Harsharan K. Singh

Polyomavirus nephropathy: morphology, pathophysiology, and clinical management.

Purpose of reviewViral nephropathies, particularly those caused by polyomaviruses of the BK-virus strain, are serious complications following renal transplantation. The review will highlight the morphological, pathophysiological and clinical aspects of BK-virus nephropathy. New patient management strategies are discussed. Recent findingsImmunosuppression with tacrolimus and mycophenolate-mofetil promotes the activation of latent BK-virus in the urinary tract and increases the odds ratio for developing BK-virus nephropathy significantly. A productive infection with BK-viruses shows viral replication in tubular epithelial cells and acute tubular injury. BK-virus nephropathy can be further complicated by concurrent acute rejection episodes contributing to graft demise. Risk assessment after transplantation and patient management during ongoing viral nephropathy have undergone revision by the introduction of real time quantitative polymerase chain reaction techniques measuring BK-virus genome load fluctuations in the serum. Treatment strategies for BK-virus nephropathy include not only low-dose immunosuppression but also drugs with antiviral effects: cidofovir and leflunomide. Transient anti-rejection therapy, including anti-lymphocytic preparations, is a therapeutic option in cases of BK-virus nephropathy and concurrent acute rejection. Recent advances in patient management strategies have resulted in markedly improved graft survival. In cases of graft loss due to BK-virus nephropathy, re-transplantation should be considered. SummaryBK-virus nephropathy is a significant complication following renal transplantation. Recent advances have improved our understanding of the morphological changes, potential risk factors and patient management strategies would be optimized. The availability of quantitative viral load measurements now offers the opportunity for a more accurate and timely clinical intervention.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d‐negative antibody‐mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor‐specific antibody tests (anti‐HLA and non‐HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i‐IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell–mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus‐based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next‐generation clinical trials.

The estrogen receptor-α A908G (K303R) mutation occurs at a low frequency in invasive breast tumors: results from a population-based study

IntroductionEvidence suggests that alterations in estrogen signaling pathways, including estrogen receptor-α (ER-α), occur during breast cancer development. A point mutation in ER-α (nucleotide A908G), producing an amino acid change from lysine to arginine at codon 303 (K303R) results in receptor hypersensitivity to estrogen. This mutation was initially reported in one-third of hyperplastic benign breast lesions, although several recent studies failed to detect it in benign or malignant breast tissues.MethodsWe screened 653 microdissected, newly diagnosed invasive breast tumors from patients in the Carolina Breast Cancer Study, a population-based case-control study of breast cancer in African American and white women in North Carolina, for the presence of the ER-α A908G mutation by using single-strand conformational polymorphism (SSCP) analysis and 33P-cycle sequencing.ResultsWe detected the ER-α A908G mutation in 37 of 653 (5.7%) breast tumors. The absence of this mutation in germline DNA confirmed it to be somatic. Three tumors exhibited only the mutant G base at nucleotide 908 on sequencing, indicating that the wild-type ER-α allele had been lost. The ER-α A908G mutation was found more frequently in higher-grade breast tumors (odds ratio (OR) 2.83; 95% confidence interval (CI) 1.09 to 7.34, grade II compared with grade I), and in mixed lobular/ductal tumors (OR 2.10; 95% CI 0.86 to 5.12) compared with ductal carcinomas, although the latter finding was not statistically significant.ConclusionThis population-based study, the largest so far to screen for the ER-α A908G mutation in breast cancer, confirms the presence of the mutant in invasive breast tumors. The mutation was associated with higher tumor grade and mixed lobular/ductal breast tumor histology.

Fine needle aspiration biopsy of soft tissue sarcomas: utility and diagnostic challenges.

The role of fine needle aspiration biopsy (FNAB) as the primary modality for the initial diagnosis of previously undiagnosed soft tissue sarcomas presents several important challenges. Most practicing pathologists are inexperienced with the wide array of soft tissue neoplasms and their morphologic heterogeneity, making them susceptible to misdiagnosis. However, in the hands of experienced cytopathologists, FNAB in conjunction with ancillary techniques has a diagnostic accuracy approaching 95% for the diagnosis of malignancy. FNAB has been shown to have a diagnostic yield nearly identical with core needle biopsy while avoiding significant clinical complications. Nevertheless, FNAB has certain limitations related to the accurate histologic grading and subtyping of certain subgroups of sarcomas. It may also be difficult to accurately distinguish between low-grade sarcomas and benign or borderline cellular lesions, especially in the spindle cell sarcoma subgroup. The aim of this review is to highlight the utility and limitations of FNAB in the primary diagnosis of soft tissue sarcomas, highlight diagnostically challenging lesions, and comment on the limitations of FNAB in providing a “definitive” diagnosis.

