal loss of MLH1 staining. We did not identify any cases with a genuine discordance in MMR status. CONCLUSION We conclude that repeat MMR IHC testing of recurrences is not warranted, as MMR status does not change relative to that of the primary tumour.

The ongoing shortage of organs for transplant combined with Manitoba having the highest prevalence of end-stage renal disease (ESRD) in Canada has resulted in long wait times on the deceased donor waitlist. Therefore, the Transplant Manitoba Adult Kidney Program has ongoing quality improvement initiatives to expand the deceased donor pool. This clinical transplant protocol describes the use of prophylactic pan-genotypic direct-acting anti-viral agents (DAA) for transplanting hepatitis C (HCV)-viremic kidneys (HCV antibody positive/nucleic acid [nucleic acid amplification testing, NAT] positive) to HCV-naïve recipients as routine standard of care. We will evaluate the provincial implementation of this protocol as a prospective observational cohort study.Scoping literature review and key stakeholder engagement with interdisciplinary health care providers and health system leaders/decision markers.Patients will be screened pre-transplant for eligibility and undergo a multilevel education and consent process to participate in this expanded donor program. Incident adult HCV-naïve recipients of an HCV-viremic kidney transplant will be treated prophylactically with glecaprevir-pibrentasvir with the first dose administered on call to the operation. Glecaprevir-pibrentasvir will be used for 8 weeks with viral monitoring and hepatology follow-up. Primary outcomes are sustained virologic response (SVR) at 12 weeks and the tolerability of DAA therapy. Secondary outcomes within the first year post-transplant are patient and graft survival, graft function, biopsy-proven rejection, HCV transmission to recipient (HCV NAT positive), and HCV nonstructural protein 5A (NS5A) resistance. Safety outcomes within the first year post-transplant include fibrosing cholestatic hepatitis, acute liver failure, primary and secondary DAA treatment failure, HCV transmission to a recipient's partner, elevated liver enzymes ≥2-fold, abnormal international normalized ratio (INR), angioedema, anaphylaxis, cirrhosis, and hepatocellular carcinoma.This program successfully advocated for and obtained hospital formulary, provincial Exceptional Drug Status (EDS), and Non-Insured Health Benefits (NIHB) to provide prophylactic DAA therapy for this indication, and this information may be useful to other provincial transplant organizations seeking to establish an HCV-viremic kidney transplant program with prophylactic DAA drug coverage.(1) Patient engagement was not undertaken during the program design phase, but patient-reported experience measures will be obtained for continuous quality improvement. (2) Only standard criteria donors (optimal kidney donor profile index [KDPI] ≤60) will be used. If this approach is safe and feasible, then higher KDPI donors may be included.The goal of this quality improvement project is to improve access to kidney transplantation for Manitobans. This program will provide prophylactic DAA therapy for HCV-viremic kidney transplant to HCV-naïve recipients as routine standard of care outside a clinical trial protocol. We anticipate this program will be a safe and effective way to expand kidney transplantation from a previously unutilized donor pool.La pénurie actuelle d’organes à transplanter, combinée au fait que le Manitoba est la province qui présente la plus forte prévalence d’insuffisance rénale terminale au Canada, entraîne de longs délais sur la liste d’attente d’un organe provenant d’un donneur décédé. Le programme de transplantation rénale pour les adultes du Manitoba (Transplant Manitoba Adult Kidney Program) a mis en place des initiatives d’amélioration continue de la qualité afin d’élargir le bassin de donneurs décédés. Ce protocole clinique de transplantation décrit l’emploi, comme traitement habituel, d’agents antiviraux directs (AAD) pan-génotypiques prophylactiques pour la transplantation de reins provenant de donneurs infectés par le virus de l’hépatite C (VHC) (individus positifs pour les anticorps VHC et acides nucléiques [NAT]) à des receveurs naïfs pour VHC. La mise en œuvre provinciale de ce protocole sera évaluée en tant qu’étude de cohorte prospective et observationnelle.Examen de la documentation et évaluation de l’engagement des principaux intervenants avec les fournisseurs de soins de santé interdisciplinaires et les dirigeants/décideurs du système de santé.L’admissibilité au programme sera évaluée avant la greffe. Pour participer à ce programme élargi de donneurs, les patients devront se soumettre à un processus d’information et de consentement à plusieurs niveaux. Les adultes incidents naïfs pour VHC devant recevoir un rein virémique-VHC seront traités de façon prophylactique par glécaprévir+pibrentasvir; la première dose administrée au moment de l’appel pour l’opération. Le traitement par glécaprévir+pibrentasvir sera administré pendant 8 semaines avec surveillance virale et suivi hépatologique. Les principaux résultats évalués seront une réponse virologique prolongée (RVP) à 12 semaines et la tolérance au traitement par AAD. Les résultats secondaires mesurés dans la première année suivant la greffe seront la survie du patient et du greffon; la fonction du greffon; le rejet avéré par biopsie; la transmission du VHC au receveur (positif pour VHC et NAT) et la résistance aux protéines non structurelles 5A (NS5A) du VHC. Les résultats relatifs à l’innocuité dans la première année suivant la greffe comprennent la cholestase hépatique fibrosante; l’insuffisance hépatique aiguë; l’échec primaire et secondaire du traitement par AAD; la transmission du VHC au partenaire d’un receveur; une élévation supérieure à 2 fois du taux d’enzymes hépatiques; un INR anormal; un angio-œdème; l’anaphylaxie; une cirrhose ou un carcinome hépatocellulaire.Le programme a recommandé et obtenu l’inscription du traitement prophylactique par AAD sur la liste de médicaments des hôpitaux pour cette indication, en plus du statut de médicament d’exception provincial et de son ajout au Programme des services de santé non assurés (SSNA). Ces renseignements pourraient être utiles à d’autres organismes provinciaux de transplantation qui cherchent à mettre en œuvre un programme de transplantation rénale virémique-VHC avec un traitement prophylactique par AAD.(1) La participation des patients n’a pas été entreprise pendant la phase de conception du programme, mais des mesures de l’expérience des patients seront obtenues pour l’amélioration continue de la qualité. (2) Seuls les donneurs satisfaisant aux critères standards (Kidney Donor Profile Index [KDPI] ≤ 60) seront inclus. Si cette approche est sécuritaire et faisable, des donneurs de KDPI plus élevés pourront être inclus.L’objectif de ce projet d’amélioration de la qualité est d’améliorer l’accès aux transplantations rénales pour les Manitobains. Ce programme offrira un traitement prophylactique aux AAD pour les greffes de reins virémiques-VHC à des receveurs naïfs pour VHC comme traitement de référence habituel en dehors d’un protocole d’essai clinique. Nous pensons que ce programme sera un moyen sûr et efficace d’étendre la transplantation rénale à partir d’un bassin de donneurs auparavant non utilisés.

