Atsuji Matsuyama

Aberrant PLAG1 expression in pleomorphic adenomas of the salivary gland: a molecular genetic and immunohistochemical study

The morphologic distinction of pleomorphic adenoma from other benign or low-grade salivary gland tumors is sometimes difficult and problematic because of their potentially overlapping histological patterns. A subset of pleomorphic adenoma harbors specific gene alterations involving PLAG1 or HMGA2, and the detection of these fusion genes and their products using formalin-fixed, paraffin-embedded (FFPE) tumor specimens may be a useful diagnostic adjunct. In the present study, gene fusions involving PLAG1 or HMGA2 were examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis, with FFPE tumor tissues and immunohistochemical expression of PLAG1 in 45 pleomorphic adenomas, using a commercially available antibody. RT-PCR analyses identified the CTNNB1-PLAG1, LIFR-PLAG1, CHCHD7-PLAG1, and HMGA2-WIF1 fusion transcripts in eight, two, one, and one case, respectively. The TCEA1-PLAG1, HMGA2-FHIT, and HMGA2-NFIB fusion transcripts were not detected. Immunohistochemically, tumor cells in all 45 pleomorphic adenomas were positive for PLAG1, irrespective of PLAG1 rearrangements, even in the case with the HMGA2-WIF1 fusion transcript. Tumor cells displaying myoepithelial or cartilaginous differentiation were almost constantly positive for PLAG1, whereas a limited expression was observed in glandular or keratinizing cells. Among the 46 tumors other than pleomorphic adenoma, 4 carcinomatous components of carcinomas ex pleomorphic adenoma were positive for PLAG1, the other 39 were negative for PLAG1, and the remaining 3 were only faintly and/or focally stained, indicating that the immunohistochemical detection of PLAG1 is diagnostically useful. The present results also suggest that overexpression of PLAG1 is essential for the tumorigenesis of pleomorphic adenomas, although the mechanisms mediating PLAG1 overexpression seem to be variable.

Association of microRNA-21 expression with its targets, PDCD4 and TIMP3, in pancreatic ductal adenocarcinoma.

Since the discovery of small non-coding RNAs, the analyses of microRNA (miRNA) expression patterns in human cancer have provided new insights into cancer biology. miRNA-21 has been suggested to be one of the miRNAs that have an important role in the development or biological behavior of a variety of malignancies, including pancreatic cancer. This study was conducted to evaluate the relationship between the expression of miRNA-21 and that of its molecular targets, programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinase (TIMP3), in pancreatic ductal adenocarcinoma. The study included 65 pancreatic ductal adenocarcinomas and 5 normal pancreatic tissue specimens for comparison. The miRNA expression profiling of five selected pancreatic ductal adenocarcinomas and five normal pancreatic specimens was performed using a microarray platform, and was evaluated by a hierarchical clustering analysis. The miRNA most highly expressed in pancreatic ductal adenocarcinomas (ie, miRNA-21) was further assessed by quantitative real-time reverse transcription PCR (RT-PCR) assays in the 65 pancreatic ductal adenocarcinoma cases. The expression pattern of its molecular targets (eg, PDCD4 and TIMP3) in pancreatic ductal adenocarcinoma was examined immunohistochemically. In the microarray analyses, 28 miRNAs were upregulated in pancreatic ductal adenocarcinoma compared with normal pancreatic tissue, whereas 48 miRNAs were downregulated. miRNA-21 was the most significantly overexpressed miRNA in the pancreatic ductal adenocarcinomas analyzed, and was also highly expressed in 75% of the 65 pancreatic ductal adenocarcinomas examined by real-time RT-PCR. High miRNA-21 expression was correlated with a worse prognosis in the pancreatic ductal adenocarcinoma patients (P=0.045). The immunohistochemical expression patterns of PDCD4 (reduced nuclear staining pattern) and TIMP3 (downregulated expression) were significantly associated with both the upregulated miR-21 expression (P<0.05) and the poor survival of the patients (P<0.001 and P=0.001, respectively). Our data suggest that an overexpression of miRNA-21 is, therefore, associated with the biological behavior of pancreatic ductal adenocarcinoma via the downregulation of the expression of tumor suppressors, PDCD4 and TIMP3, thus resulting in tumor progression and the adverse clinical course of pancreatic ductal adenocarcinoma.

