Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII.

Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited nodular glomerular involvement. Although prior in vitro studies have shown that Apo-CII can form amyloid fibrils and that certain mutations in this protein promote amyloid fibrillogenesis, there are no reports of this type of amyloidosis in humans. We propose that this study reveals a new form of hereditary amyloidosis (AApoCII) that is derived from the Apo-CII protein and appears to manifest in the elderly and preferentially affect the kidneys.

Clinical, biopsy, and mass spectrometry characteristics of renal apolipoprotein A-IV amyloidosis.

Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis with only a single reported case. Here we describe the clinical, biopsy, and mass spectrometry characteristics of 11 cases of renal AApoAIV amyloidosis encompassing 9 men and 2 women with a mean age at diagnosis of 63.5 years. Progressive chronic kidney disease (mean serum creatinine 2.9 mg/dl) was the most common cause for biopsy with proteinuria absent or minimal in all except one. Hematological and serological evaluation was negative in 9 patients, while 2 had a monoclonal gammopathy. The renal biopsy findings were striking and showed large amounts of eosinophilic Congo-red positive amyloid deposits restricted to the renal medulla with sparing of the renal cortex. In 6 cases, peritubular amyloid was noted in addition to the interstitial involvement. Immunofluorescence studies were negative for immunoglobulins. Electron microscopy showed nonbranching fibrils measuring 7 to 10 nm in diameter. Laser microdissection of the amyloid deposits followed by mass spectrometry showed large spectra number (a semiquantitative measure of abundance) for AApoAIV protein ranging from 49 to 169 (average 85), serum amyloid protein (average 19), and apolipoprotein E (average 48). Importantly, no peptides were detected for any other forms of known amyloidogenic precursor proteins. Thus, renal AApoAIV amyloidosis typically presents with progressive chronic kidney disease and histologically exhibits extensive medullary involvement with sparing of the cortex. The diagnosis is best established by mass spectrometry. Hence, a high degree of suspicion and examination of the renal medulla is required to make the diagnosis.

Young investigator challenge: Validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens.

BACKGROUND: The objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system. METHODS: Thirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin-fixed, paraffin-embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions. RESULTS: Thirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC-31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber-EP4, D2-40, and paired box gene 8 [PAX8]) were not successfully validated. CONCLUSIONS: Nearly one-half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol-fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs.

Neurohistiocytosis of the Cerebellum: A Rare Cause of Ataxia.

Erdheim-Chester disease (ECD) is a non-Langerhans cell histiocytosis that affects multiple body organs, notably the skeletal system. We examined a 58-year-old man who presented with ataxia and T2 hyperintensity of the middle cerebellar peduncles and dentate nuclei without contrast enhancement on MRI brain. Workup for malignancy revealed "hairy kidneys" on CT scan of the abdomen, and excisional biopsy of the retroperitoneal mass for concerns of lymphoma revealed foamy histiocytes that tested positive for CD68 and negative for CD1a, confirming the diagnosis of ECD. Further genetic testing on excised tissue revealed BRAF (V600E) gene mutation that is present in 50% of ECD patients. Treatment was initiated with targeted therapy using the BRAF inhibitor Dabrafenib. X-ray of the lower extremities did not reveal sclerosis of the long bones, and bone scan with technetium 99 was negative except for a nonspecific tracer uptake in left calvarial bone with no corresponding CT changes or T1/T2 signal changes on MRI. His MRI brain revealed classic cerebellar involvement in ECD without other central nervous system (CNS) involvement. It has been postulated that bone involvement is almost universal in ECD; however, our patient with ECD had ataxia and cerebellar involvement without significant bone involvement, as evidenced by bone scan. This is a rare presentation of ECD affecting the CNS and sparing the skeletal system. It confirms the wide spectrum of presentation this multisystem disease can have.

Gastric amyloidosis: clinicopathological correlations in 79 cases from a single institution.