Purpose: Gene fusions involving neuregulin 1 (NRG1) have been noted in multiple cancer types and have potential therapeutic implications. Although varying results have been reported in other cancer types, the efficacy of the HER-family kinase inhibitor afatinib in the treatment of NRG1 fusion–positive pancreatic ductal adenocarcinoma is not fully understood. Experimental Design: Forty-seven patients with pancreatic ductal adenocarcinoma received comprehensive whole-genome and transcriptome sequencing and analysis. Two patients with gene fusions involving NRG1 received afatinib treatment, with response measured by pretreatment and posttreatment PET/CT imaging. Results: Three of 47 (6%) patients with advanced pancreatic ductal adenocarcinoma were identified as KRAS wild type by whole-genome sequencing. All KRAS wild-type tumors were positive for gene fusions involving the ERBB3 ligand NRG1. Two of 3 patients with NRG1 fusion–positive tumors were treated with afatinib and demonstrated a significant and rapid response while on therapy. Conclusions: This work adds to a growing body of evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in pancreatic cancers. Based on the clinical outcomes described here, patients with KRAS wild-type tumors harboring NRG1 gene fusions may benefit from treatment with afatinib. See related commentary by Aguirre, p. 4589

AimsSerous cystadenomata (SCAs) are benign pancreatic cystic neoplasms that present a diagnostic challenge despite many investigational approaches. Notwithstanding the promise of molecular diagnostics, these tests have limited accessibility in day-to-day surgical pathology practices. We aim to corroborate and build on recent evidence which suggests that positive α-inhibin immunohistochemistry (IHC) is a helpful adjunct in the biopsy confirmation of pancreatic SCA.MethodsWe retrospectively reviewed 22 fine-needle aspirates/biopsies from 14 patients (mean age 65 years, 47–83 years) with pancreatic multicystic lesions radiologically suspicious for SCA (location: 6 body, 2 head, 4 tail, 1 neck, 1 uncinate; cyst size: mean 3.7 cm, 2.0–7.6 cm), as well as an additional 10 pancreatic resection specimens with confirmed SCA; α-inhibin IHC was performed on all cell blocks, biopsy slides and representative resection specimen sections. Where available, associated cyst fluid was analysed for correlative vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen levels.ResultsAn α-inhibin IHC sensitivity of 80% was observed in the cases with resection confirmed SCA. Of the fine-needle aspirate/biopsy specimens, 59% (13/22) contained epithelial cells strongly positive for α-inhibin. When selecting for specimens that exhibited distinct strips of epithelium, the α-inhibin strong positivity rate increased to 73% (8/11). VEGF-A values were supportive of false-negative α-inhibin IHC in three cases and true-negative α-inhibin IHC in one case.ConclusionThis study postulates a diagnostic algorithm to confirm pancreatic SCA which may help to decrease unnecessary follow-up endoscopy/surgical resection and would decrease the associated morbidity, mortality and financial costs in patients with this otherwise benign condition.

Background The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. Methods Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. Results In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. Conclusions There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

The recent literature has shown that vulvar squamous cell carcinoma (VSCC) can be stratified into two prognostically relevant groups based on human papillomavirus (HPV) status. The prognostic value of p53 for further sub-stratification, particularly in the HPV-independent group, has not been agreed upon. This disagreement is likely due to tremendous variations in p53 immunohistochemical (IHC) interpretation. To address this problem, we sought to compare p53 IHC patterns with TP53 mutation status. We studied 61 VSCC (48 conventional VSCC, 2 VSCC with sarcomatoid features, and 11 verrucous carcinomas) and 42 in situ lesions (30 differentiated vulvar intraepithelial neoplasia [dVIN], 9 differentiated exophytic vulvar intraepithelial lesions [deVIL], and 3 high-grade squamous intraepithelial lesions or usual vulvar intraepithelial neoplasia [HSIL/uVIN]). IHC for p16 and p53, and sequencing of TP53 exons 4-9 were performed. HPV in situ hybridization (ISH) was performed in selected cases. We identified six major p53 IHC patterns, two wild-type patterns: (1) scattered, (2) mid-epithelial expression (with basal sparing), and four mutant patterns: (3) basal overexpression, (4) parabasal/diffuse overexpression, (5) absent, and (6) cytoplasmic expression. These IHC patterns were consistent with TP53 mutation status in 58/61 (95%) VSCC and 39/42 (93%) in situ lesions. Cases that exhibited strong scattered staining and those with a weak basal overexpression pattern could be easily confused. The mid-epithelial pattern was exclusively observed in p16-positive lesions; the basal and parabasal layers that had absent p53 staining, appeared to correlate with the cells that were positive for HPV-ISH. This study describes a pattern-based p53 IHC interpretation framework, which can be utilized as a surrogate marker for TP53 mutational status in both VSCC and vulvar in situ lesions.

Background NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown. Patients and methods Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization. Results Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4–NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1–NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib. Conclusion These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin. Clinical trial information Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

Biomarkers are needed that identify patients with antibody-mediated rejection (AMR). The goal of this study was to evaluate the utility of urinary metabolomics for early noninvasive detection of AMR in pediatric kidney transplant recipients.Urine samples (n = 396) from a prospective, observational cohort of 59 renal transplant patients with surveillance or indication biopsies were assayed for 133 unique metabolites by quantitative mass spectrometry. Samples were classified according to Banff criteria for AMR and partial least squares discriminant analysis was used to identify associated changes in metabolite patterns by creating a composite index based on all 133 metabolites.Urine samples of patients with (n = 40) and without AMR (n = 278) were analyzed and a classifier for AMR was identified (area under receiver operating characteristic curve = 0.84; 95% confidence interval, 0.77-0.91; P = 0.006). Application of the classifier to "indeterminate" samples (samples that partially fulfilled Banff criteria for AMR; n = 65) yielded an AMR score of 0.19 ± 0.15, intermediate between scores for AMR and No AMR (0.28 ± 0.14 and 0.10 ± 0.13 respectively, P ≤ 0.001). The AMR score was associated with the presence of donor-specific antibodies, biopsy indication, Banff ct, t, ah and cg scores, and retained accuracy when applied to subclinical cases (creatinine,25% increase from baseline) or had minimal or no transplant glomerulopathy (Banff cg0-1). Exploratory classifiers that segregated samples based on concurrent T cell-mediated rejection (TCMR) identified overlapping metabolite signatures between AMR and TCMR, suggesting similar pathophysiology of tissue injury.These preliminary findings identify a urine metabolic classifier for AMR. Independent validation is needed to verify its utility for accurate, noninvasive AMR detection.

Adenoid cystic carcinoma is a rare malignant tumor that usually arises in the major and minor salivary glands and other locations containing secretory glands, including the lower female genital tract. Lower female genital tract carcinomas with adenoid cystic differentiation can be subclassified into 2 distinct groups based on the presence or absence of high-risk HPV. Cervical mixed carcinomas with some adenoid cystic differentiation are high-risk HPV-related but pure adenoid cystic carcinomas of vulvar and cervical origin appear to be unrelated to high-risk HPV. Mechanisms by which normal cells give rise to an HPV-unrelated adenoid cystic carcinoma remain largely unknown. Studies demonstrate that chromosomal translocation involving the genes encoding the transcription factors MYB and NFIB functions as a driving force of adenoid cystic carcinomas development regardless of anatomic site. The current study used fluorescence in situ hybridization with 3 different probes including MYB break-apart probe, NFIB break-apart probe, and MYB-NFIB fusion probe to assess for the presence of gene rearrangements in adenoid cystic carcinomas of the vulva. Six (66.7%) of 9 vulvar adenoid cystic carcinomas demonstrated NFIB rearrangement. Of these 6 cases with a disturbed NFIB, only 2 cases (33.3%) were positive for a MYB rearrangement that was also confirmed by a positive MYB-NFIB fusion pattern. NFIB-associated gene rearrangement is a frequent genetic event in vulvar adenoid cystic carcinomas. Chromosome translocations involving NFIB but with an intact MYB indicate the presence of novel oncogenic mechanisms for the development of adenoid cystic carcinomas of the vulva.

De novo donor-specific antibodies (dnDSAs) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dnDSA C1q status or titer at the time of first detection can independently predict allograft loss. In a consecutive cohort of 508 renal transplant recipients, 70 developed dnDSAs. Histologic and clinical outcomes were correlated with the C1q assay or dnDSA titer. C1q positivity correlated with dnDSA titer (p < 0.01) and mean fluorescence intensity (p < 0.01) and was more common in class II versus class I dnDSAs (p < 0.01). C1q status correlated with tubulitis (p = 0.02) and C4d status (p = 0.03) in biopsies at the time of dnDSA development, but not T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR). De novo DSA titer correlated with Banff g, i, t, ptc, C4d scores, TCMR (p < 0.01) and ABMR (p < 0.01). Post-dnDSA graft loss was observed more frequently in recipients with C1q-positve dnDSA (p < 0.01) or dnDSA titer ≥ 1:1024 (p ≤ 0.01). However, after adjustment for clinical phenotype and nonadherence in multivariate models, neither C1q status nor dnDSA titer were independently associated with allograft loss, questioning the utility of these assays at the time of dnDSA development.

