Highly differentiated cytotoxic T cells in inclusion body myositis.

Inclusion body myositis is a late onset treatment-refractory autoimmune disease of skeletal muscle associated with a blood autoantibody (anti-cN1A), an HLA autoimmune haplotype, and muscle pathology characterized by cytotoxic CD8+ T cell destruction of myofibres. Here, we report on translational studies of inclusion body myositis patient muscle compared with a diverse set of other muscle disease samples. Using available microarray data on 411 muscle samples from patients with inclusion body myositis (n = 40), other muscle diseases (n = 265), and without neuromuscular disease (normal, n = 106), we identified a signature of T-cell cytotoxicity in inclusion body myositis muscle coupled with a signature of highly differentiated CD8 T-cell effector memory and terminally differentiated effector cells. Further, we examined killer cell lectin-like receptor G1 (KLRG1) as a marker of this population of cells, demonstrated the correlation of KLRG1 gene expression with lymphocyte cytotoxicity across 28 870 human tissue samples, and identified the presence of KLRG1 on pathogenic inclusion body myositis muscle invading T cells and an increase in KLRG1 expressing T cells in inclusion body myositis blood. We examined inclusion body myositis muscle T-cell proliferation by Ki67 immunohistochemistry demonstrating that diseased muscle-invading T cells are minimally or non-proliferative, in accordance with known properties of highly differentiated or terminally differentiated T cells. We found low expression of KLRG1 on infection-protective human lymphoid tissue central memory T cells and autoimmune-protective human blood regulatory T cells. Targeting highly differentiated cytotoxic T cells could be a favourable approach to treatment of inclusion body myositis.

Association of inclusion body myositis with T cell large granular lymphocytic leukaemia.

SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present on follow-up testing in 15 patients a median of 350 days later. T cell aberrant loss of CD5 or gain of expression of CD16 and CD94 were common (19/42, 45%). In comparison, 2/15 (14%) age-matched patients with dermatomyositis, polymyositis, or necrotizing myopathy, and 0/20 (0%) age-matched healthy subjects had large granular lymphocyte expansions, with none of these patients having T cell aberrant expression of CD5, CD16 or CD94. Reduced blood CD4/CD8 ratio, increased blood CD8 count, and lymphocytosis were additional biomarkers highly correlated with flow cytometry-measured large granular lymphocyte expansions. Cross-sectional data suggested more aggressive disease in patients with such expansions than without. Muscle immunohistochemistry demonstrated invasion of large granular lymphocytes into muscle in 15/15 inclusion body myositis patients but in only 1/28 patients with dermatomyositis or polymyositis. The extent of CD8+ and CD57+ cells in inclusion body myositis muscle correlated with the size of blood large granular lymphocyte populations. Myofibre-invading cells expressed CD57, a marker of persistent T cell exposure to antigen and T cell aggressiveness. In many patients with inclusion body myositis, the autoimmune T cell expansion has evolved into a neoplastic-like or overtly neoplastic disorder, perhaps contributing to its relative refractoriness to immune-directed therapies previously reported.

α-Hemoglobin-stabilizing Protein: An Effective Marker for Erythroid Precursors in Bone Marrow Biopsy Specimens.

Accurate analysis of the erythroid lineage is essential in evaluating bone marrow biopsies and can be particularly challenging in settings of dyserythropoiesis. α-Hemoglobin-stabilizing protein (AHSP) is an erythroid-specific chaperone protein and represents a potential specific marker for erythroid elements. This study defines the immunohistochemical profile of AHSP, as compared with an established erythroid marker CD71, in 101 bone marrow biopsies including normal marrows and cases of acute pure erythroid leukemia, acute erythroid/myeloid leukemia, other types of acute myeloid leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, other types of myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, plasma cell neoplasm, and metastatic carcinoma. In acute pure erythroid leukemia, blasts in 7 of 11 cases showed similar reactivity for CD71 and AHSP, whereas less extensive reactivity was observed for AHSP as compared with CD71 in the remaining 4 cases. In normal marrows and other various disorders, reactivity for AHSP was similar to CD71 and was restricted to the erythroid lineage. Mature erythrocytes were negative for AHSP as were myeloblasts, lymphoblasts, nonerythroid hematopoietic marrow elements, plasma cells, and carcinoma cells. AHSP is an effective marker for detection of normal or abnormal erythroid precursors in bone marrow biopsies and is a useful addition to an immunohistochemical panel for assessment of neoplastic cells of possible erythroid derivation.

