Eiji Kudo

High expression of Toll-like receptor 4/myeloid differentiation factor 88 signals correlates with poor prognosis in colorectal cancer

Background:The Toll-like receptor (TLR) 4 signalling pathway has been shown to have oncogenic effects in vitro and in vivo. To demonstrate the role of TLR4 signalling in colon tumourigenesis, we examined the expression of TLR4 and myeloid differentiation factor 88 (MyD88) in colorectal cancer (CRC).Methods:The expression of TLR4 and MyD88 in 108 CRC samples, 15 adenomas, and 15 normal mucosae was evaluated by immunohistochemistry, and the correlations between their immunoscores and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analysed.Results:Compared with normal mucosae and adenomas, 20% cancers displayed high expression of TLR4, and 23% cancers showed high expression of MyD88. The high expression of TLR4 and MyD88 was significantly correlated with liver metastasis (P=0.0001, P=0.0054). In univariate analysis, the high expression of TLR4 was significantly associated with shorter OS (hazard ratio (HR): 2.17; 95% confidence interval (95% CI): 1.15–4.07; P=0.015). The high expression of MyD88 expression was significantly associated with poor DFS and OS (HR: 2.33; 95% CI: 1.31–4.13; P=0.0038 and HR: 3.03; 95% CI: 1.67–5.48; P=0.0002). The high combined expression of TLR4 and MyD88 was also significantly associated with poor DFS and OS (HR: 2.25; 95% CI: 1.27–3.99; P=0.0053 and HR: 2.97; 95% CI: 1.64–5.38; P=0.0003). Multivariate analysis showed that high expressions of TLR4 (OS: adjusted HR: 1.88; 95% CI: 0.99–3.55; P=0.0298) and MyD88 (DFS: adjusted HR: 1.93; 95% CI: 1.01–3.67; P=0.0441; OS: adjusted HR: 2.25; 95% CI: 1.17–4.33; P=0.0112) were independent prognostic factors of OS. Furthermore, high co-expression of TLR4/MyD88 was strongly associated with both poor DFS and OS.Conclusion:Our findings suggest that high expression of TLR4 and MyD88 is associated with liver metastasis and is an independent predictor of poor prognosis in patients with CRC.

Overexpression of HMGA2 relates to reduction of the let-7 and its relationship to clinicopathological features in pituitary adenomas

High-mobility group A2 is highly expressed during embryogenesis and in various benign and malignant tumors. Recent studies report that high-mobility group A2 is negatively regulated by the let-7 microRNAs (miRNAs) family in vitro. The development of pituitary adenomas in high-mobility group A2 transgenic mice showed that high-mobility group A2 may be involved in pituitary tumorigenesis. However, no studies have investigated the clinical significance of high-mobility group A2 and its relationship to the let-7 miRNA family in human pituitary adenomas. Using immunohistochemistry, we analyzed high-mobility group A2 expression with respect to various clinicopathologic factors in 98 pituitary adenomas. Overexpression of high-mobility group A2 was observed in 39% (38/98) of pituitary adenomas compared with normal adenohypophysial tissue and was frequently found in adenomas including prolactin (PRL), adrenocorticotrophic hormone, or follicle-stimulating hormone/luteinizing hormone and in null cell adenomas, but relatively rare in growth hormone (GH) and mixed GH/PRL adenomas. High-mobility group A2 expression was significantly associated with tumor invasion (P<0.05) and was significantly higher in grade IV than in grades I, II, and III adenomas (P<0.05). High levels of high-mobility group A2 expression were more frequently observed in macroadenomas than in microadenomas (P<0.05). High levels of high-mobility group A2 expression also significantly correlated with the proliferation marker Ki-67 (P<0.0001). Real-time quantitative RT-PCR analysis was carried out to evaluate the expression of let-7 in 55 pituitary adenomas. Subsequently, decreased expression of let-7 was confirmed in 23 of 55 (42%) adenomas and was correlated with high-grade tumors (P<0.05). An inverse correlation between let-7 and high-mobility group A2 expression was evident (R=−0.33, P<0.05). These findings support a causal link between let-7 and high-mobility group A2 whereby loss of let-7 expression induces high-mobility group A2 upregulation that represents an important mechanism in pituitary tumorigenesis and progression.