Introduction Acute kidney injury (AKI) is a potentially fatal complication of cardiac surgery. The inability to predict cardiac surgery-associated AKI is a major barrier to prevention and early treatment. Current clinical risk models for the prediction of cardiac surgery-associated AKI are insufficient, particularly in patients with preexisting kidney dysfunction. Methods To identify intraoperative variables that might improve the performance of a validated clinical risk score (Cleveland Clinic Score, CCS) for the prediction of cardiac surgery-associated AKI, we conducted a prospective cohort study in 289 consecutive elective cardiac surgery patients at a tertiary care center. We compared the area under the receiver operator characteristic curve (AUC) of a base model including only the CCS with models containing additional selected intraoperative variables including mean arterial pressure, hematocrit, duration of procedure, blood transfusions, and fluid balance. AKI was defined by the Kidney Disease Improving Global Outcomes 2012 criteria. Results The CCS alone gave an AUC of 0.72 (95% confidence interval, 0.62–0.82) for postoperative AKI. Nadir intraoperative hematocrit was the only variable that improved AUC for postoperative AKI when added to the CCS (AUC = 0.78; 95% confidence interval, 0.70–0.87; P = 0.002). In the subcohort of patients without preexisting chronic kidney disease (n = 214), where the CCS underperformed (AUC, 0.60 [0.43–0.76]), the improvement with the addition of nadir hematocrit was more marked (AUC, 0.74 [0.62–0.86]). Other variables did not improve discrimination. Discussion Nadir intraoperative hematocrit is useful in improving discrimination of clinical risk scores for AKI, and may provide a target for intervention.

Mesonephric carcinoma is a rare malignancy, thought to derive from Wolffian remnants. To date, no targeted molecular therapeutic options have been identified. On the basis of limited case reports, c-KIT immunohistochemical expression has been reported in female adnexal tumors of Wolffian origin, and targeted therapy with Imatinib has been attempted with mixed success. Currently, it is unclear whether c-KIT immunohistochemical expression is seen in mesonephric carcinoma, a tumor that is thought to be related to female adnexal tumors of Wolffian origin, and how this correlates with KIT mutational status. In this study, we assessed the immunohistochemical expression of c-KIT and KIT mutational status, in a series of 13 mesonephric neoplasms (5 cervical [including 2 cervical carcinosarcomas], 3 uterine corpora, 4 ovarian, and 1 vaginal/pelvic). The intensity of staining and proportion of cells showing cytoplasmic/membranous staining for c-KIT were recorded. KIT was sequenced using a next-generation sequencing panel that targeted 120 hotspots and 17 exons in 33 known actionable cancer genes. This panel included KIT exons 9, 11, and 13, and 6 hotspots (T670, D816, D820, N822, Y823, A829). Although c-KIT immunohistochemical expression was observed in the majority of mesonephric carcinomas (10/12 cases; 83%), no KIT mutations were detected. This cautions pathologists against the use of c-KIT immunohistochemistry as a surrogate marker for KIT-activating mutations in this setting. Consistent with previous studies, the majority of mesonephric neoplasms (10/13; 77%) harbored KRAS mutations. Additional mutations were found in CTNNB1 (2/13, 15%), TP53 (2/13, 15%), and PIK3CA (1/13, 8%).

AIMS Dedifferentiated chondrosarcoma (DDCHS) is an aggressive type of chondrosarcoma that results from high-grade transformation of a low-grade chondrosarcoma. Mutations in the isocitrate dehydrogenase (IDH) 1 gene and the IDH2 gene that lead to increased d-2-hydroxyglutarate (2HG) oncometabolite production, promoting tumorigenesis, have been recently described in low-grade cartilaginous neoplasms. The aims of this study were to examine the prevalence of IDH mutations in a single-institution cohort of DDCHS cases and correlate 2HG levels with mutation status. METHODS AND RESULTS We examined a series of 21 primary DDCHS cases by using Sanger sequencing and quantitative polymerase chain reaction genotyping to look for IDH1/IDH2 mutations, and evaluated the 2HG levels in formalin-fixed paraffin-embedded tumour and matched normal tissue samples by using a fluorometric assay. Seventy-six per cent of DDCHS cases (16/21) harboured a heterozygous IDH1 or IDH2 mutation. Six of 14 IDH-mutated DDCHS cases showed elevated 2HG levels in tumour tissue relative to matched normal tissue. There were no consistent histological or disease-specific survival differences between IDH-mutated tumours and wild-type tumours. CONCLUSIONS Our study confirms the frequent presence of a variety of IDH1 and IDH2 mutation variants, indicating that a sequencing-based approach is required for DDCHS if IDH is to be used as a diagnostic marker. Similarly to other IDH-mutated tumour types, IDH-mutated DDCHS cases show elevated 2HG levels, indicating that the oncometabolite activity of 2HG may contribute to DDCHS oncogenesis and progression.

We performed phylogenetic analysis of high-grade serous ovarian cancers (68 samples from seven patients), identifying constituent clones and quantifying their relative abundances at multiple intraperitoneal sites. Through whole-genome and single-nucleus sequencing, we identified evolutionary features including mutation loss, convergence of the structural genome and temporal activation of mutational processes that patterned clonal progression. We then determined the precise clonal mixtures comprising each tumor sample. The majority of sites were clonally pure or composed of clones from a single phylogenetic clade. However, each patient contained at least one site composed of polyphyletic clones. Five patients exhibited monoclonal and unidirectional seeding from the ovary to intraperitoneal sites, and two patients demonstrated polyclonal spread and reseeding. Our findings indicate that at least two distinct modes of intraperitoneal spread operate in clonal dissemination and highlight the distribution of migratory potential over clonal populations comprising high-grade serous ovarian cancers.