Profile of CD103 expression in T-cell neoplasms: immunoreactivity is not restricted to enteropathy-associated T-cell lymphoma.

Intestinal intraepithelial T lymphocytes express the α E subunit of integrin αEß7, which is detected by antibodies to CD103. Accordingly, within T-cell neoplasms, CD103 reactivity has most frequently been reported in enteropathy-associated T-cell lymphomas, which are postulated to arise from intestinal intraepithelial T lymphocytes. However, prior studies of CD103 expression in T-cell neoplasms have been limited by the requirement for fresh or frozen tissue, given the historic lack of an antibody to CD103 for use in paraffin-embedded sections. Thus, a thorough assessment of CD103 expression in a broad spectrum of T-cell neoplasms as categorized by the current classification system has not yet been performed. This study uses a newly described antibody to define the profile of CD103 immunoreactivity in paraffin sections of a wide variety of T-cell neoplasms (184 cases). Overall, 22 T-cell neoplasms (12%) were CD103 positive, including 7 of 15 gastrointestinal lymphomas (3.8% of total cases; 46% of gastrointestinal cases). In intestinal cases, CD103 positivity did not correlate with morphology, presence or absence of enteropathy, or immunohistochemical profile. A history of celiac disease was not documented in any case. Frequent but inconsistent reactivity was also noted for adult T-cell leukemia/lymphoma with 4 of 10 cases (40%) positive. In the remaining T-cell neoplasms representing most entities within the current World Health Organization classification, CD103 reactivity was sporadically observed in 11 of 159 cases (6.9%). CD103 positivity is an unusual feature in T-cell neoplasms and tends to occur in gastrointestinal lymphomas and adult T-cell leukemia/lymphoma but is not a consistent characteristic of these neoplasms.

Abnormal distribution of heterogeneous nuclear ribonucleoproteins in sporadic inclusion body myositis.

Previous histopathologic studies of sporadic inclusion body myositis (sIBM) identified sarcoplasmic aggregation and myonuclear depletion of the predominantly nuclear heterogeneous nuclear ribonucleoprotein (hnRNP) TDP-43 in sIBM myofibers. Here, we examined sIBM muscle for abnormalities in two other hnRNPs hnRNPA1 and hnRNPA2B1, mutations in which cause multisystem proteinopathy associated with rimmed-vacuolar myopathies. Muscle biopsy specimens from 13 patients with sIBM and 13 patients without sIBM (dermatomyositis N=3, polymyositis N=3, muscular dystrophy N=3, motor neuron disease N=2, non-neuromuscular disease N=2) underwent immunohistochemistry for hnRNPA1, hnRNPA2B1, and TDP-43. Muscle transcriptional microarray data from 27 patients with sIBM and 12 patients without neuromuscular disease was analyzed. Depletion of hnRNPA1 and hnRNPA2B1 was present in 15% and 7% of sIBM myonuclei, respectively, compared with 1% and 0% of myonuclei in non-sIBM muscle. Sarcoplasmic aggregates of hnRNPA1 and hnRNPA2B1 distinct from TDP-43 aggregates were also found in sIBM. hnRNPA1 and hnRNPA2B1, as well as other hnRNPs, gene expression was unaltered in sIBM compared to normal muscle. Along with TDP-43, other hnRNPs, including hnRNPA1 and hnRNPA2B1, are depleted from sIBM myonuclei at the protein but not transcript level. The depletion of multiple hnRNPs from sIBM myonuclei together with their sarcoplasmic aggregation suggests that one aspect of sIBM pathophysiology may involve abnormal RNA metabolism that includes hyperassembly of ribonucleoprotein granules mediated by prion-like domains in hnRNPs, evolving into pathological aggregates.

Cytosolic 5'-nucleotidase 1A autoimmunity in sporadic inclusion body myositis.