Clinicopathological features of growth hormone-producing pituitary adenomas: Difference among various types defined by cytokeratin distribution pattern including a transitional form

Pituitary adenomas producing almost exclusively growth hormones (GH) have been ultrastructurally classified into two distinct types: densely granulated somatotroph (DG) adenomas and sparsely granulated (SG) adenomas. Fibrous body (FB), an intracytoplasmic globular aggregation of cytokeratin (CK) filaments, is a hallmark of SG adenomas. Under light microscope, FB could be identified by CK immunohistochemistry as a dot-pattern immunoreaction versus a perinuclear pattern for cells without FB. However, it has been noted that numerous adenomas contain mixed populations of the two patterns. To clarify clinicopathological characteristics of the adenomas with mixed populations (“intermediate type” adenomas) and to confirm clinicopathological differences between strictly defined DG-type and SG-type adenomas, we performed this study on 104 GH cell adenomas. Having segregated “intermediate-type” adenomas (26 cases), we found significant differences between typical DG-type (47 cases) and SG-type adenomas (31 cases); SG-type adenomas had younger ages (44 vs. 50), higher frequency of macroadenomas (86% vs. 58%), invasiveness (65% vs. 38%), advanced grades (3 or 4) in Knosp’s classification (50% vs. 24%), and weaker immunoreaction for GH, β-TSH, α-subunit, E-cadherin, and β-catenin. Clinicopathological characteristics of “intermediate-type” adenomas were identical to those of DG-type adenomas. These findings confirm that SG-type adenoma is a distinct section of GH cell adenomas with special properties and biological behavior, and suggest that intermediate-phenotype adenomas are enrolled in DG-type adenomas. Special properties and biological behavior of SG-type adenomas may appear after the majority of tumor cells possess a fully developed fibrous body.

Malignant peripheral nerve sheath tumor (MPNST) showing perineurial cell differentiation.

We report a malignant peripheral nerve sheath tumor (MPNST) showing the unusual immunohistochemical feature of epithelial membrane antigen (EMA) immunoreactivity in a 52-year-old man. The tumor, located in the left paravertebral muscles, was composed of both cellular and myxoid areas. Spindle-shaped tumor cells were arranged in longitudinal cell cords, in a storiform pattern, and in whorled structures. Ultrastructurally, the tumor cells had many interdigitating cell processes, primitive cell junctions, and discontinuous external laminae. These histological and ultrastructural features were most consistent with those of an MPNST; but on immunohistochemistry, the tumor cells reacted for epithelial membrane antigen rather than for S-100 protein or Leu-7. Because EMA-immunoreactivity was recently demonstrated in perineurial cells, we concluded that the tumor was an MPNST mainly composed of tumor cells showing perineurial cell differentiation.

Malignant peripheral nerve sheath tumors : an immunohistochemical study in relation to ultrastructural features

The constituent cells in malignant peripheral nerve sheath tumors were examined by studying the expression of immunohistochemical markers for Schwann cells and perineurial cells in relation to ultrastructural features in 12 malignant peripheral nerve sheath tumors. Ultrastructural studies demonstrated mixed proliferation of Schwann cells, perineurial cells, fibroblastic cells, and primitive cells in many malignant peripheral nerve sheath tumors. Expression of S-100 protein was well correlated with Schwann cell-like differentiation of tumor cells. However, Leu-7 and epithelial membrane antigen, which have been considered to be specific to Schwann cells and perineurial cells, respectively, were common to Schwann cells, perineurial cells, and primitive cells. The common immunophenotypic expression suggests a close relationship among these cell types. The unusual expression of cytokeratin could be explained by the plasticity of intermediate filament expression.