Identification of Altered MicroRNA Expression Patterns in Synovial Sarcoma

MicroRNAs (miRNAs) are noncoding small RNAs that function as an endogenous regulator of gene expression. Their dysregulation has been implicated in the development of several cancers. However, the status of miRNA in soft tissue sarcomas has not yet been thoroughly investigated. This study examined the global miRNA expression in synovial sarcoma and compared the results to those in another translocation‐associated sarcoma, the Ewing family of tumors, and in normal skeletal muscle. The 3D‐Gene miRNA microarray platform (Toray, Kamakura, Japan) and unsupervised hierarchical clustering revealed a distinct expression pattern of miRNAs in synovial sarcoma from Ewing tumors and skeletal muscle. Thirty‐five of the more than 700 miRNAs analyzed were differentially expressed in synovial sarcomas in comparison to other tissue types. There were 21 significantly up‐regulated miRNAs, including some miRNAs, such as let‐7e, miR‐99b, and miR‐125a‐3p, clustered within the same chromosomal loci. Quantitative reverse transcription‐polymerase chain reaction also demonstrated that these miRNAs were over‐expressed in synovial sarcomas. The down‐regulation of let‐7e and miR‐99b by anti‐miR miRNA inhibitors resulted in the suppression of the proliferation of synovial sarcoma cells, and modulated the expression of their putative targets, HMGA2 and SMARCA5, suggesting that these molecules have a potential oncogenic role. The unique miRNA expression pattern including the over‐expressed miRNA clusters in synovial sarcoma warrants further investigation to develop a better understanding of the oncogenic mechanisms and future therapeutic strategies for synovial sarcoma.

Peculiar histiocytic lesions with massive lanthanum deposition in dialysis patients treated with lanthanum carbonate.

Pathologic lesions caused by lanthanum carbonate (LC), a recently developed phosphate-binding agent, have not been recorded. A peculiar gastroduodenal histiocytic lesion associated with a mucosal lanthanum overload was reported. Our routine gastrointestinal biopsy series included 6 cases with heavy lanthanum burden in the gastroduodenal mucosa. In addition to routine histopathologic examinations, a series of immunohistochemical analysis and electron microscopic examinations associated with x-ray diffraction and elemental analysis were performed. Six cases, 3 of male and 3 of female individuals with ages from 59 to 69 years, were all patients of end-stage renal diseases managed under dialysis and treated with LC for >21 months. Endoscopic examinations demonstrated gastric erosions in 3, gastric polyps in 2, and duodenal ulcer in 1. In the mucosal layer, there were numerous non-Langerhans cell histiocytes, stained with CD68 but not S100 protein, engulfing a large amount of mineral-like materials. An electron microscopic and elemental analysis revealed a similar distribution of lanthanum and phosphorus in the histiocytes. Long-standing LC administration can cause massive mucosal accumulation of lanthanum in the tissue histiocytes associated with several forms of gastroduodenal lesions. A long-standing outcome is not clear at present; hence, careful follow-up studies of these patients may be needed.

