The clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)-based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens. (Blood. 2009;114: 4957-4959)

BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on posttranslational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events -mediated through separate signaling processes and involving different target sites -in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERKinducible versus ERK-independent sites.

C3 Glomerulonephritis (C3GN) is a recently described disorder that typically results from abnormalities in the alternative pathway of complement. Here, we describe the clinical features, kidney biopsy findings, alternative pathway abnormalities, glomerular proteomic profile, and follow-up in 12 cases of C3GN. This disorder equally affected all ages, both genders, and typically presented with hematuria and proteinuria. In both the short and long term, renal function remained stable in the majority of patients with native kidney disease. In two patients, C3GN recurred within one year of transplantation and resulted in a decline in allograft function. Kidney biopsy mainly showed a membranoproliferative pattern; although both mesangial proliferative and diffuse endocapillary proliferative glomerulonephritis were noted. Alternative pathway abnormalities were heterogeneous; both acquired and genetic. The most common acquired abnormality was the presence of C3 nephritic factors, while the most common genetic finding was the presence of H402 and V62 alleles of Factor H. In addition to these risk factors, other abnormalities included Factor H auto-antibodies and mutations in CFH, CFI and CFHR genes. Laser dissection and mass spectrometry of glomeruli from patients with C3GN showed accumulation of alternative pathway and terminal complement complex proteins. Thus, C3GN results from diverse abnormalities of the alternative complement pathway leading to subsequent glomerular injury.

SummaryBackground and objectives The kidney is the organ most commonly involved in systemic amyloidosis. This study reports the largest clinicopathologic series of renal amyloidosis.

We describe a kindred with slowly progressive gastrointestinal symptoms and autonomic neuropathy caused by autosomal dominant, hereditary systemic amyloidosis. The amyloid consists of Asp76Asn variant β(2)-microglobulin. Unlike patients with dialysis-related amyloidosis caused by sustained high plasma concentrations of wild-type β(2)-microglobulin, the affected members of this kindred had normal renal function and normal circulating β(2)-microglobulin values. The Asp76Asn β(2)-microglobulin variant was thermodynamically unstable and remarkably fibrillogenic in vitro under physiological conditions. Previous studies of β(2)-microglobulin aggregation have not shown such amyloidogenicity for single-residue substitutions. Comprehensive biophysical characterization of the β(2)-microglobulin variant, including its 1.40-Å, three-dimensional structure, should allow further elucidation of fibrillogenesis and protein misfolding.

TLE1, a transcriptional repressor essential in hematopoiesis, neuronal differentiation and terminal epithelial differentiation, has recently been shown in a single tissue microarray study to be a highly sensitive and relatively specific marker of synovial sarcomas. Expression of TLE1 has not, however, been studied in standard sections of soft tissue and bone tumors. We investigated TLE1 expression in a large series of well-characterized mesenchymal tumors, to more fully characterize the range of TLE1 expression. Standard sections of 163 bone and soft tissue tumors were immunostained for TLE1 (sc-9121, 1:100; Santa Cruz Biochemicals) using the Dako Dual Envision þ detection system. Nuclear positivity was scored as negative (o5% of cell positive), 1 þ (5-25% of cells positive), 2 þ (25-50% of cells positive), and 3 þ (450% of cells positive). Overall, TLE1 was expressed by 18 of 20 (90%) of synovial sarcoma, with 16 cases (89%) showing 2-3 þ positivity. However, TLE1 expression was also seen in 53 of 143 (37%) non-synovial sarcoma, with 36 such cases (25%) showing 2-3 þ positivity. TLE1 expression was commonly seen in peripheral nerve sheath tumors, including 33% of neurofibromas, 100% of schwannomas, and 30% of malignant peripheral nerve sheath tumors. Among non-neoplastic tissues, nuclear TLE1 expression was variably present in basal keratinocytes, adipocytes, perineurial cells, endothelial cells and mesothelial cells. Our study confirms the excellent sensitivity of TLE1 for synovial sarcoma. However, TLE1 expression is by no means specific for synovial sarcoma, being present in a number of tumors, which enter its differential diagnosis, in particular tumors of peripheral nerve sheath origin. Heterogeneity of TLE1 expression likely explains the differences between the present standard section study and the earlier TMA study. TLE1 may be of value in the differential diagnosis of synovial sarcoma, but should be used only in the context of a panel of antibodies. Morphology, ancillary immunohistochemistry for traditional markers such as cytokeratins and CD34, and molecular confirmation of synovial sarcoma-associated fusion genes should remain the 'gold standards' for this diagnosis.

Background C3 Glomerulonephritis (C3GN) is a proliferative glomerulonephritis resulting from glomerular deposition of complement factors due to dysregulation of the alternative pathway of complement. The dysregulation of the alternative pathway of complement may occur as a result of mutations or functional inhibition of complement regulating proteins. Functional inhibition of the complement regulating proteins may result from a monoclonal gammopathy. Study Design Case series Setting and Participants 32 Mayo Clinic patients of C3GN, of which 10 patients (31.25%) had evidence of a monoclonal immunoglobulin in the serum. Outcomes Clinical features, hematological and bone marrow biopsy findings, kidney biopsy findings, renal outcomes, complement pathway abnormalities, treatment and follow-up of patients with C3GN that was associated with a monoclonal gammopathy. Results The mean age of patients with C3GN associated with monoclonal gammopathy was 54.5 years. Bone marrow biopsy done in 9 patients revealed monoclonal gammopathy of undetermined significance (MGUS) in 5 patients, small lymphocytic lymphoma/chronic lymphocytic leukemia (CLL) in one patient ,and no abnormal clones in the remaining 3 patients. Kidney biopsy showed a membranoproliferative glomerulonephritis with bright capillary wall C3 staining in all 10 patients. Evaluation of the alternative pathway of complement showed abnormalities in 7 of the 9 patients tested. None of the patients had a mutation in the complement regulating proteins. As an index case, one patient with C3GN and CLL was treated with Rituximab, cyclophosphamide, vincristine and prednisone and one patient with C3GN and MGUS was treated with dexamethasone and bortezomib. Both patients showed significant decrease in hematuria and proteinuria, and stabilization of renal function. Limitations Studies to show evidence of direct activation of the alternative pathway by the monoclonal immunoglobulin were not done. Conclusions The study highlights the association of C3GN and monoclonal gammopathy, in particular in the older population, and the importance of targeting the underlying hematologic malignancy as an approach to treating C3GN.

Accurate diagnosis and typing of renal amyloidosis is critical for prognosis, genetic counseling, and treatment. Laser microdissection and mass spectrometry are emerging techniques for the analysis and diagnosis of many renal diseases. Here we present the results of laser microdissection and mass spectrometry performed on 127 cases of renal amyloidosis during 2008–2010. We found the following proteins in the amyloid deposits: immunoglobulin light and heavy chains, secondary reactive serum amyloid A protein, leukocyte cell–derived chemotaxin-2, fibrinogen-α chain, transthyretin, apolipoprotein A-I and A-IV, gelsolin, and β-2 microglobulin. Thus, laser microdissection of affected areas within the kidney followed by mass spectrometry provides a direct test of the composition of the deposit and forms a useful ancillary technique for the accurate diagnosis and typing of renal amyloidosis in a single procedure.