Gastrointestinal involvement by amyloidosis is common, but large clinicopathological studies specifically addressing gastric amyloidosis are lacking. Seventy-nine patients with biopsy-proven, gastric amyloidosis were identified by a retrospective review of our pathology archives, from 2007 to 2013. Amyloid typing was performed by laser microdissection/mass spectrometry (in 44 patients), immunohistochemistry, immunofluorescence, and/or genetic testing. The median age at diagnosis was 62years, with 61% being males. The amyloid was derived from immunoglobulin light chain (67%), transthyretin (ATTR) (18%), serum amyloid A (9%), and apolipoprotein A1 (3%). When other gastrointestinal sites were biopsied, amyloid was demonstrated in the small bowel (89%), colon (81%), and esophagus (33%). The most common gastrointestinal manifestations were weight loss (37%), abdominal pain/dyspepsia (23%), and nausea/vomiting (23%). Endoscopic findings included normal (35%), erythema (33%), erosions (18%), and nodularity (15%) and were not related to amyloid type. No case showed gastric lymphoma. The most common location of amyloid was the muscularis mucosae regardless of the type of amyloid. Lamina propria involvement was less frequent in ATTR than other types. In 22% of patients, the first diagnosis of amyloid was based on the gastric biopsy. Patients' survival at 3years was 60% and was not different by type of amyloid. Our study shows that light-chain amyloidosis is the most common form of gastric amyloidosis, followed by ATTR. Type of amyloid cannot be predicted based on clinical or endoscopic findings, and therefore, biopsy with amyloid typing, preferably by laser microdissection/mass spectrometry, is critical to establish the correct diagnosis, prognosis, and appropriate treatment.

ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes.

Anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a CD30-positive T-cell non-Hodgkin lymphoma that morphologically resembles ALK-positive ALCL but lacks chromosomal rearrangements of the ALK gene. The genetic and clinical heterogeneity of ALK-negative ALCL has not been delineated. We performed immunohistochemistry and fluorescence in situ hybridization on 73 ALK-negative ALCLs and 32 ALK-positive ALCLs and evaluated the associations among pathology, genetics, and clinical outcome. Chromosomal rearrangements of DUSP22 and TP63 were identified in 30% and 8% of ALK-negative ALCLs, respectively. These rearrangements were mutually exclusive and were absent in ALK-positive ALCLs. Five-year overall survival rates were 85% for ALK-positive ALCLs, 90% for DUSP22-rearranged ALCLs, 17% for TP63-rearranged ALCLs, and 42% for cases lacking all 3 genetic markers (P < .0001). Hazard ratios for death in these 4 groups after adjusting for International Prognostic Index and age were 1.0 (reference group), 0.58, 8.63, and 4.16, respectively (P = 7.10 × 10(-5)). These results were similar when restricted to patients receiving anthracycline-based chemotherapy, as well as to patients not receiving stem cell transplantation. Thus, ALK-negative ALCL is a genetically heterogeneous disease with widely disparate outcomes following standard therapy. DUSP22 and TP63 rearrangements may serve as predictive biomarkers to help guide patient management.

Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.

Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method for the diagnosis of systemic amyloidosis. Here we describe the development and implementation of a clinical assay using mass spectrometry-based proteomics to type amyloidosis in subcutaneous fat aspirates. First, we validated the assay comparing amyloid-positive (n=43) and -negative (n=26) subcutaneous fat aspirates. The assay classified amyloidosis with 88% sensitivity and 96% specificity. We then implemented the assay as a clinical test, and analyzed 366 amyloid-positive subcutaneous fat aspirates in a 4-year period as part of routine clinical care. The assay had a sensitivity of 90%, and diverse amyloid types, including immunoglobulin light chain (74%), transthyretin (13%), serum amyloid A (%1), gelsolin (1%), and lysozyme (1%), were identified. Using bioinformatics, we identified a universal amyloid proteome signature, which has high sensitivity and specificity for amyloidosis similar to that of Congo red staining. We curated proteome databases which included variant proteins associated with systemic amyloidosis, and identified clonotypic immunoglobulin variable gene usage in immunoglobulin light chain amyloidosis, and the variant peptides in hereditary transthyretin amyloidosis. In conclusion, mass spectrometry-based proteomic analysis of subcutaneous fat aspirates offers a powerful tool for the diagnosis and typing of systemic amyloidosis. The assay reveals the underlying pathogenesis by identifying variable gene usage in immunoglobulin light chains and the variant peptides in hereditary amyloidosis.