OBJECTIVE: We previously identified a circulating autoantibody against a 43 kDa muscle autoantigen in sporadic inclusion body myositis (IBM) and demonstrated the feasibility of an IBM diagnostic blood test. Here, we sought to identify the molecular target of this IBM autoantibody, understand the relationship between IBM autoimmunity and muscle degeneration, and develop an IBM blood test with high diagnostic accuracy. METHODS: IBM blood samples were screened using mass spectrometry and a synthetic human peptidome. Plasma and serum samples (N=200 patients) underwent immunoblotting assays, and results were correlated to clinical features. Muscle biopsy samples (n=30) were examined by immunohistochemistry and immunoblotting. Exome or whole genome sequencing was performed on DNA from 19 patients. RESULTS: Both mass spectrometry and screening of a 413,611 human peptide library spanning the entire human proteome identified cytosolic 5'-nucleotidase 1A (cN1A; NT5C1A) as the likely 43 kDa IBM autoantigen, which was then confirmed in dot blot and Western blot assays using recombinant cN1A protein. Moderate reactivity of anti-cN1A autoantibodies was 70% sensitive and 92% specific, and high reactivity was 34% sensitive and 98% specific for the diagnosis of IBM. One to 3 major cN1A immunodominant epitopes were identified. cN1A reactivity by immunohistochemistry accumulated in perinuclear regions and rimmed vacuoles in IBM muscle, localizing to areas of myonuclear degeneration. INTERPRETATION: Autoantibodies against cN1A are common in and highly specific to IBM among muscle diseases, and may provide a link between IBM's dual processes of autoimmunity and myodegeneration. Blood diagnostic testing is feasible and should improve early and reliable diagnosis of IBM.

Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS.

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Immunohistochemical detection of hairy cell leukemia in paraffin sections using a highly effective CD103 rabbit monoclonal antibody.

Detection of the integrin subunit CD103 is a useful diagnostic tool in the diagnosis of hairy cell leukemia (HCL). Currently, flow cytometric analysis (FC) and frozen section immunohistochemistry (IHC) represent the only available methods of detection. This study is the first to describe the successful use of a CD103 antibody to identify HCL and HCL-variant in paraffin sections of formalin- or Bouin solution- fixed specimens (n = 68) using an immunoperoxidase technique. In other B-cell lymphoproliferative disorders that morphologically may resemble HCL, including chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 32), mantle cell lymphoma (n = 23), lymphoplasmacytic lymphoma (n = 27), follicular lymphoma (n = 7), and marginal zone lymphoma (n = 13), lymphoid cells are nonreactive for CD103. In HCL, the CD103 staining pattern is predominantly membranous with delineation of delicate cytoplasmic projections. This CD103 antibody is an extremely valuable addition to the IHC panel for the diagnosis of HCL, especially in cases lacking FC analysis.

The immunohistochemical characterization of sarcomatoid malignant mesothelioma of the pleura.

The immunohistochemical characteristics of epithelioid malignant mesothelioma are well described. However, immunohistochemical analyses of sarcomatoid mesothelioma, the less common type, are limited and its distinction from other tumors of the chest wall, lung and pleura is often problematic. We evaluated 24 patients with pleural sarcomatoid mesothelioma who had surgery (12 extrapleural pneumonectomies, 9 pleurectomies and 3 large biopsies) between 1989 and 2005. Clinicopathologic features and demographic data were recorded. We describe immunohistochemical results for 10 antibodies: AE1/AE3, CAM5.2 and MNF-116 keratins, calretinin, WT-1 protein, bcl-2, CD34, desmin, D2-40 and podoplanin. The patients were 23 men and one woman with a median age at diagnosis of 64.7 years (range 47 to 76). Tumor cells were positive for the keratin proteins AE1/AE3 in 18/24 cases, CAM 5.2 in 23/24 cases and MNF-116 in 21/21 cases. Calretinin was positive in 6/24 cases, WT-1 (nuclear) in 8/24 cases, bcl-2 in 0/24 cases, CD34 in 0/24 cases, desmin in 0/24 cases, D2-40 in 24/24 cases and podoplanin in 24/24 cases. This panel of antibodies may be helpful in establishing a pathologic diagnosis of sarcomatoid mesothelioma. In our study, D2-40 and podoplanin are highly sensitive immunohistochemical markers for sarcomatoid mesothelioma. Additional studies are required to define their role in the differential diagnosis of other spindle cell tumors.

Permissive environment for B-cell maturation in myositis muscle in the absence of B-cell follicles.