The urinary CXC chemokine ligand (CXCL)10 detects renal transplant inflammation noninvasively, but has limited sensitivity and specificity. In this study, we performed urinary proteomic analysis to identify novel biomarkers that may improve the diagnostic performance of urinary CXCL10 for detecting alloimmune inflammation in renal transplant patients.In preliminary studies, adult renal transplant patients with normal histology (n = 5), interstitial fibrosis and tubular atrophy (n = 6), subclinical (n = 6) and clinical rejection (n = 6), underwent in-depth urine protein compositional analysis with LC-MS/MS, and matrix metalloproteinase-7 (MMP7) were identified as a potential candidate for the diagnosis of renal allograft inflammation. Urine MMP7 performance was then studied in a larger, prospective adult renal transplant population (n = 148 urines from n = 133 patients) with matched surveillance/indication biopsies. The diagnostic performance of urinary MMP7 and CXCL10 in combination was next evaluated using concordance (C-) statistics, net reclassification improvement and integrated discrimination improvement indices, to determine whether it was better than CXCL10 alone.Urinary MMP7:creatinine (Cr) was lower in normal transplants compared to those with inflammation: glomerulonephritis (P = 0.009), viral nephropathies (P = 0.002), interstitial fibrosis and tubular atrophy and inflammation (P = 0.04), borderline (P = 0.08), subclinical (P = 0.01) and clinical rejection (P = 0.0006), and acute tubular necrosis (P0.0001). Urinary MMP7:Cr and CXCL10:Cr significantly distinguished noninflamed from inflamed biopsies (area under the curve, 0.74 and 0.70, respectively). The addition of urinary MMP7:Cr to CXCL10:Cr improved the diagnostic performance for subclinical and clinical inflammation/injury by integrated discrimination improvement (P = 0.002) and net reclassification improvement (P = 0.006) analyses.Urinary MMP7:Cr improves the overall diagnostic performance of urinary CXCL10:Cr for distinguishing normal histology from subclinical and clinical inflammation/injury, but not subclinical inflammation alone.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Objectives Iron regulation is an important modifier of renal ischemia–reperfusion injury, but the role of iron-binding proteins during cardiopulmonary bypass remains unclear. The goal was to characterize iron-binding proteins throughout ischemia–reperfusion injury to determine their association with acute kidney injury development. Methods A prospective observational cohort of adult patients who underwent cardiac surgery (n = 301) was obtained, and acute kidney injury was defined by Kidney Disease Improving Global Outcomes. Serum ferritin, transferrin saturation, and urine hepcidin-25 were measured. Results Intraoperative serum ferritin was lower at the start of cardiopulmonary bypass (P = .005) and 1-hour cardiopulmonary bypass (P = .001) in patients with acute kidney injury versus patients without acute kidney injury. Lower serum ferritin and higher transferrin saturation at 1-hour cardiopulmonary bypass were independent predictors of acute kidney injury (serum ferritin odds ratio, 0.66; 95% confidence interval [CI], 0.48-0.91; transferrin saturation odds ratio, 1.26; 95% CI, 1.02-1.55) and improved model discrimination (area under the curve [AUC], 0.76; 95% CI, 0.67-0.85) compared with clinical prediction alone (AUC, 0.72; 95% CI, 0.62-0.81; ΔAUC and net reclassification index, P = .01). Lower ferritin, higher transferrin saturation at 1-hour cardiopulmonary bypass, and lower urine hepcidin-25 at postoperative day 1 were also independent predictors for acute kidney injury development, and this model demonstrated an AUC of 0.80 (0.72-0.87), which was superior to clinical prediction (ΔAUC P = .002, integrated discrimination improvement and net reclassification index P = .003). Conclusions Our findings suggest that lower levels of intraoperative iron-binding proteins may reflect an impaired capacity to rapidly handle catalytic iron released during cardiopulmonary bypass, leading to kidney injury. These data highlight the importance of iron homeostasis in human ischemia–reperfusion injury and suggest it is a potentially modifiable risk during cardiac surgery. Intraoperative detection of incipient acute kidney injury may be feasible and could be used as an enrichment strategy for clinical trials.

The current immunosuppressive pipeline in kidney transplantation is limited. In part, this is due to excellent one-year allograft outcomes with the current standard of care (ie, calcineurin inhibitor in combination with anti-proliferative agents). Despite this success, a recent Federal government-sponsored systematic review has identified gaps/limits in the evidence of what constitutes optimal calcineurin inhibitor use in the short- and long-term. Moreover, recent empiric approaches to minimize/withdraw/convert from calcineurin inhibitors have come with the price of increased alloreactivity. As the time horizon to replace calcineurin inhibitors on a global scale may be distant, the transplant community should seize the opportunity to develop ways to personalize calcineurin inhibitor immunosuppression to the individual-transitioning from empiricism to precision. The authors argue in this viewpoint that the path to precision will require measures capable of detecting subclinical alloreactivity to define adequacy of immunosuppression, as well as novel genetic analytics to accurately define alloimmune risk at the individual level-both approaches will require validation in clinical trials.

BackgroundEarly accurate detection of acute kidney injury (AKI) occurring after cardiac surgery may improve morbidity and mortality. Although several novel biomarkers have been developed for the early detection of AKI, their clinical utility in the critical intraoperative and immediate postoperative period remains unclear.Study DesignSystematic review and meta-analysis.Setting & PopulationAdult patients having cardiac surgery.Selection Criteria for StudiesEMBASE, CINAHL, Cochrane Library, Scopus, and PubMed from January 1990 until January 2015 were systematically searched for cohort studies reporting the utility of novel biomarkers for the early diagnosis of AKI after adult cardiac surgery. Reviewers extracted data for study design, population, timing of biomarker measurement and AKI occurrence, biomarker performance (area under the receiver operating characteristic curve [AUROC]), and risk of bias.Index TestsNovel urine, plasma, and serum AKI biomarkers, measured intraoperatively and in the early postoperative period (

Understanding rates and determinants of clinical pathologic progression for recipients with de novo donor-specific antibody (dnDSA), especially subclinical dnDSA, may identify surrogate endpoints and inform clinical trial design. A consecutive cohort of 508 renal transplant recipients (n = 64 with dnDSA) was studied. Recipients (n = 388) without dnDSA or dysfunction had an eGFR decline of -0.65 mL/min/1.73 m(2) /year. In recipients with dnDSA, the rate eGFR decline was significantly increased prior to dnDSA onset (-2.89 vs. -0.65 mL/min/1.73 m(2) /year, p

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Background Urinary CXC chemokine ligand 10 (CXCL10) is a promising biomarker for subclinical tubulointerstitial inflammation, but limited data exist regarding its correlation with (micro)vascular inflammation. Furthermore, no study has evaluated whether concomitant serum CXCL10 improves the discrimination for (micro)vascular inflammation. Methods We investigated whether serum/urinary CXCL10 reflect subclinical inflammation within different renal compartments. Patients (n = 107) with 107 surveillance biopsies were classified as: normal histology (n = 47), normal histology with polyomavirus BK (BKV) or cytomegalovirus (CMV) viremia (n = 17), moderate-severe tubulointerstitial inflammation (tubulitis ≥2, n = 18), pure microvascular inflammation (n = 15), and isolated v lesions (n = 10). Serum and urinary CXCL10 Enzyme-linked Immunosorbent Assay was performed. An independent validation set was evaluated for urine CXCL10: normal histology (n = 14), normal histology with BKV or CMV viremia (n = 19), tubulitis ≥2 (n = 15), pure microvascular inflammation (n = 41), and isolated v lesions (n = 14). Results Elevated urinary CXCL10 reflected inflammation within the tubulointerstitial (urinary CXCL10/creatinine, 1.23 ng/mmol vs 0.46 ng/mmol; P = 0.02; area under the curve, 0.69; P = 0.001) and microvascular compartments (urinary CXCL10/creatinine, 1.72 ng/mmol vs 0.46 ng/mmol; P = 0.03; area under the curve, 0.69; P = 0.02) compared to normal histology. Intriguingly, urinary CXCL10 was predominantly elevated with peritubular capillaritis, but not glomerulitis (P = 0.04). Furthermore, urinary CXCL10 corresponded with BKV, but not CMV viremia (P = 0.02). These urine CXCL10 findings were confirmed in the independent validation set. Finally, serum CXCL10 was elevated with BKV and CMV viremia but was not associated with microvascular or vascular inflammation (P ≥ 0.19). Conclusions Urinary CXCL10 reflects subclinical inflammation within the tubulointerstitial and peritubular capillary spaces, but not the vascular/systemic compartments; this was consistent with BKV (tubulointerstitial) and CMV viremia (systemic). Serum CXCL10 was not a useful marker for (micro)vascular inflammation.

A 60-year-old woman presented to the emergency department with difficulty breathing and a nonproductive cough that worsened when she lay flat. The symptoms progressed from a previous mild respiratory tract illness despite the patient having received an empiric five-day course of azithromycin that

Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis.We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in microdissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase-chain-reaction (PCR) assay for recurrent activating KRAS mutations.Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe-Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P=0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions.We found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions.