Leukocyte cell-derived chemotaxin 2 (LECT2)-associated amyloidosis is a frequent cause of hepatic amyloidosis in the United States.

Using laser microdissection and mass spectrometry (MS)-based proteomics, we subtyped amyloid deposits from 130 cases of hepatic amyloidosis. Although we confirmed that immunoglobulin light chain amyloidosis was the most frequent cause of hepatic amyloidosis, leukocyte cell-derived chemotaxin 2 (LECT2) amyloidosis (ALect2) accounted for 25% of cases. This novel finding was associated with Hispanic ancestry, incidental discovery of amyloid in liver specimens sampled for other unrelated conditions, and a characteristic pattern of hepatic amyloid deposition. Although ALect2 patients had a common LECT2 polymorphism, pathogenic mutations were not discovered, suggesting that constitutive or compensatory LECT2 overexpression led to ALect2 deposition. These findings indicate that ALect2 is a common cause of hepatic amyloidosis in the population of the United States, and subtyping hepatic amyloid deposits by an accurate analytic method such as MS is required for optimal clinical management of hepatic amyloidosis patients and to avoid incorrect and unnecessarily toxic therapies.

Islet-1 is a sensitive but not entirely specific marker for pancreatic neuroendocrine neoplasms and their metastases.

Islet-1 (Isl1) is a transcription factor involved in the embryogenesis of islets of Langerhans. Immunohistochemically, Isl1 is a sensitive lineage-specific marker for pancreatic neuroendocrine neoplasms (NENs) and their metastases. Its specificity has not been documented, nor have large numbers of NENs from other parts of the gut or other organs been studied. We examined Isl1 expression in 203 primary NENs (gastroenteropancreatic, lung, breast, and ovarian neoplasms) and 40 hepatic NEN metastases (enteropancreatic and lung neoplasms) from known primaries. The correlation between Isl1 and CDX2 expression was studied using a tissue microarray containing 46 pancreatic NENs. Immunostaining for Isl1 and CDX2 was also performed in selected NENs from other sites. Isl1 was positive in 90% of pancreatic, 89% of duodenal, 100% of rectal, 38% of colonic, 14% of appendiceal, and 6% of ileal primaries. Isl1 was negative in all other NENs. Among metastatic neoplasms, 76% of pancreatic and 2 of 2 rectal NEN metastases were Isl1 positive, whereas all other tested metastases were negative. The overall sensitivity and specificity of Isl1 in identifying primary pancreatic NENs was 88% and 80%, respectively. Thirty-six of 46 pancreatic NENs in the tissue microarray were Isl1 positive; 4 were Isl1 negative but CDX2 positive. Our findings confirm that Isl1 is a sensitive marker of pancreatic origin in cases of metastatic NEN. However, Isl1 does not distinguish pancreatic NEN from duodenal and colorectal NEN, even when used in association with CDX2.

Nodular pulmonary amyloidosis is characterized by localized immunoglobulin deposition and is frequently associated with an indolent B-cell lymphoproliferative disorder.