Immunophenotypic heterogeneity in osteosarcomas

Eighteen osteosarcomas were studied immunohistochemically. The tumors were classified into the following six histologic subtypes: five osteoblastic, four chondroblastic, four malignant fibrous histiocytoma-like, two telangiectatic, two low-grade central, and one giant cell-rich. Variable amounts of osteocalcin immunoreactivity were found in all tumors. Factor XIIIa-positive cells, which may be of fibrohistiocytic lineage, were present in three tumors of the malignant fibrous histiocytoma-like type, one of the telangiectatic type, one of the low-grade central type, and the tumor of the giant cell-rich type. One tumor of the osteoblastic type showed cytokeratin and epithelial membrane antigen immunoreactivities. The positive reactions for desmin in four tumors, for alpha-smooth muscle actin in 11 tumors, and for type IV collagen in one tumor seemed to indicate myofibroblastic differentiation of some tumor cells. S-100 protein-positive tumor cells were detected not only in all four tumors of the chondroblastic type, but also in three of the osteoblastic type, one of the low-grade central type, and in the tumor of the giant cell-rich type. These immunohistochemical results suggest that osteosarcomas are composed of heterogeneous cell populations, such as those of the osteoblastic, chondroblastic, myofibroblastic, and fibrohistiocytic types, and occasionally also of cells with epithelial features.

Cytoskeletal characteristics of myofibroblasts in benign neoplastic and reactive fibroblastic lesions

The characteristics of the cytoskeleton of myofibroblasts were examined immunohistochemically in 10 extra-abdominal desmoid tumours, 3 palmar and 2 plantar fibromatoses and 5 nodular fasciites; in the cultured cells of one desmoid tumour, and also ultrastructurally in 3 desmoid tumours. Polyclonal anti-desmin antibody reacted with the cells in 7 extra-abdominal desmoid tumours, 1 palmar fibromatosis, 1 plantar fibromatosis and 3 nodular fasciites. Monoclonal antidesmin antibody reacted with cells in only 2 desmoid tumours. Desmin-positive spindle cells were scattered throughout these lesions. There were no marked ultrastructural differences between desmin-positive and desmin-negative desmoids. All specimens except one specimen of nodular fasciitis showed immunoreactivity for alpha-smooth muscle actin and vimentin. Muscle actin-positive cells were observed in all specimens. Cultured cells gave positive reactions with polyclonal desmin antibody as well as to vimentin antibodies and two preparations of actin antibodies, whereas the original tumour did not react with desmin antibody. The present studies suggested that the cytoskeleton of some myofibroblasts in both neoplastic and reactive lesions resembles that of smooth muscle cells.

Expression of intermediate filaments in malignant fibrous histiocytomas

The expression of intermediate filaments (IFs) in 34 malignant fibrous histiocytomas (MFHs) was studied immunohistochemically and ultrastructurally. Using the avidin-biotin-peroxidase method, positive reactions were detected as follows: for desmin in 12 tumors, for neurofilament in two tumors, for cytokeratin in one tumor, and for vimentin in 30 tumors. Desmin immunoreactivity was found in tumors of all four histologic subtypes and cytokeratin immunoreactivity was found in one tumor of the myxoid type. Because of the cross-reactivity of anti-neurofilament antibody with reactive histiocytes, the immunoreactivity for neurofilament seemed to be non-specific. Ultrastructurally, five of 13 tumors studied contained some tumor cells showing myofibroblastic or smooth muscle cell differentiation. A few tumor cells in one cytokeratin-positive tumor had tonofilaments in their cytoplasm. Desmin expression in some MFHs seemed to be due to myofibroblastic or smooth muscle cell differentiation of some tumor cells. Cytokeratin expression seemed to indicate epithelial differentiation in some MFHs. This varied expression of IFs in MFHs may reflect the heterogeneous nature of MFHs, and suggests that MFHs represent the final stages of dedifferentiation of several different types of sarcomas or, alternatively, represent forms of poorly differentiated sarcoma with the potential of developing into more differentiated sarcomas of heterogeneous origin.