Diagnostic significance of peritubular capillary basement membrane multilaminations in kidney allografts: Old concepts revisited

Background Injury to peritubular capillaries and capillary basement membrane multilamination (PTCL) is a hallmark of antibody-mediated chronic renal allograft rejection. However, the predictive diagnostic value of PTCL is incompletely studied. Methods We analyzed the diagnostic significance of PTCL and propose diagnostic strategies. We evaluated 360 diagnostic native and 187 transplant kidney specimens by electron microscopy (terminology: PTCL-C, severe; PTCL subgroup C3, very severe multilamination; see Materials and Methods for definitions). Results PTCL was not pathognomonic for any specific disease. PTCL-C/C3 was rare in native kidneys (C, 6%; C3, 1%), associated mainly with late thrombotic microangiopathy (C: 78%; C3: 11% of cases). In allografts, PTCL-C/C3 was significantly more common, especially in specimens more than 24 months after transplantation (C, 47%; C3, 31%). PTCL-C/C3 was found in acute (C, 20%; C3, 7%) and chronic T-cell rejection (C, 67%; C3, 29%), calcineurin inhibitor toxicity (C, 36%; C3, 18%), or C4d+ specimens (C, 61%; C3, 50%) with odds ratios between 4 and 36. PTCL-C3 was more predominant in cases with antibody-mediated injury. Highest odds ratios (81–117) for PTCL-C/C3 were noted in combined injuries, that is, mixed chronic T-cell and concurrent chronic antibody–mediated rejection. Positive predictive values of PTCL-C and C3 are the following: all rejection types, 89% and 93%; all Banff chronic rejection types, 69% and 71%; and chronic presumptive antibody rejection, 37% and 49%, respectively. Corresponding negative predictive values of C and C3 for different Banff rejection categories are between 50% and 94%. Conclusions The presence of PTCL-C3 is a helpful adjunct finding to diagnose rejection-induced tissue injury but cannot precisely predict the Banff rejection category. Conversely, the absence of PTCL-C3 is helpful in excluding chronic, Banff category II antibody–mediated rejection.

The oncogenic potential of BK‐polyomavirus is linked to viral integration into the human genome

It has been suggested that BK‐polyomavirus is linked to oncogenesis via high expression levels of large T‐antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK‐polyomavirus, large T‐antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high‐throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK‐polyomavirus strain, named CH‐1, is integrated into exon 26 of the myosin‐binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK‐polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T‐antigen; (b) preclusion of viral replication; and (c) deletions in the non‐coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK‐polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T‐antigen, with persistent over‐expression in non‐lytic cells, promotes cell growth, genetic instability and neoplastic transformation.

The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell–mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i‐IFTA) and its relationship to T cell–mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody‐mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i‐IFTA is associated with reduced graft survival. Furthermore, these groups presented that i‐IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i‐IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor‐specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next‐generation clinical trials.

Polyomaviruses and disease: is there more to know than viremia and viruria?

Purpose of reviewPolyomavirus nephropathy (PVN) mainly caused by BK virus (BKV) remains the most common productive viral infection of the kidney. Over the past decade, clinical interest often focused on BK viremia and viruria as the diagnostic mainstays of patient management. The purpose of this review is to discuss viral nephropathy in the context of BK viremia and viruria and new strategies to optimize diagnostic accuracy and patient management. The emerging roles of polyomaviruses in oncogenesis, salivary gland disease, and post-bone marrow transplantation as well as novel Polyomavirus strains are highlighted. Recent findingsAreas of investigation include proposals by the Banff working group on the classification of PVN and studies on PVN progression and resolution, including the role cellular immune responses may play during reconstitution injury. New noninvasive strategies to optimize the diagnosis of PVN, that is, the urinary ‘polyomavirus-haufen’ test and mRNA expression levels for BKV in the urine, hold great promise to accurately identify patients with viral nephropathy. Tools are now available to separate ‘presumptive’ from ‘definitive’ disease in various patient cohorts including individuals post-bone marrow transplantation. Recent observations also point to a currently underrecognized role of polyomaviruses in oncogenesis post-transplantation and salivary gland disease in patients with HIV-AIDS. SummaryThis review summarizes recent studies on PVN and the significance of the BKV strain in disease. Current paradigms for patient management post-(renal) transplantation are discussed in the setting of new observations. Issues that still require clarification and further validation are highlighted.