Nodular pulmonary amyloidosis, a rare localized form of amyloidosis, has been associated with immunoglobulin light chains (AL type) and variably with low-grade lymphoma. The clinicopathologic features of 18 cases were investigated; 5 of 14 had autoimmune disease. In 14 cases monotypic plasma cells could be demonstrated by immunohistochemistry. By mass spectrometry analysis of the amyloid deposits, all 18 cases showed a peptide profile with an abundance of immunoglobulin light chains (12 κ, 4 λ, and 2 mixed κ and λ), with 13 also showing significant codeposition of heavy chains (10 γ, 2 α, 1 δ). Of 14 patients with follow-up, 3 developed recurrent pulmonary amyloidoma, 2 had pulmonary recurrence plus cutaneous extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type with and without amyloid, and 1 had a history of parotid gland MALT lymphoma. This study highlights the unique features of this localized form of amyloidosis. AL κ type is more frequent than AL λ type, with a ratio of 3:1, in contrast to the AL λ predominance that characterizes systemic AL amyloidosis. In addition, the majority of nodular pulmonary amyloid is of mixed AL/AH type, a rare finding in systemic amyloidosis. The association of nodular pulmonary amyloidoma with autoimmune disease and lymphoma indicate the majority of these lesions relate to an underlying lymphoplasmacytic neoplasm in the spectrum of MALT lymphoma.

Clinicopathologic features of B-Cell lineage neoplasms with aberrant expression of CD3: a study of 21 cases.

CD3 expression by immunohistochemistry was historically considered restricted to T-lineage or NK-lineage neoplasms but recently has been reported in rare cases of mature B-cell neoplasms, frequently in association with Epstein-Barr virus. Here, we describe the pathologic features of 21 B-cell lineage neoplasms that express CD3 protein by immunohistochemistry: 12 diffuse large B-cell lymphomas (DLBCLs); 2 plasmablastic lymphomas (PBLs); 4 plasma cell neoplasms; 2 Burkitt lymphomas; and 1 nodal follicular lymphoma, grade 3A. CD20 expression was negative or only partially positive in 13/21 cases. Epstein-Barr virus was positive in 3/20 tested cases (2 PBLs and 1 DLBCL). All tested neoplasms (14/14) had clonal immunoglobulin gene rearrangements, and no clonal T-cell gene rearrangements were detected (0/14). The 12 DLBCLs segregated into 2 main groups: 7 demonstrated features of plasmacytic differentiation but did not meet criteria for PBL, and 5 had anaplastic features. In addition to morphology, other features shared among the DLBCLs with plasmacytic differentiation, the plasma cell neoplasms, and the PBLs included extranodal presentation, cytoplasmic localization of CD3, and lack of expression of other T-cell antigens in most cases. In contrast, DLBCLs with anaplastic features and the single follicular lymphoma coexpressed multiple T-cell antigens in a predominantly membranous pattern and presented with nodal disease in a relatively younger patient population. Our data expand the spectrum of morphologic, phenotypic, and clinical features of B-cell neoplasms aberrantly expressing CD3. As these neoplasms often lack typical expression of B-cell antigens, knowledge of these features will help avoid misclassification.

Clusterin is expressed in normal synoviocytes and in tenosynovial giant cell tumors of localized and diffuse types: diagnostic and histogenetic implications.

Tenosynovial giant cell tumors arise from synovium of joints, bursae, or tendon sheaths, and are classified into localized and diffuse types based on the growth pattern and clinical behavior. The mononuclear component of these tumors includes small histiocytoid cells and large mononuclear cells, which are positive for desmin in about 50% of cases. This study seeks to further characterize the immunophenotype of these tumors, and investigates the utility of clusterin as a diagnostic marker. Immunostaining for clusterin was performed on 40 cases of tenosynovial giant cell tumor (11 localized and 29 diffuse). Most cases were also stained for desmin, CD163, CD21, and CD35. Four cases were stained for podoplanin/D2-40 and CXCL13. Clusterin staining was diffuse and strong in the large mononuclear cells in all cases. Desmin positivity in the large cells was identified in 24 out of 34 cases (71%), but was seen in only a subset of cells (<5% to 80%), with 19 out of 24 cases (79%) showing positivity in 10% or less. The large cells were positive for podoplanin in 4 out of 4 cases, but negative for CD163, CD21, CD35, and CXCL13. The smaller histiocytoid cells were positive for CD163 and negative for all other markers. When present, non-neoplastic synoviocytes were positive for clusterin and podoplanin, and focally positive for desmin. Clusterin is a highly sensitive marker for tenosynovial giant cell tumors, which has diagnostic utility in challenging cases. The observed staining patterns provide evidence linking the large mononuclear cells with normal synoviocytes and support that tenosynovial giant cell tumors are neoplasms showing synovial differentiation.