Despite more than two decades of use, the optimal maintenance dose of tacrolimus for kidney transplant recipients is unknown. We hypothesized that HLA class II de novo donor-specific antibody (dnDSA) development correlates with tacrolimus trough levels and the recipient’s individualized alloimmune risk determined by HLA-DR/DQ epitope mismatch. A cohort of 596 renal transplant recipients with 50,011 serial tacrolimus trough levels had HLA-DR/DQ eplet mismatch determined using HLAMatchmaker software. We analyzed the frequency of tacrolimus trough levels below a series of thresholds 6 months before dnDSA development in the same patients. Recipients with a high-risk HLA eplet mismatch score were less likely to tolerate low tacrolimus levels without developing dnDSA. We conclude that HLA-DR/DQ eplet mismatch and tacrolimus trough levels are independent predictors of dnDSA development. Recipients with high HLA alloimmune risk should not target tacrolimus levels

Despite modern immunosuppression, renal allograft rejection remains a major contributor to graft loss. Novel biomarkers may help improve posttransplant outcomes through the early detection and treatment of rejection. Our objective is to provide an overview of proteomics, review recent discovery-based rejection studies, and explore innovative approaches in biomarker development.Urine MMP7 was identified as a biomarker of subclinical and clinical rejection using two-dimensional liquid chromatography tandem-mass spectrometry (LC-MS/MS) and improved the overall diagnostic discrimination of urine CXCL10 : Cr alone for renal allograft inflammation. A novel peptide signature to classify stable allografts from acute rejection, chronic allograft injury, and polyoma virus (BKV) nephropathy was identified using isobaric tag for relative and absolute quantitation (TRAQ) and label-free MS, with independent validation by selected reaction monitoring mass spectrometry (SRM-MS). Finally, an in-depth exploration of peripheral blood mononuclear cells identified differential proteoform expression in healthy transplants versus rejection.There is still much in the human proteome that remains to be explored, and further integration of renal, urinary, and exosomal data may offer deeper insight into the pathophysiology of rejection. Functional proteomics may be more biologically relevant than protein/peptide quantity alone, such as assessment of proteoforms or activity-based protein profiling. Discovery-based studies have identified potential biomarker candidates, but external validation studies are required.

Introduction The fact that acute kidney injury (AKI) is associated with worse clinical outcomes forms the basis of most AKI prognostic scoring systems. However, early reversibility of renal dysfunction in acute illness is not considered in such systems. We sought to determine whether early (≤ 24 hours after shock documentation) reversibility of AKI was independently associated with in-hospital mortality in septic shock. Methods Patient information was derived from an international database of septic shock cases from 28 different institutions in Canada, the United States and Saudi Arabia. Data from a final cohort of 5443 patients admitted with septic shock between Jan 1996 and Dec 2009 was analyzed. The following 4 definitions were used in regards to AKI status: (1) reversible AKI = AKI of any RIFLE severity prevalent at shock diagnosis or incident at 6 hours post-diagnosis that reverses by 24 hours, (2) persistent AKI = AKI prevalent at shock diagnosis and persisting during the entire 24 hours post-shock diagnosis, (3) new AKI = AKI incident between 6 and 24 hours post-shock diagnosis, and (4) improved AKI = AKI prevalent at shock diagnosis or incident at 6 hours post followed by improvement of AKI severity across at least one RIFLE category over the first 24 hours. Cox proportional hazards were used to determine the association between AKI status and in-hospital mortality. Results During the first 24 hours, reversible AKI occurred in 13.0%, persistent AKI in 54.9%, new AKI in 11.7%, and no AKI in 22.4%. In adjusted analyses, reversible AKI was associated with improved survival (HR, 0.64; 95% CI, 0.53-0.77) compared to no AKI (referent), persistent AKI (HR, 0.99; 95% CI, 0.88-1.11), and new AKI (HR, 1.41; 95% CI, 1.22-1.62). Improved AKI occurred in 19.1% with improvement across any RIFLE category associated with a significant decrease in mortality (HR, 0.53; 95% CI, 0.45-0.63). More rapid antimicrobial administration, lower Acute Physiology and Chronic Health Evaluation II score, lower age, and a smaller number of failed organs (excluding renal) on the day of shock as well as community-acquired infection were independently associated with reversible AKI. Conclusion In septic shock, reversible AKI within the first 24 hours of admission confers a survival benefit compared to no, new, or persistent AKI. Prognostic AKI classification schemes should consider integration of early AKI reversibility into the scoring system.

Background. The urine C-X-C motif chemokine 10 (CXCL10) is a promising screening biomarker for renal allograft rejection. The aim of the study was to investigate important technical and biological aspects as well as potential confounders when measuring urine CXCL10. Methods. We analyzed 595 urine samples from 117 patients, who participated in a randomized controlled trial investigating the clinical utility of urine CXCL10 monitoring for posttransplant management. Urine CXCL10 was measured by an immunoassay using electrochemiluminescence. Results. Intraassay coefficient of variation was 2.5%, and interassay coefficient of variation was 10%. Urine CXCL10 remained stable (ie

Acute kidney injury (AKI) is associated with pro-longed hospitalization, substantial health care re-source consumption, high mortality, and can lead toprogressive chronic kidney disease (CKD), includingchronic kidney failure, in survivors. The CanadianSociety of Nephrology (CSN) believes there is a needto develop clinical practice guidelines for patientswith AKI; however, efforts to synthesize knowledgefrom clinical studies evaluating the prevention andtreatmentofAKIandtogenerateguidelineshavebeenlimited. One barrier has been the absence of consen-sus on the definition of AKI, with more than 35definitionsofAKIpublishedtodate.Further,thereisaperceivedlackofeffectiveprophylacticandtreatmentstrategies for AKI, and it is challenging to developguidelines that involve multiple stakeholders fromdiversedisciplinesincludingnephrology,criticalcare,and radiology, all of which are important end users ofguidelines forAKI. In this context, the KDIGO (Kid-neyDisease:ImprovingGlobalOutcomes)AKIwork-ing group has recently published new criteria for thedefinition and classification of AKI, as well as aclinicalpracticeguidelineaddressingbothAKIpreven-tion and treatment.

After heart surgery, acute kidney injury (AKI) confers substantial long-term risk of death and chronic kidney disease. We hypothesized that small changes in serum creatinine (SCr) levels measured within a few hours of exit from the operating room could help discriminate those at low versus high risk of AKI.Prospective cohort of 350 elective cardiac surgery patients (valve or coronary artery bypass grafting) recruited in Winnipeg, Canada. Baseline SCr level was obtained at the preoperative visit 2 weeks before surgery. The postoperative SCr level was drawn within 6 hours of completion of surgery and then daily while the patient was in the hospital.Immediate (ie,6 hours) postoperative SCr level change (ΔSCr), categorized as within 10% (reference), decrease10%, or increase10% relative to baseline.AKI, defined according to the new KDIGO (Kidney Disease: Improving Global Outcomes) consensus definition as an increase in SCr level0.3 mg/dL within 48 hours or1.5 times baseline within 1 week.We compared the C statistic of logistic models with and without inclusion of immediate postoperative ΔSCr.After surgery, 176 patients (52%) experienced a decrease10% in SCr level, 26 (7.4%) experienced an increase10%, and 143 had ΔSCr within ±10% of baseline. During hospitalization, 53 (14%) developed AKI. Bypass pump time, baseline estimated glomerular filtration rate, and European System for Cardiac Operative Risk Evaluation (euroSCORE) were associated with AKI in a parsimonious base logistic model. Added to the base model, immediate postoperative ΔSCr was associated strongly with subsequent AKI and significantly improved model discrimination over the base model (C statistic, 0.78 [95% CI, 0.71-0.85] vs 0.69 [95% CI, 0.62-0.77]; P0.001). A ≥10% SCr level decrease predicted significantly lower AKI risk (OR, 0.37; 95% CI, 0.18-0.76), whereas a ≥10% SCr level increase predicted significantly higher (OR, 6.38; 95% CI, 2.37-17.2) AKI risk compared with the reference category.We used a surrogate marker of AKI. External validation of our results is warranted.In elective cardiac surgery patients, measurement of immediate postoperative ΔSCr improves prediction of AKI.