PLAG1 expression in mesenchymal tumors: An immunohistochemical study with special emphasis on the pathogenetical distinction between soft tissue myoepithelioma and pleomorphic adenoma of the salivary gland

PLAG1, a proto‐oncogene activated in several types of tumors including pleomorphic adenoma of the salivary gland and lipoblastoma, is usually overexpressed because of chromosomal aberrations resulting in fusion genes. Myoepithelial tumors in soft tissue are morphologically similar to pleomorphic adenoma, but the genetic profiles of these tumors have not been fully examined. In the present study, we immunohistochemically evaluated the expression of PLAG1 in a series of 243 mesenchymal tumors. We determined that 14 tumors, including eight of 10 lipoblastomas, two of seven gastrointestinal stromal tumors, one of two angiomyofibroblastomas, one of five synovial sarcomas, one of seven leiomyomas and one of 12 myxofibrosarcomas were positive for PLAG1, whereas all seven soft tissue myoepitheliomas were PLAG1 negative. We examined two soft tissue myoepitheliomas, whose paraffin blocks were available, for fusion gene transcripts involving PLAG1 or HMGA2 specific for pleomorphic adenoma by a reverse transcription‐polymerase chain reaction assay, and no fusion transcripts were detected. Our results suggest that soft tissue myoepithelioma may be a pathogenetically distinct tumor entity from pleomorphic adenoma based on the absence of PLAG1 overexpression and characteristic fusion genes. On the other hand, PLAG1 immunohistochemistry is useful for distinguishing lipoblastoma from other lipomatous tumors including liposarcoma.

TLE1 expression in malignant mesothelioma

Malignant mesothelioma, an aggressive and often lethal tumor commonly associated with asbestos exposure, has been morphologically classified into epithelial, biphasic, and sarcomatoid subtypes. Histological distinction between biphasic or sarcomatoid mesothelioma and synovial sarcoma may be problematic in certain circumstances of intrathoracic location because of their similar clinicopathologic features, including not only their morphology but also occasional positive immunoreaction of mesothelioma markers. TLE1, which plays an important role in Wnt pathway, has been shown to be a specific marker for synovial sarcoma and diagnostically is useful; however, TLE1 expression in malignant mesotheliomas has not been fully evaluated. We immunohistochemically examined the expression of TLE1, factors related to the Wnt pathway including β-catenin and cyclin D1, and mesothelioma markers including calretinin, HBME-1, cytokeratin 5/6, and thrombomodulin in 29 malignant mesotheliomas. TLE1 was variably expressed in 28 malignant mesotheliomas regardless of histomorphological subtype with >25% of positive cells in 20 cases (69.0%). There was no evidence of association of TLE1 expression with immunoreactivity to other markers. Our study showed no or limited value of the immunohistochemical TLE1 expression in distinguishing malignant mesothelioma and synovial sarcoma.

CAMTA1 is a useful immunohistochemical marker for diagnosing epithelioid haemangioendothelioma

The diagnosis of epithelioid haemangioendothelioma (EHE) is usually straightforward, based on characteristic histological features. However, it is sometimes difficult to differentiate EHE from a variety of other tumours with epithelioid morphology. The WW domain‐containing transcription regulator 1–calmodulin‐binding transcription activator 1 (WWTR1–CAMTA1) fusion gene, resulting in the overexpression of CAMTA1, is demonstrated in approximately 90% of EHEs, and the yes‐associated protein 1–transcription factor E3 (YAP1–TFE3) fusion gene, associated with the strong and diffuse nuclear expression of TFE3, is present in another small subset of EHEs. The aim of our study was to examine CAMTA1 expression in EHEs and a variety of other tumours to evaluate its diagnostic utility, and to analyse TFE3 expression status in EHEs.