BK polyoma virus nephropathy in the native kidney

BACKGROUND While BK polyoma virus nephropathy (PVN) is a well-recognized cause of renal allograft dysfunction, PVN of native kidneys is likely under-recognized. METHODS We present the pathologic features, risk factors and outcomes of eight cases of PVN in native kidneys. RESULTS The cohort included eight males aged 16-73 years (mean 47.4) with an immunocompromised state (mean duration 3.15 years) attributable to: hematologic malignancies (n = 6), for which three had undergone bone marrow transplant; lung transplant (n = 1) and combined tuberculosis and diabetes (n = 1). Seven patients were receiving specific immunosuppressive therapies. Patients were biopsied for acute kidney injury (AKI) with rise in mean creatinine levels from baseline 1.6 to 2.8 mg/dL. Pathology showed BK PVN with characteristic intranuclear inclusions staining positive for SV40 T antigen and negative for JC virus (JCV), with positive serum and/or urine PCR for BK virus. One patient had focal medullary JCV co-infection. Two patients also had renal infiltration by chronic lymphocytic leukemia (CLL). Six patients received specific therapy directed to PVN (cidofovir or leflunomide). Follow-up ranged from 2 to 20 (mean 10) months. Despite marked decrease in serum BK viral copy numbers, creatinine continued to rise in six cases (mean 3.7 mg/dL in four, requiring dialysis in two) and three patients died of malignancy, opportunistic infection or renal failure. Advanced histologic stage of PVN, ineffective antiviral therapy, co-morbidities and persistent immunocompromised state likely contributed to the poor outcomes. CONCLUSION A high level of suspicion in immunocompromised patients is needed to diagnose PVN in an early stage that may respond more favorably to antiviral therapy.

Polyomavirus Nephropathy: Quantitative Urinary Polyomavirus-Haufen Testing Accurately Predicts the Degree of Intrarenal Viral Disease

Background A qualitative highly predictive urinary test for polyomavirus nephropathy (PVN) is the PV-Haufen test. This article evaluates whether a quantitative PV-Haufen analysis, that is, the number of PV-Haufen shed per milliliter urine, predicts PVN disease grades and the severity of intrarenal PV replication. Methods Polyomavirus-Haufen were counted in 40 urine samples from patients with biopsy-proven definitive PVN. The number of PV-Haufen was correlated with both histologic PVN disease grades 1 to 3 and the number of SV40-T–expressing cells as indicators of intrarenal PV replication in corresponding renal allograft biopsies (manual counts and automated morphometry). Findings from quantitative PV-Haufen analyses were compared to conventional laboratory test results, that is, BK viremia (quantitative polymerase chain reaction [PCR]) and BK viruria (quantitative PCR and decoy cell counts). Results Polyomavirus-Haufen counts showed excellent correlation (&agr;0.77–0.86) with the severity of intrarenal PV replication and disease grades. In particular, low PV-Haufen numbers strongly correlated with early PVN grade 1 and minimal intrarenal expression of SV40-T antigen (P < 0.001). In comparison, BK viremia and viruria levels by PCR showed only modest correlations with histologic SV40-T expression (&agr;0.40–0.49) and no significant correlation with disease grades or minimal intrarenal PV replication. No correlations were seen with urinary decoy cell counts. In contrast to conventional quantitative PCR assays or decoy cell counts, quantitative urinary PV-Haufen testing accurately reflects the severity of PV replication, tissue injury, and PVN disease grades. Conclusions Quantitative PV-Haufen testing is a novel noninvasive approach to patient management for the diagnosis and prediction of PVN disease grades and monitoring of disease course during therapy.