Incidence of TCR and TCL1 gene translocations and isochromosome 7q in peripheral T-cell lymphomas using fluorescence in situ hybridization.

Translocations involving the T-cell receptor (TCR) and TCL1 genes occur in T-cell precursor lymphoblastic leukemia/lymphoma and prolymphocytic leukemia; isochromosome 7q has been associated with hepatosplenic T-cell lymphoma. However, the incidence of these abnormalities in peripheral T-cell lymphomas (PTCLs) as a whole has not been well defined. We studied genetic abnormalities in 124 PTCLs seen at the Mayo Clinic, Rochester, MN, between 1987 and 2007. Tissue microarrays were screened using 2-color break-apart fluorescence in situ hybridization probes flanking the TCRalpha (TCRA, 14q11), TCRbeta (TCRB, 7q35), and TCRgamma (TCRG, 7p15) genes and the TCL1 gene (14q32). Isochromosome 7q was analyzed by using a 2-color probe to 7p and 7q32.1. Translocations involved TCRA in 3 (2.9%) of 102 cases and TCRB in 1 (1%) of 88. Isochromosome 7q was detected in 2 cases of extranodal NK/T-cell lymphoma, nasal type, and 2 cases of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. One of the latter cases also had a translocation of TCRA, and further studies confirmed a novel t(5;14) translocation.

Primary cutaneous CD4-positive small/medium-sized pleomorphic T-cell lymphoma: a clonal T-cell lymphoproliferative disorder with indolent behavior.

Primary cutaneous CD4-positive small/medium-sized pleomorphic T-cell lymphoma, a provisional entity in the 2005 WHO-EORTC classification for cutaneous lymphomas, is not well characterized. Fifteen cases meeting the definition of this entity were identified. Fourteen represented solitary lesions on the head/neck (n=9), upper extremity (n=4), or trunk (n=1). One patient presented with multiple lesions on the trunk and extremities. Histologically, the infiltrate showed a nodular pattern in the dermis and subcutis without epidermotropism, and had a polymorphous composition with a predominance of small to medium-sized CD4-positive T cells. Most cases showed normal T-cell antigen expression; diminished/absent expression of CD7 was seen in three cases and CD2 expression was absent in one case. All cases showed a notable reactive infiltrate including frequent B cells, plasma cells, and histiocytes. Clonal TCR gene rearrangements were detected in each case. No clonal Ig gene rearrangements were detected. Out of the 11 patients with follow-up, none showed systemic disease. The majority resolved without relapse, one without treatment, four with excision, and four with radiation therapy. One patient developed local recurrence. The patient with multiple lesions had disease progression despite chemotherapy and stem cell transplant. These cases highlight the polymorphous histology and prominent reactive B-cell component of this entity. Diagnosis requires molecular genetic analysis, as prominent cytologic atypia and immunophenotypic aberrancy are rare. The differential diagnosis includes reactive lymphoid hyperplasia, mycosis fungoides and cutaneous B-cell lymphomas. In patients with isolated cutaneous lesions, the indolent behavior of this rare T-cell neoplasm should be recognized to avoid unnecessary treatment.

CD33 detection by immunohistochemistry in paraffin-embedded tissues: a new antibody shows excellent specificity and sensitivity for cells of myelomonocytic lineage.