Aberrant MAD2 expression in soft-tissue sarcoma

Soft‐tissue sarcomas can be divided into translocation‐associated (TA) and non‐TA sarcomas, the latter of which is often characterized by pleomorphic cytomorphology and aneuploidy. The aberrant expression of MAD2, an essential component of the mitotic spindle checkpoint, has been recently shown to promote aneuploidy. The aim of the present paper was to assess MAD2 status on immunohistochemistry in 50 TA sarcomas with known fusion genes and 50 non‐TA pleomorphic sarcomas. MAD2 was overexpressed in 26 TA (52%) and 33 non‐TA sarcomas (66%). Notably, the MAD2 overexpression was frequently detected in TA sarcomas with atypical or high‐grade morphology, such as round cell liposarcoma and fibrosarcomatous dermatofibrosarcoma protuberans. The MAD2 overexpression was significantly frequent in non‐TA sarcomas compared with TA tumors without such atypical or high grade morphology (P = 0.012). In addition, sarcomas with MAD2 overexpression were significantly rich in abnormal mitotic figures, including multipolar mitoses and anaphase bridges, compared to MAD2‐negative tumors (P = 0.003), although the overall mitotic activity was equivalent between the sarcomas with or without the MAD2 overexpression. These data suggest that the aberrant MAD2 expression is potentially associated with pleomorphic morphology and abnormal mitosis in soft‐tissue sarcomas, as well as with high‐grade tumor progression in its TA subset.

Sclerosing mucoepidermoid carcinoma with eosinophilia of the salivary gland: Case report and review of the literature

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMCE) of the salivary gland is a rare variant of mucoepidermoid carcinoma. We report a case of SMCE in the right submandibular gland of a 79‐year‐old man. Fine needle aspiration cytology revealed cohesive clusters of atypical squamous epithelial cells admixed with cells containing intracytoplasmic mucin and eosinophils. Histologically, the tumor was composed of epithelial nests with keratinizing cells occasionally present at the center, as well as peripherally located atypical basaloid cells, and some mucin‐containing cells embedded in a fibrosclerotic stroma, which were accompanied by a prominent lymphoplasmacytic and eosinophilic infiltrate. Inflammatory infiltrate and stromal fibrosclerosis also were seen in the non‐neoplastic salivary gland tissue adjacent to the tumor. Immunohistochemically, many plasma cells were IgG4‐positive. The postoperative serum IgG4 level was elevated. Our reverse transcription‐polymerase chain reaction using formalin‐fixed, paraffin‐embedded tumor tissue failed to detect any fusion‐gene transcripts which are specifically identified in ordinary mucoepidermoid carcinoma. The findings of the present case suggest that this rare type of salivary gland carcinoma may be associated with a chronic inflammatory condition such as IgG4‐related sclerosing disease. Only 23 cases of sclerosing mucoepidermoid carcinoma with or without eosinophilic infiltratie have been reported to date in such an anatomical location.

Aberrant Calreticulin Expression Is Involved in the Dedifferentiation of Dedifferentiated Liposarcoma

Liposarcomas are a representative group of soft tissue sarcomas with variably hampered adipogenesis, which is most exemplified by its dedifferentiated subtype. However, the factor(s) responsible for inhibiting adipocyte differentiation remains unknown. A recent gene expression profiling study identified several unique genes that were highly expressed in dedifferentiated liposarcoma, and the gene encoding calreticulin (CALR), a major Ca(2+)-buffering protein that can inhibit adipocyte differentiation, was found to be overexpressed. Thus, we investigated the expression of calreticulin in 45 cases of liposarcomas, including 15 dedifferentiated tumors, at both the protein and mRNA levels. Immunohistochemically, calreticulin was consistently expressed in the dedifferentiated areas of dedifferentiated liposarcomas and commonly observed in atypical stromal cells and/or lipoblasts in the well-differentiated areas (87%), whereas large vacuolated adipocytic cells in either the tumors or normal fat were essentially negative. These results were further supported by the findings of Western blot and quantitative RT-PCR analyses. Although abnormalities in 19p13.1-13.2 where CALR is localized were uncommon in the dedifferentiated liposarcomas examined by fluorescence in situ hybridization, expression of miR-1257, a putative microRNA that targets calreticulin, was suppressed in the dedifferentiated subtype. The down-regulation of calreticulin by small-interfering RNA could induce adipogenesis in dedifferentiated liposarcoma cells and reduce cell proliferation. Our results therefore suggest that aberrantly expressed calreticulin in dedifferentiated liposarcoma is involved in its dedifferenitation and/or tumor progression.