Urinary CXCL10 is a promising noninvasive biomarker for tubulo-interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real-life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2-3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo-interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1-3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10-guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.

BACKGROUND: It is unknown whether favorable long-term data on the safety of living kidney donation can be extrapolated to populations at higher risk of chronic kidney disease. Indigenous people (i.e., Aboriginals) have a high prevalence of risk factors for chronic kidney disease and Aboriginal living donor outcomes need to be defined. METHODS: We performed a retrospective cohort study of all 38 Aboriginal donors donating at our center since 1970 and 76 randomly selected white donor controls to determine the long-term rates of hypertension, diabetes, and renal function postdonation. RESULTS: Follow-up was obtained for 91% of both Aboriginal and white donors (mean follow-up approximately 14 years). Hypertension has been diagnosed more frequently among Aboriginal donors (Ab 42% vs. white 19%, P=0.02). Notably, all 11 Aboriginal donors more than 20 years postdonation have developed hypertension. Diabetes has also been diagnosed more frequently among Aboriginal donors (Ab 19% vs. white 2%, P=0.005), including 5 of 11 (45%) more than 20 years postdonation. Follow-up estimated glomerular filtration rate was higher in Aboriginal donors (Ab 77+/-17 vs. white 67+/-13 mL/min/1.73 m, P=0.002) but not significantly different in adjusted analyses. One Aboriginal donor developed end-stage renal disease 14 years postdonation. CONCLUSIONS: Aboriginal living kidney donors at our center have high rates of hypertension and diabetes on long-term follow-up, although renal function is preserved to date. This profile is similar to that of the general unselected Aboriginal population despite detailed medical evaluation before donation. These findings have important implications for donor counseling and may have implications for other high-risk donor populations.

Intravesical Bacillus Camlette-Guérin (BCG) is the treatment of choice for non-muscle invasive bladder cancer, and has been used successfully for over 40 years. A rare and potentially fatal complication of intravesical BCG therapy is BCG-induced sepsis. We report a rare case in which a patient with end-stage renal disease secondary to chronic granulomatous interstitial nephritis underwent remote, pre-transplant intravesical BCG treatment for high-grade non-invasive papillary bladder carcinoma. The patient subsequently received a deceased donor kidney transplant 5 years after BCG therapy, with thymoglobulin induction therapy and standard triple maintenance immunosuppression. Two years post-transplant, he developed BCG-induced sepsis confirmed by cultures from urine, blood, and left native kidney biopsy. He died from disseminated BCG-induced sepsis and failure of his renal allograft. This case highlights the potential adverse reactions associated with intravesical BCG therapy that may occur years after bladder cancer therapy is completed, and should heighten physician awareness for BCG-related infections during pre-transplant assessment and post-transplant care of solid organ transplants recipients.

Pediatric kidney transplantation is lifesaving, but long-term allograft survival is still limited by injury processes mediated by alloimmune inflammation that may otherwise be clinically silent. Chemokines associated with alloimmune inflammation may offer prognostic value early post-transplant by identifying patients at increased risk of poor graft outcomes. We conducted a single-center prospective cohort study of consecutive pediatric kidney transplant recipients (

Acute kidney injury (AKI) following cardiac surgery leads to increased morbidity and mortality. Characterization and validation of early biomarkers of AKI may ultimately facilitate early therapeutic intervention. We have previously identified that elevated urinary hepcidin-25 is inversely and independently associated with the development of AKI in adult cardiac surgery patients. Hepcidin-25 is an antimicrobial peptide that sequesters iron intracellularly, and its elevation following human ischemia reperfusion injury may represent a renoprotective response to minimize renal injury.Our goal was to validate urinary hepcidin-25 as a non-invasive biomarker in an independent cardiac surgery cohort, within the context of clinical AKI predictors.Prospective observational cohort study.Adult cardiac surgery program at St. Boniface Hospital, Winnipeg, Manitoba, Canada.Adult cardiac surgery patients undergoing cardiopulmonary bypass (CPB), n = 306.Urine hepcidin-25, measured on post-operative day (POD) 1.A prospective, observational cohort of adult CPB patients (n = 306) was collected with serial perioperative urine samples. Urine hepcidin-25 at POD 1 was measured by competitive ELISA. Its diagnostic performance was evaluated in conjunction with clinical parameters and the Thakar clinical prediction score, using multivariate logistic regression.Urinary hepcidin-25 is elevated following cardiac surgery in AKI and non-AKI patients. Elevated urinary hepcidin-25 concentration was inversely associated with AKI on both univariate (odds ratio [OR]: 0.61, 95% confidence interval [CI]: 0.45-0.83,(1) A single-center observational study. (2) Polyclonal antibody-based competitive ELISA.Hepcidin-25 is inversely associated with AKI in a multivariate model when combined with eGFR and diabetes mellitus, with an overall AUC of 0.82. Notably, urinary hepcidin-25 improves on clinical AKI prediction compared to the Thakar score alone.L’insuffisance rénale aiguë (IRA) qui survient à la suite d’une chirurgie cardiaque est associée à une augmentation du taux de morbidité et de mortalité. La caractérisation et la validation de biomarqueurs précoces d’une IRA permettraient éventuellement une intervention thérapeutique plus opportune. Nous avions antérieurement déterminé qu’un taux élevé d’hepcidine-25 est inversement et indépendamment associé au développement d’une IRA à la suite d’une chirurgie cardiaque chez les patients adultes. L’hepcidine-25 est un peptide antimicrobien qui capte le fer intracellulaire. L’élévation de sa concentration à la suite d’une lésion d’ischémie-reperfusion pourrait être une réponse rénoprotectrice permettant de limiter les lésions inflammatoires.Nous avions pour objectif de valider la qualité de biomarqueur non invasif de l’hépcidine-25 urinaire dans une cohorte indépendante de patients subissant une chirurgie cardiaque, dans le contexte des prédicteurs cliniques de l’IRA.Il s’agit d’une étude de cohorte observationnelle prospective.L’étude a été réalisée au Canada, dans le cadre du programme de chirurgie cardiaque du St. Boniface Hospital de Winnipeg, au Manitoba.La cohorte était constituée de 306 patients adultes subissant un pontage cardiopulmonaire.Le taux d’hepcidine-25 urinaire a été mesuré le jour suivant l’intervention.Une série d’échantillons d’urine périopératoires a été collectée chez une cohorte prospective et observationnelle constituée de 306 patients adultes subissant un pontage cardiopulmonaire. Le taux d’hepcidine-25 a été mesuré à l’aide d’un test ELISA compétitif, et sa performance diagnostique évaluée conjointement avec les paramètres cliniques et le score prédictif de Thakar en utilisant une régression logistique multivariée.Le taux d’hepcidine-25 urinaire était élevé en post-opératoire chez tous les patients de la cohorte, qu’ils soient ou non atteints d’insuffisance rénale. Une concentration élevée d’hepcidine-25 urinaire a été inversement associée à la survenue d’une IRA tant selon l’analyse univariée (RC=0,61; IC à 95 % 0,45-0,83; p=0,002) que selon l’analyse multivariée (RC=0,67; IC à 95 % 0,50-0,95; p=0,002). Un modèle combinant certains facteurs de risque cliniques a démontré que le DFG initial, le diabète sucré et la concentration d’hepcidine-25 urinaire présentaient une surface sous la courbe (SSC) de 0,82 (0,75-0,88) pour la prédiction du développement d’une IRA à la suite d’une chirurgie cardiaque. Ce niveau d’exactitude s’est avéré supérieur à la prédiction clinique déterminée par le score de Thakar.Deux principaux facteurs limitent la portée de l’étude : d’abord, le fait qu’il s’agit d’une étude observationnelle menée dans un seul centre, puis, la variabilité inhérente au test ELISA compétitif pour les anticorps polyclonaux.Dans une analyse multivariée, lorsque combinée au DFG et au diabète sucré, l’hepcidine-25 urinaire est inversement associée à la survenue d’une IRA (SSC globale de 0,82). Ainsi, comparativement au score de Thakar utilisé seul, la mesure du taux d’hepcidine-25 urinaire prédit plus exactement le risque d’IRA.