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas

The gene products of CDH13 and CDH1, H-cadherin and E-cadherin, respectively, play a key role in cell–cell adhesion. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancers, indicating that the CDH13 and CDH1 function as tumor suppressor and invasion suppressor genes. In this study, we analyzed the expression of H-cadherin mRNA and E-cadherin protein in 5 normal pituitary tissues and 69 primary pituitary adenomas including all major types by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry, respectively. Reduced expression of H-cadherin was detected in 54% (28/52) of pituitary tumors and was significantly associated with tumor aggressiveness (P<0.05). E-cadherin expression was lost in 30% (21 of 69) and significantly reduced in 32% (22 of 69) of tumors. E-cadherin expression was significantly lower in grade II, III, and IV than in grade I adenomas (P=0.015, P=0.029, and P=0.01, respectively). Using methylation-specific PCR (MSP), promoter hypermethylation of CDH13 and CDH1 was detected in 30 and 36% of 69 adenomas, respectively, but not in 5 normal pituitary tissues. Methylation of CDH13 was observed more frequently in invasive adenomas (42%) than in non-invasive adenomas (19%) (P<0.05) and methylation of CDH1 was more frequent in grade IV adenomas compared with grade I adenomas (P<0.05). Methylation of either CDH13 or CDH1 was identified in 35 cases (51%) and was more frequent in grade IV invasive adenomas than in grade I non-invasive adenomas (P<0.05 and P<0.05, respectively). Downregulation of expression was correlated with promoter hypermethylation in CDH13 and CDH1. In conclusion, the tumor-specific downregulation of expression and methylation of CDH13 and CDH1, alone or in combination, may be involved in the development and invasive growth of pituitary adenomas.

Frequent overexpression of HMGA1 and 2 in gastroenteropancreatic neuroendocrine tumours and its relationship to let-7 downregulation.

The molecular pathogenesis of gastroenteropancreatic (GEP) neuroendocrine tumours (NETs) remains to be elucidated. High-mobility group A (HMGA) proteins play important roles in the regulation of transcription, differentiation, and neoplastic transformation. In this study, the expression of HMGA1 and HMGA2 was studied in 55 GEP NETs. Overexpression of HMGA1 and 2 was frequently detected in GEP NETs compared with normal tissues. Nuclear immunostaining of HMGA1 and 2 was observed in GEP NETs (38 of 55, 69%; 40 of 55, 73%, respectively). High-mobility group A2 expression increased from well-differentiated NET (WNET) to well-differentiated neuroendocrine carcinoma (WNEC) and poorly differentiated NEC (PNEC) (P<0.005) and showed the highest level in stage IV tumours (P<0.01). In WNECs, the expression of HMGA1 and 2 was significantly higher in metastatic tumours than those without metastasis (P<0.05). Gastroenteropancreatic NETs in foregut showed the highest level of HMGA1 and 2 expressions. MIB-1 labelling index (MIB-1 LI) correlated with HMGA1 and 2 overexpression (R=0.28, P<0.05; R=0.434, P<0.001; respectively) and progressively increased from WNETs to WNECs and PNECs (P<0.001). Let-7 expression was addressed in 6 normal organs, 30 tumour samples, and 24 tumour margin non-tumour tissues. Compared with normal tissues, let-7 downregulation was frequent in NETs (19 of 30, 63%). Higher expression of HMGA1 and 2 was frequently observed in tumours with let-7 significant reduction (53, 42%, respectively). The reverse correlation could be detected between HMGA1 and let-7 (P<0.05). Our findings suggested that HMGA1 and 2 overexpression and let-7 downregulation might relate to pathogenesis of GEP NETs.