Clinical and laboratory data were obtained by review of the medical record. a plasma cell neoplasm in all cases. In 10 (77%) cases, CSH represented more than 50% of the neoplastic infiltrate. By immunohistochemical studies, histiocytes were positive for monotypic kappa in 5 (50%), lambda in 4 (40%) cases; in 1 (10%) case, results were equivocal. Rabbit Polyclonal to MLKL MS analysis of the histiocyte contents in all 3 tested cases showed predominance of variable-region fragments of Ig light and/or heavy chains. == Conclusions == CSH is frequently associated with an underlying lymphoplasmacytic neoplasm. MS findings suggest that Ig alterations and/ Edasalonexent or possibly defects in the ability of histiocytes to process Ig play a role in pathogenesis. Keywords:crystal-storing histiocytosis, mass spectrometry, proteomic analysis, immunoglobulin, lymphoplasmacytic neoplasm == Introduction == Crystal-storing histiocytosis (CSH) is a rare lesion that results from the intra-lysosomal accumulation of immunoglobulins (Ig) as crystals within histiocytes. It can involve a variety of sites, such as bone marrow, lymph nodes, liver, spleen, gastrointestinal tract and kidney.1,2There is a localized form of CSH, which is slightly more frequent, that is confined to a single site. A generalized form that involves multiple organ sites also can occur. Crystal-storing histiocytosis is frequently associated with various types of B-cell lymphomas, plasma cell neoplasms, or rarely inflammatory disorders.1,3-7Often, extensive involvement by CSH can obscure the underlying neoplasm making the recognition of the neoplasm challenging. In the literature, CSH has been reported to show a prominent association with lymphoplasmacytic neoplasms expressing Ig kappa light chain without any association with a specific type of Ig heavy chain.8-11These findings suggest that Ig may have a role in the pathogenesis of CSH. However, extensive studies to analyze the protein content of these crystals have not been performed. In this study, we present the clinicopathologic characteristics of 13 patients with localized or generalized Edasalonexent CSH. We also report the protein contents of the Ig crystals in 3 cases analyzed by mass spectrometry. == Materials and Methods == == Study Group == We retrieved all CSH cases from the pathology files of our institution between 1999 and 2015. In addition, we obtained one case from a collaborating institution. Clinical and laboratory data were obtained by review of the medical record. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of each participating center. The overall collaboration was approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center in Houston, Texas, USA. == Case Review and Immunohistochemical Methods == We reviewed hematoxylin-eosin stained slides prepared from formalin-fixed, paraffin-embedded tissue sections. In cases with Edasalonexent bone marrow involvement, Wright-Giemsastained aspirate smears, touch imprints, and hematoxylin-eosin stained slides of aspirate clot and biopsy specimens were reviewed. Immunohistochemical analysis was Edasalonexent performed using 4-mm-thick formalin-fixed, paraffin-embedded tissue sections using heat-induced antigen retrieval, an avidin biotin-peroxidase complex method and an automated immunostainer (Ventana Biotech, Tucson, AZ, USA). A variable panel of antibodies was used as appropriate for establishing the diagnosis as described elsewhere.12The Edasalonexent antibodies used were reactive with the following antigens: CD3 (1:100) (Lab Vision; Thermo Fisher Scientifics, Fremont, CA); CD20 (1:200), CD68 (KP-1; 1:900;), CD138 (M1-15; 1:600), IgG (1:40000), IgM (1:3000), IgA (1:5000), kappa (1:20,000) and lambda (1:20,000) (Dako North America, Inc., Carpenteria, CA). Detection was performed with biotinylated secondary antibody, horseradish peroxidase and 3,3-diaminobenzidine as a chromogen. Slides were counterstained with haematoxylin. Appropriate negative and positive control slides were run in parallel. == IgH sequencing == DNA was extracted from fixed, paraffin-embedded tissue sections using with the QIAamp DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). PCR amplification of Ig heavy chain variable region (IGVH) genes was performed using consensus primers for variable and conserved regions: 5-TGG(A/G)TCCG(A/C/G)CAG(G/C)C(T/C)CC(A/C/G/T)GG-3 (FRII), 5-TCGGATCCACGGC(T/C)(C/G)TGTATTACTGT-3 (FRIII) and 5-AACTGCAGAGGAGACGGTGACC-3 (conserved region of JH segment). The details of the PCR conditions are described elsewhere.13Amplification of -actin gene was performed to confirm that the DNA quality was adequate. Following gel electrophoresis, the PCR products from the rearranged bands underwent sequence analysis using the fluorescence dye terminator (Sanger) method performed by SeqWright DNA Technology Services (Houston, TX, USA). == Laser Capture Microdissection and Mass Spectrometry == Three cases of CSH with sufficient tissue were assessed by proteomic analysis. Areas to be analyzed were selected under bright-field microscopy from fixed, paraffin-embedded tissue sections using laser capture microdissection (Leica DM6000B Microdissection System, Wetzler, Germany). The tissue was collected into 0.5 ml microcentrifuge tube caps containing 10mM Tris/1mM EDTA/0.002% Zwittergent 316 (Calbiochem, San Diego, CA, USA) as described elsewhere.14Following heating at 98C (x 90 minutes) and sonication in a waterbath (x 60 mts), samples underwent overnight digestion with 1.5 ml of 1 1 mg/ml trypsin (Promega, Madison, WI, USA) at 37C..