BACKGROUND: Endometriosis affects ~10% of reproductive–aged women and is characterized by extra–uterine biphasic growth of uterine endometrial epithelium and stroma. Endometriosis is widely considered to be a hormonally–dependent inflammatory condition and is classified into three anatomically defined types: deep infiltrating endometriosis that locally invades pelvic organs; ovarian endometriotic cysts, and superficial peritoneal endometriosis. A clonal “precursor–cancer” link to co–occurring clear cell and endometrioid ovarian cancers has been established and somatic mutations occur in these cancer–associated endometriotic cysts, including distant benign–appearing lesions. The spectrum of genetic alteration in non–cancer associated endometriosis is not known. METHODS: We screened a pilot series of 10 deep–infiltrating [pelvic] endometriosis lesions, without co–occurrence or history of cancer, for somatic alterations using laser captured endometrial stroma and epithelium. Two overlapping amplicon panels and digital PCR were used to validate mutations. RESULTS: In four of ten cases we validated the presence of activating codon 12 KRAS mutations. Mutations were restricted to the epithelium and no evidence of any alterations was found in the stromal fraction. CONCLUSIONS: We provide incontrovertible evidence of activating KRAS mutations in 40% of deep infiltrating endometriosis. This frequency of KRAS mutations is substantially higher than what has been observed in clear cell (CCOC) or endometrioid (ENOC) ovarian cancers and the occurrence of non–ovarian primary CCOC/ENOC is largely anecdotal. Our evidence suggests that these mutations represent core features of endometriosis itself, rather than a risk of malignant transformation. Overall we propose this as an opportunity to fundamentally shift the study of endometriosis to a spectrum of neoplasms. This may lead to the generation of a molecularly informed classification system and ultimately to improved prognostication and treatment. Citation Format: Michael S. Anglesio, Tayyebeh M. Nazeran, Hugo M. Horlings, Vivian Lac, Amy Lum, Janine Senz, Julie Ho, Amy Wang, Fontayne Wong, Catherine Allaire, Christina Williams, C. Blake Gilks, Paul J. Yong, and David G. Huntsman. GENOMIC ALTERATIONS IN ENDOMETRIOSIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP09.

BACKGROUND We have demonstrated that 6-month urinary CCL2: Cr is a predictor of interstitial fibrosis and tubular atrophy (IFTA) on 24-month biopsy and death-censored graft loss. However, IFTA is no longer considered prognostically significant, whereas patients with graft loss frequently have interstitial fibrosis and inflammation (IF+i=ci>0+i>0). As early CCL2: Cr predicts late graft loss, the goal of this study was to determine if 6-month urinary CCL2: Cr was a predictor of IF+i at 24 months. METHODS Urinary CCL2 at 6 months was measured with ELISA and correlated with IF+i on 24-month surveillance biopsies from a prospective, multicenter adult renal transplant cohort (n=111). RESULTS Six-month urinary CCL2: Cr was significantly higher in IF+i and transplant glomerulopathy patients compared with normal histology at 24 months. By multivariate analysis, 6-month urinary CCL2: Cr was independently correlated with IF+i at 24 months (OR 2.78, 95% CI 1.38-6.12, AUC 0.695, P=0.003). Six-month urinary CCL2: Cr was also an independent correlate of 6-month IF+i (OR 1.99, 95% CI 1.03-4.18, AUC 0.63, P=0.04). Six-month urinary CCL2: Cr distinguished noninflamed renal tissue (normal, fibrosis) from IF+i with a sensitivity/specificity of 0.71/0.62 at a cutoff of 15 ng CCL2/mmol Cr (AUC 0.695, P=0.003, n=91). CONCLUSIONS Urinary CCL2: Cr may be useful for the noninvasive identification of patients with or at risk for IF+i. These patients may benefit from avoidance of drug minimization/withdrawal protocols and more intensive post-transplant surveillance. Furthermore, urinary CCL2: Cr may also identify individuals who may benefit from novel interventional trials targeting IF+i.

Tissue hypoxia has been demonstrated, in both the renal cortex and medulla, during the acute phase of reperfusion after ischemia induced by occlusion of the aorta upstream from the kidney. However, there are also recent clinical observations indicating relatively well preserved oxygenation in the nonfunctional transplanted kidney. To test whether severe acute kidney injury can occur in the absence of widespread renal tissue hypoxia, we measured cortical and inner medullary tissue Po2 as well as total renal O2 delivery (Do2) and O2 consumption (V̇o2) during the first 2 h of reperfusion after 60 min of occlusion of the renal artery in anesthetized rats. To perform this experiment, we used a new method for measuring kidney Do2 and V̇o2 that relies on implantation of fluorescence optodes in the femoral artery and renal vein. We were unable to detect reductions in renal cortical or inner medullary tissue Po2 during reperfusion after ischemia localized to the kidney. This is likely explained by the observation that V̇o2 (−57%) was reduced by at least as much as Do2 (−45%), due to a large reduction in glomerular filtration (−94%). However, localized tissue hypoxia, as evidence by pimonidazole adduct immunohistochemistry, was detected in kidneys subjected to ischemia and reperfusion, particularly in, but not exclusive to, the outer medulla. Thus, cellular hypoxia, particularly in the outer medulla, may still be present during reperfusion even when reductions in tissue Po2 are not detected in the cortex or inner medulla.

cute kidney injury (AKI) is a serious andpotentially devastating complication inhospitalized patients. In the setting of cardiacsurgery with cardiopulmonary bypass (CPB),acute kidney failure requiring dialysis occurs inapproximately 1%-2% of patients and is associ-ated with a mortality in excess of 60%.

Background Secondary hyperparathyroidism is a common complication of end-stage renal disease (ESRD), which occurs as a result of hyperphosphataemia, hypovitaminosis D and hypocalcaemia. Chronic hyperparathyroidism can result in osteitis fibrosis cystica, also known as brown tumours. Spinal cord compression from a brown tumour is a rare emergency and of the eight reported cases in the literature, this represents the first case while undergoing treatment with sevelamer and cinacalcet. Although cinacalcet may have a role in the metabolic control of hyperparathyroidism, caution needs to be taken in delay or avoidance of parathyroidectomy in severe cases, as it may cause delays in necessary therapy.

A single, recurrent somatic point mutation (402C→G) in FOXL2 has been described in almost all adult-type granulosa cell tumors but not other ovarian neoplasms. Histopathological features of adult-type granulosa cell tumors can be mimicked by a variety of other tumors, making diagnosis of adult-type granulosa cell tumor challenging. It has been suggested that molecular testing for FOXL2 mutation might be a useful tool in the diagnosis of adult-type granulosa cell tumors. The aim of this study was to demonstrate how testing for the FOXL2 mutation can be used in a gynecological pathology consultation service and to establish clear procedural guidelines for FOXL2 testing. Immunohistochemistry for FOXL2 was done using an anti-FOXL2 polyclonal antiserum. If immunohistochemistry was positive, FOXL2 mutation status was subsequently analyzed using a TaqMan assay. A dilution experiment was done to assess the sensitivity and minimum tumor cellularity requirements for our TaqMan assay. Twenty problematic cases were assessed, where the differential diagnosis after the initial investigations included adult-type granulosa cell tumors. Differential diagnoses included: thecoma, Sertoli–Leydig cell tumor, juvenile granulosa cell tumor, endometrial stromal sarcoma and others. In all cases, FOXL2 immunohistochemistry was positive and in six samples the FOXL2 mutation was detected, thus confirming a diagnosis of adult-type granulosa cell tumor. The TaqMan assay was able to reliably detect the FOXL2 mutation with input DNA in the range of 2.5–20 ng, and with a minimum of 25% tumor cell nuclei. The analysis of the FOXL2 mutational status in clinical samples is a useful diagnostic tool in situations where the differential diagnosis is between adult-type granulosa cell tumor and other ovarian tumors. The TaqMan assay requires a minimum of 2.5 ng DNA, with optimal assay performance for 5 to 10 ng DNA input. Laser capture or needle-macrodissection should be undertaken to enrich samples with tumor cell content below 25%.