Myositis muscle contains antigen-matured B-cells and plasma cells. Myositis muscle biopsy specimens were examined for nodular collections of T-cells, B-cells, myeloid dendritic cells, plasma cells, and follicular dendritic cells. Immunoglobulin and B-cell-activating factor (BAFF) transcripts were quantitated. Laser-capture microdissection was used to isolate single plasma cells, and their immunoglobulin transcripts were sequenced. Dense inflammatory infiltrates contained histological elements of ectopic lymphoid tissue but not B-cell follicles. Immunoglobulin transcript sequence analysis demonstrated spatially distributed, clonally related B-cells and plasma cells, suggesting local maturation of B-cells into plasma cells in myositis muscle. Regions of dense cellular infiltrates in myositis muscle are sometimes areas of B-cell maturation into antibody-producing plasma cells. An atypical lymphoid histology, lacking concentrated collections of germinal-center-like B-cell follicles, is capable of antigen-stimulated clonal maturation of antibody-producing plasma cells.

Pathology of the liver in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis is a rare, rapidly progressive disorder characterized by an activation of the immune system resulting in a systemic proliferation of lymphocytes and histiocytes. The disease is genetically heterogeneous and maps to at least 4 loci including the gene encoding perforin, a protein critical for the cytotoxic and regulatory functions of T lymphocytes and natural killer (NK) cells. Hepatic dysfunction often occurs early in the clinical course, but the pathology of the liver is not well characterized. The clinical history, laboratory data, and pathologic material (25 hepatic specimens) from 19 children (11 boys, 7 girls, 1 unknown, 12 d to 11 mo of age, median 3 mo) with FHL were reviewed. Routine and immunohistochemical stains were carried out in all cases, and perforin gene sequencing in a subset. Common to all specimens was a portal and sinusoidal infiltrate of CD3, CD8, granzyme B+ lymphocytes admixed with CD68, CD1a- histiocytes that exhibited hemophagocytosis. There was endothelialitis of portal and central veins and lymphocyte-mediated bile duct injury. The degree of portal and sinusoidal lymphohistiocytic infiltrate and endothelialitis varied from mild to marked and correlated with clinical severity. In some specimens, histiocytic cells predominated and in others, there was extensive hepatocellular giant cell transformation. Accordingly, 4 histopathologic patterns were observed: (1) chronic hepatitis-like, (2) leukemia-like, (3) histiocytic storage disorder-like, and (4) neonatal giant cell hepatitis-like. Two siblings homozygous for a 50delT nucleotide deletion had no perforin immunoreactive cells, 1 compound heterozygote for a deletion and missense mutation had cells with markedly diminished perforin expression, and 1 infant hemizygous for a perforin missense mutation had intact expression. Recognizing the morphologic changes in the liver and the immunophenotypic features of the infiltrate are critical for a rapid diagnosis and a prompt institution of treatment.

Interferon-stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy.

OBJECTIVE: We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. METHODS: We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. RESULTS: Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. INTERPRETATION: A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin.

Utility of fascin and JunB in distinguishing nodular lymphocyte predominant from classical lymphocyte-rich Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and lymphocyte-rich classical Hodgkin lymphoma (LRCHL), although clinically and morphologically similar, differ biologically and in prognosis. Immunolabeling of Reed-Sternberg (RS) cells in LRCHL and lymphocytic and/or histiocytic variants (L&H cells) in NLPHL is often required to help distinguish between the 2 variants. Our aim was to evaluate fascin (a distinct 55-kd actin-bundling protein) and JunB (an activator protein-1 family transcription factor) to differentiate NLPHL from LRCHL. A total of 35 archival cases of NLPHL (n = 24) and LRCHL (n = 11) from adults and children were studied. Slides were reviewed for all cases and clinical, morphologic, and immunohistochemical features were evaluated. Each case was immunostained for fascin and JunB, and immunoreactivity of RS cells, L&H cells, and background lymphocytes were recorded. Whereas occasional L&H cells were weakly positive for fascin in 3 out of 24 (12.5%) cases of NLPHL, RS cells in LRCHL were positive for fascin in 11 out of 11 (100%) cases with a strong cytoplasmic staining pattern. JunB was positive in 10 out of 24 (41.7%) of NLPHL cases, and 11 out of 11 (100%) of LRCHL cases, showing a stippled and/or diffuse nuclear staining pattern. In addition to L & H Cells, JunB also stained small background lymphocytes, particularly in areas of progressively transformed germinal centers of NLPHL. Either stains when tested alone, if negative, or with rare L&H cell weak positivity for fascin, is indicative of NLPHL. The L&H cells of NLPHL cases were negative for concomitant staining in 24 out of 24 (100%) cases. Concomitant positive staining of classic RS cells for fascin and JunB was found in 11 out of 11 (100%) of LRCHL cases. Although fascin positivity alone supports the diagnosis of LRCHL, concomitant positivity offers stronger support and is less likely to lead to a false conclusion if aberrant fascin staining were to be encountered in a case of NLPHL. Staining for fascin and JunB provides a basis for distinguishing NLPHL from LRCHL and offers an alternative to other antibody profiles.