A new monoclonal antibody to CD33 that reacts in paraffin-embedded tissue samples was evaluated. The expected reactivity in granulocytic and monocytic cells was found in a tissue microarray composed of multiple tissue sites. There was no unexpected reactivity found in a wide variety of hematolymphoid and nonhematolymphoid disorders. In cases of acute leukemia, the CD33 antibody showed equivalent results by immunohistochemical analysis compared with flow cytometric analysis. The CD33 antibody was also found to be a useful marker in the workup of myeloid sarcomas. This anti-CD33 antibody will be a useful marker in the workup of acute leukemias and myeloid sarcomas on paraffin-embedded tissue samples.

Spectrum of bone marrow findings in patients with angioimmunoblastic T-cell lymphoma.

Bone marrow (BM) biopsy is often performed early in the evaluation of patients with angioimmunoblastic T-cell lymphoma (AITL), and may be the first diagnostic tissue sample; yet the BM histopathology associated with this disease has not been well described. In this study, BM specimens from 13 patients with AITL were reviewed. Seven (54%) were involved by AITL, which was characterised by paratrabecular and interstitial polymorphous infiltrates containing cytologically atypical lymphocytes, histiocytes and eosinophils. The neoplastic cells were positive for CD10 and CXCL13 by immunohistochemistry in a subset of cases. As in lymph nodes, the lymphomatous infiltrate in some BMs contained numerous small or scattered large B cells, resembling either benign lymphoid aggregates or T cell rich large B cell lymphoma, respectively. Secondary haematological changes were frequent and presented independent of BM involvement by AITL; these included trilineage haematopoietic hyperplasia and plasmacytosis. When BM biopsy preceded the diagnosis of AITL, these secondary changes were misinterpreted as chronic myeloproliferative disease (n = 2), or plasma cell dyscrasia (n = 2). In two cases, these changes obscured the presence of BM involvement by AITL. The spectrum of BM findings in AITL patients is important to recognise for early and accurate diagnosis in this disease.

Utility of ALK-1 protein expression and ALK rearrangements in distinguishing inflammatory myofibroblastic tumor from malignant spindle cell lesions of the urinary bladder.

Inflammatory myofibroblastic tumor of the urinary bladder is an unusual spindle cell neoplasm that displays cytologic atypia, infiltrative growth and mitotic activity mimicking malignant tumors, such as leiomyosarcoma, rhabdomyosarcoma and sarcomatoid carcinoma. The objective of this study was to determine if anaplastic lymphoma kinase (ALK-1) protein expression detected by immunohistochemistry and ALK rearrangements detected by fluorescence in situ hybridization (FISH) were useful in distinguishing inflammatory myofibroblastic tumor from malignant spindle cell tumors of the urinary bladder. In inflammatory myofibroblastic tumor, ALK-1 expression was identified in 13 of 21 cases (62%) and ALK rearrangements in 14 of 21 cases (67%). All cases of inflammatory myofibroblastic tumor demonstrating ALK-1 expression, carried ALK rearrangements. One case negative for ALK-1 expression exhibited ALK rearrangement. ALK rearrangements were more common in women (P=0.0032). Leiomyosarcoma, sarcomatoid carcinoma, embryonal rhabdomyosarcoma and reactive myofibroblastic proliferations were negative for ALK-1 protein and ALK rearrangements. Immunohistochemistry using markers of muscle, epithelial, neural, and follicular dendritic cell differentiation showed overlap between inflammatory myofibroblastic tumor with and without ALK gene rearrangements, and between inflammatory myofibroblastic tumor and spindle cell malignancies. However, coexpression of cytokeratin and muscle-specific antigens was unique to inflammatory myofibroblastic tumor, observed in approximately half the tumors. This study indicates that detection of ALK protein and ALK gene rearrangements are useful in distinguishing inflammatory myofibroblastic tumor from spindle cell malignancies in the urinary bladder. Additionally, our findings suggest that ALK rearrangement is the primary mechanism for ALK activation and that inflammatory myofibroblastic tumor likely represents a heterogeneous group of spindle cell proliferations with the majority associated with ALK translocations, and the remaining associated with other etiologies.