Pediatric acute kidney injury (AKI) is associated with increased morbidity, mortality and associated healthcare costs. Unfortunately, there are currently no effective therapies available, and this has been attributed in part to the late diagnosis of AKI. Therefore, significant efforts have been made to develop early diagnostic tools for AKI in the hope that early identification of renal injury will allow for effective therapeutic intervention. Different transcriptomic, proteomic and metabolomic technologies offer unbiased approaches to identifying novel biomarkers of AKI. This review will provide an overview of non-invasive pediatric AKI biomarkers. It will focus on unbiased technologies by using examples of biomarkers identified with “-omic” technologies and different methodological and implementation challenges will be highlighted. Finally, emerging proteomic techniques that may be applicable to biomarker discovery will be presented. Ultimately, the development of novel biomarkers of AKI may lead to the early diagnosis and effective therapeutic intervention of AKI to improve patient outcomes.

Acute kidney injury (AKI) complicating cardiopulmonary bypass (CPB) results in increased morbidity and mortality. Urinary hepcidin-25 has been shown to be elevated in patients who do not develop AKI after CPB using semiquantitative mass spectrometry (SELDI TOF-MS). The goals of this study were to quantitatively validate these findings with ELISA and evaluate the diagnostic performance of hepcidin-25 for AKI.A nested, case-control analysis of urinary hepcidin-25 in AKI (n = 22) and non-AKI (n = 22) patients was conducted to validate the SELDI TOF-MS data at the following times: preoperatively; the start of CPB; 1 hour on CPB; on arrival to the intensive care unit; and postoperative days (POD) 1 and 3 to 5. The diagnostic performance of hepcidin-25 was then evaluated in the entire prospective observational cohort (n = 338) at POD 1. AKI was defined as Cr50% from baseline, within 72 hours postoperatively.Urinary hepcidin-25/Cr ratio was significantly elevated in all patients at POD 1 compared with baseline (P0.0005) and was also significantly elevated in non-AKI versus AKI patients at POD 1 (P0.0005). Increased log(10) hepcidin-25/Cr ratio was strongly associated with avoidance of AKI on univariate analysis. On multivariate analysis, the log(10) hepcidin-25/Cr ratio (P0.0001) was associated with avoidance of AKI with an area under the curve of 0.80, sensitivity 0.68, specificity 0.68, and negative predictive value 0.96.Elevated urinary hepcidin-25 on POD 1 is a strong predictor of avoidance of AKI beyond postoperative day 1.

Renal allograft injury secondary to subclinical and clinical tubulitis remains an important cause of allograft fibrosis and loss despite modern immunosuppression. The goal of this study was to validate the previously reported use of urinary CXCL10 (interferon-γ-induced protein of 10 kDa) as a noninvasive marker of tubulitis in an independent clinical cohort.Urine samples (n=102) from 91 patients with protocol or indication biopsies were assayed for urinary CXCL10 using ELISA. The groups analyzed were as follows: normal histology (n=22); interstitial fibrosis and tubular atrophy (IFTA) (n=20); IFTA and borderline tubulitis (n=13); borderline (n=13), subclinical (n=17); and clinical tubulitis (n=17) without IFTA.The ratio of urinary CXCL10 to creatinine (CXCL10: Cr) was found to distinguish borderline, subclinical and clinical tubulitis from normal histology, and IFTA. The area under the curve receiver operating characteristic curve to distinguish normal versus borderline and subclinical tubulitis was 0.845 (OR 1.407, P=0.0184); normal versus borderline, subclinical and clinical tubulitis was 0.835 (OR 1.400, P=0.0127). CXCL10: Cr demonstrated a sensitivity of 73.3% and specificity of 72.7% for normal versus borderline and subclinical tubulitis at a cut-off of 1.97 ng CXCL10/mmol Cr.This study validates urinary CXCL10 as a noninvasive, sensitive, and specific marker for tubulitis in an independent cohort. The straightforward urine processing is accessible to clinical laboratories. We propose that CXCL10 may be useful as a supplementary noninvasive screening test for tubulitis in renal transplant patients, with a level more than 1.97 ng CXCL10/mmol Cr being a threshold to consider biopsy.

Background Chronic renal allograft injury resulting in progressive interstitial fibrosis and tubular atrophy (IFTA) is a leading cause of graft loss. The goal of this study was to identify early urinary predictors for the subsequent development of IFTA in a prospective cohort of patients (n=111) who underwent serial protocol biopsies at 0, 6, and 24 months. Methods The urinary proteins evaluated were CCL2, CXCL9, CXCL10, and alpha1-microglobulin (alpha1M) using ELISA and immunonephelometry. Results We first evaluated urines obtained at 1 to 3 months and found that alpha1M and CXCL10 were associated with IFTA at 6 months but not at 24 months. Next, we evaluated urines at 6 months and found that CCL2 was associated with both IFTA and graft dysfunction at 24 months. On univariate analysis, 6-month urinary CCL2 was a risk factor for developing 24-month IFTA, defined as ci+ct score more than 0 (odds ratio 1.045, 95% confidence interval: 1.005-1.084, P=0.028). Furthermore, CCL2 remained an independent predictor of IFTA on multivariate analysis (odds ratio 1.049, 95% confidence interval: 1.006-1.094, P=0.024) when adjusted for donor age, delayed graft function, deceased donation, and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker exposure. In comparison, alpha1M, CXCL9, and CXCL10 were not associated with late graft outcomes. Conclusion This study demonstrates that early urinary CCL2 is an independent predictor for the subsequent development of IFTA at 24 months.

Anti-glomerular basement membrane (anti-GBM) disease is an aggressive form of glomerulonephritis, usually mediated by immunoglobulin G (IgG) autoantibodies to the noncollagenous (NC1) domain of alpha 3(IV) collagen. Less is known about the target antigen(s) in patients with atypical anti-GBM disease involving IgA autoantibodies. We report a new case of IgA anti-GBM disease in a patient with a history of proliferative lupus nephritis who presented with increasing creatinine levels, proteinuria, and hematuria, but no clinical or serological evidence of lupus recurrence. Renal biopsy showed focal and segmental necrotizing glomerulonephritis with strong linear capillary loop IgA staining by means of immunofluorescence. Serological test results were negative for IgG or IgA autoantibodies against the alpha 3NC1 domain. By means of immunoblotting, IgA from patient serum bound to 38- to 48-kd antigens collagenase-solubilized from human GBM, but not to purified NC1 domains of GBM collagen IV. The target of patient's IgA autoantibodies thus was identified as a novel GBM antigen, distinct from the alpha 3NC1 domain or other known targets of anti-GBM IgA autoantibodies. Clinical resolution was attained by means of conventional treatment with steroids and cyclophosphamide. The diversity of antigens recognized by anti-GBM IgA autoantibodies highlights the importance of renal biopsy for the reliable diagnosis of this rare condition because conventional serological immunoassays likely would yield false-negative results.