Interferon-gamma-dependent infiltration of human T cells into neuroblastoma tumors in vivo.

PURPOSE: To investigate the impact of interferon-gamma-mediated upregulation of major histocompatibility complex class I expression on tumor-specific T-cell cytotoxicity and T-cell trafficking into neuroblastoma tumors in vivo. EXPERIMENTAL DESIGN: Restoration of major histocompatibility complex class I expression by interferon-gamma treatment enhances killing of neuroblastoma cells. To understand the potential of this approach in vivo, we developed a novel model of neuroblastoma in which NOD/scid/IL2R gamma(null) immunodeficient mice are engrafted with both human T cells and tumor cells. RESULTS: Here, we show enhanced killing of neuroblastoma cells by patient-derived, tumor-specific T cells in vitro. In addition, interferon-gamma treatment in vivo induces efficient upregulation of major histocompatibility complex class I expression on neuroblastoma tumor cells, and this is accompanied by significantly enhanced infiltration of T cells into the tumor. In a pilot clinical trial in patients with high-risk neuroblastoma, we similarly observed augmented T-cell trafficking into neuroblastoma nests in tumor biopsy specimens obtained from patients after 5 days of systemic interferon-gamma therapy. CONCLUSIONS: Interferon-gamma overcomes critical obstacles to the killing of human neuroblastoma cells by specific T cells. Together, these findings provide a rationale for the further testing of interferon-gamma as an approach for improving the efficacy of T cell-based therapies for neuroblastoma and other major histocompatibility complex class I-deficient malignancies. In addition, we describe a model that may expedite the preclinical screening of approaches aimed at augmenting T-cell trafficking into human tumors.

Nature of "Tau" immunoreactivity in normal myonuclei and inclusion body myositis.

Sarcoplasmic accumulation of phosphorylated-tau has been widely stated to occur in and contribute to the pathogenesis of muscle disease in inclusion body myositis. Twenty inflammatory myopathy and 10 normal muscle samples along with a range of other tissues were stained with anti-"tau" antibodies (tau-5, pS422, and SMI-31). Myonuclear and sarcoplasmic fractions were prepared using differential solubilization and laser-capture microdissection, and immunoblots were performed using pS422 and SMI-31 antibodies. All three antibodies demonstrated anti-tau immunoreactivity in myonuclei from normal and diseased muscle, but not in nuclei from other tissues. Western blots showed pS422 and SMI-31 immunoreactivity against nuclear proteins outside the region expected for phosphorylated-tau. Antibodies previously reported to indicate abnormal accumulation of phosphorylated-tau in IBM myofibers react to normal myonuclei and recognize proteins other than tau. Normal myonuclei contain neurofilament H or other unidentified 200 kDa proteins with similar phosphorylated motifs accounting for SMI-31 immunoreactivity.

Sarcoplasmic redistribution of nuclear TDP-43 in inclusion body myositis.

The nucleic acid binding protein TDP-43 was recently identified in normal myonuclei and in the sarcoplasm of inclusion body myositis (IBM) muscle. Here we found TDP-43 sarcoplasmic immunoreactivity in 23% of IBM myofibers, while other reported IBM biomarkers were less frequent, with rimmed vacuoles in 2.8%, fluorescent Congo red material in 0.57%, SMI-31 immunoreactivity in 0.83%, and focal R1282 beta-amyloid immunoreactivity in 0.00% of myofibers. The presence of as little as >1% of myofibers with nonnuclear sarcoplasmic TDP-43 was highly sensitive (91%) and specific (100%) to IBM among 50 inflammatory myopathy patient samples, although some patients with hereditary inclusion body myopathies and myofibrillar myopathy also had sarcoplasmic TDP-43. TDP-43 mutations were sought, and none were identified. TDP-43 could be one of many nucleic acid binding proteins that are abnormally present in IBM sarcoplasm. They could potentially interfere with the normal function of extranuclear RNAs that maintain myofiber protein production.

Fast-twitch sarcomeric and glycolytic enzyme protein loss in inclusion body myositis.

Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.

Histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease): lesional cells exhibit an immature dendritic cell phenotype.

Histiocytic necrotizing lymphadenitis (HNL) is a rare benign disorder characterized histologically by nodal lesions composed of histiocytes, lymphoid cells, and so-called plasmacytoid T cells/plasmacytoid monocytes, with associated karyorrhexis. It has been proposed that plasmacytoid monocytes represent immature myeloid and lymphoid (plasmacytoid) early-committed dendritic cells (DCs). Monoclonal antibodies are now available for the detection of myeloid (CD1c [BDCA-1]+) and plasmacytoid (CD303 [BDCA-2]+) dendritic cells. With an extensive panel of antibodies to immature and mature DCs and interferon-alpha (IFN-alpha), cryostat section studies of 6 cases of HNL revealed that the morphologically distinctive mononuclear cells in lesional areas consisted of 2 populations of immature DCs: myeloid DCs immunoreactive for CD1c with coexpression of myeloid antigens CD13 and CD33 and plasmacytoid DCs immunoreactive for CD303 and CD123. These cells were CD68+, strongly expressed the IFN-alpha inducible protein MxA, and were nonreactive for fascin, a mature DC marker.

Primary cutaneous ALCL with phosphorylated/activated cytoplasmic ALK and novel phenotype: EMA/MUC1+, cutaneous lymphocyte antigen negative.

Primary cutaneous anaplastic large-cell lymphoma (ALCL) ordinarily is distinguished from systemic ALCL by clinical presentation, absence of anaplastic lymphoma kinase (ALK) expression, and immunophenotype (CLA+, EMA/MUC1-). We present an exceptional case of an elderly man with primary cutaneous ALCL and no systemic disease for a 13-year period. Recurrent skin tumors in this patient were characterized by anaplastic, often multinucleated, cells infiltrating the lymphatics and associated with pseudoepitheliomatous hyperplasia. Cutaneous lymphocyte antigen was absent and EMA/MUC1, typical of systemic ALCL, was strongly expressed by the tumor cells. Remarkably, the tumor cells expressed a cytoplasmic-only variant of ALK protein, as reported in 3 previous cases of primary cutaneous ALCL. Fluorescence in situ hybridization revealed lack of rearrangements of the chromosome 2 ALK gene locus usually involved by translocation t(2;5) or other chromosomal rearrangements that generate nucleophosmin-ALK or the variant ALK fusions that occur in systemic ALCL. Nonetheless, the cytoplasmic ALK protein in the patient's tumor cells was shown to be phosphorylated/activated, suggesting a novel mechanism of ALK activation. Primary cutaneous ALCL of this novel subtype should be distinguished from systemic ALCL to ensure proper clinical management.

Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma.

BACKGROUND: To the authors' knowledge, there are no reliable markers able to identify patients with nonsmall cell lung cancer (NSCLC) that will develop metastases to the brain. The authors investigated associations between immunohistochemical markers and the development of brain metastases in patients with NSCLC. METHODS: This was a hospital-based, case-control study of patients who were newly diagnosed with NSCLC between 1989 and 2003, developed brain metastases, and had pathology material available from both the primary NSCLC and the brain metastases. These patients were compared with a control group of patients who had NSCLC and no evidence of brain metastases. NSCLC was examined for expression levels of Ki-67, caspase-3, vascular endothelial growth factor A (VEGF-A), VEGF-C, E-cadherin, and epidermal growth factor receptor (EGFR) in 54 surgical pathology specimens using immunohistochemistry, and associations were evaluated between those markers and the development of brain metastases. RESULTS: Brain metastases developed after a median of 12.5 months (range, 1.7-89.4 months) after the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients with NSCLC who had primary tumors with high Ki-67 levels (adjusted odds ratio [OR] of 12.2; 95% confidence interval [95% CI], 2.4-70.4 [P < .001]), low caspase-3 expression (adjusted OR of 43; 95% CI, 5.3 to >100 [P < .001]), high VEGF-C expression (adjusted OR of 14.6; 95% CI, 2.0 to >100 [P < .001]), and low E-cadherin (adjusted OR of 3.6; 95% CI, 0.9-16.4 [P = .05]). No significant risk was associated with VEGF-A or EGFR expression. High Ki-67 expression also was associated with a shorter overall survival (P = .04). CONCLUSIONS: The results of the current study indicated that patients with NSCLC who had high Ki-67 expression, low caspase-3 expression, high VEGF-C expression, and low E-cadherin expression in their tumors may benefit from close surveillance because they may have an increased risk of developing brain metastases.