Chikashi Kobayashi

Frequent alteration of p16INK4a/p14ARF and p53 pathways in the round cell component of myxoid/round cell liposarcoma: p53 gene alterations and reduced p14ARF expression both correlate with poor prognosis

In myxoid/round cell liposarcoma (MLS/RCLS), the presence of a round cell (RC) component has been reported to correlate with a worse prognosis for the patients. However, little is known about the molecular genetic differences between conventional myxoid (MX) components and RC components in this tumour. The aim of this study was to investigate the possible implications of molecular alterations of G1 to S‐phase check‐point genes, especially in the RC component. We evaluated the immunohistochemical expression of p53, MDM2, p14 and p16 protein and assessed proliferative activities using MIB‐1 in 29 RC components and 81 MX components from 90 cases. Mutation of the p53 gene, amplification of the MDM2 gene, homozygous deletion, methylation status and mutation of the p16INK4a/p14ARF genes were also investigated, using concordant paraffin‐embedded and frozen material. The data were analysed together with clinicopathological factors to assess their prognostic implications in MLS/RCLS. Immunohistochemically, the over‐expression of p53 protein (p = 0.01366) and the reduced expression of p14 (p < 0.0001) and p16 (p < 0.0001) proteins were significantly more frequently observed in RC components than in MX components. Reduced expression of p14 protein correlated significantly with hypermethylation of the p14ARF gene promoter (p = 0.0176) and over‐expression of p53 protein (p = 0.00837). By univariate analysis, reduced expression of p14 and p53 missense mutation were found to reduce the rate of survival significantly (p < 0.05). Multivariate analysis, including clinicopathological factors, revealed that tumour site (p = 0.0251), the presence of an RC component (p = 0.0113), high MIB‐1 labelling index (p = 0.0005) and p53 missense mutation (p = 0.0036) were adverse prognostic factors. In MLS/RCLS, reduction of p14 protein expression and p53 mutation were related to poor prognosis. Accordingly, the p14ARF/p53 pathway may contribute to the presence of an RC component and malignant progression in this tumour. Copyright

DNA hypermethylation status of multiple genes in soft tissue sarcomas

The aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-related genes. To determine the clinicopathological significance of gene promoter methylation in soft tissue sarcomas, we examined the promoter methylation status of 10 tumor-related genes in 65 soft tissue sarcomas and 19 adjacent non-neoplastic tissues by methylation-specific PCR. The methylation frequencies of tumor-related genes tested in soft tissue sarcomas were 17 (26%) for RASSF1A, 11 (17%) for DAP kinase, 10 (15%) for MGMT, nine (14%) for GSTP1, eight (12%) for PTEN, six (9%) for p16 and hMLH1, five (8%) for hMSH2, two (3%) for p14, and one (2%) for RB. Promoter methylation of these genes was not recognized in non-neoplastic tissues. All those cases of soft tissue sarcoma that had MGMT methylation, with the exception of one case of malignant peripheral nerve sheath tumor, showed large tumor size (≥10 cm) or recurrence. Moreover, eight of 10 cases with MGMT methylation revealed high American Joint Committee on Cancer stage. Seven of 10 cases (70%) with MGMT methylation showed a loss of MGMT expression by immunohistochemistry. In addition, MGMT methylation status had a statistically significant correlation with a loss of MGMT expression (P=0.014). In conclusion, although methylation of tumor-related genes was a relatively rare event in soft tissue sarcomas, methylation was tumor-specific. Of 10 tumor-related genes, cases with MGMT methylation had a tendency to be aggressive behavior. Moreover, MGMT methylation was closely associated with a loss of MGMT expression. Although our findings need to be extending to a large series, promoter methylation of tumor-related genes is likely to have an association with the pathogenesis of soft tissue sarcomas. Furthermore, MGMT methylation may be associated with tumor aggressiveness and the inactivation of MGMT gene.

Malignant perivascular epithelioid cell tumor of the colon : Report of a case with molecular analysis

Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm, and malignant cases are extremely rare. A case of malignant PEComa arising in the colon is described herein. The patient was a 43‐year‐old Japanese woman without a history of tuberous sclerosis complex. The tumor occurred in the abdominal cavity attached to the serosal side of the descending colon. Histologically, the tumor consisted of sheets or closely packed nests of epithelioid cells with clear or eosinophilic cytoplasms. The tumor cells were positive for HMB‐45 but negative for S‐100 protein and cytokeratins by immunohistochemical staining. Ki‐67 labeling index was 2.9%. Peritoneal dissemination of tumor occurred at 20 months and the patient died of tumor at 38 months after the initial operation. This was considered to be a case of malignant PEComa, based on the histological and clinical features. Tumor cells showed overexpression of cyclin D1 but lacked the loss of heterozygosity of the TSC1 and TSC2 genes. The result suggests that the overexpression of cyclin D1 may play an important role in the tumorigenesis of PEComa. Because PEComas can behave in an aggressive manner, careful follow up is warranted.

Prognostic significance of dysadherin expression in epithelioid sarcoma and its diagnostic utility in distinguishing epithelioid sarcoma from malignant rhabdoid tumor

Dysadherin is a cancer-associated cell membrane glycoprotein, which downregulates E-cadherin and promotes metastasis. We studied the clinicopathological features in 72 cases of epithelioid sarcoma and in six cases of malignant rhabdoid tumor, and also assessed the immunohistochemical expression of dysadherin, E-cadherin and MIB-1 in epithelioid sarcoma and malignant rhabdoid tumor cases. In addition, we compared dysadherin mRNA expression between epithelioid sarcoma and malignant rhabdoid tumor cell lines, using RT-PCR and real-time quantitative RT-PCR analysis. Immunohistochemical dysadherin expression was more frequently observed in proximal-type epithelioid sarcoma (71%) in comparison with distal-type epithelioid sarcoma (36%) (P=0.037). Furthermore, seven proximal-type epithelioid sarcoma cases mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied by frequent rhabdoid cells and located in deep soft tissue) were all positive for dysadherin (100%), whereas dysadherin expression was not detected at all in any of the true six malignant rhabdoid tumors (0%). Cell lines established from proximal-type epithelioid sarcoma revealed significantly higher levels of dysadherin mRNA expression, compared with the levels seen in malignant rhabdoid tumor cell lines by real-time quantitative RT-PCR (P=0.0433). Epithelioid sarcoma patients with dysadherin expression survived for a significantly shorter time than those without dysadherin expression (P=0.001). In multivariate analysis, dysadherin immunopositivity (P=0.0004) was one of the two independent adverse prognostic factors. We conclude that dysadherin expression in epithelioid sarcoma is a significant poor prognostic factor and that it is a powerful diagnostic marker for distinguishing epithelioid sarcoma, including the proximal-type epithelioid sarcoma, from malignant rhabdoid tumor. In epithelioid sarcoma, especially in proximal-type epithelioid sarcoma, increased cell disadhesion and motility by dysadherin plays an important role to acquire aggressive biological behavior. However, in malignant rhabdoid tumor, cell growth cycle that is regulated by hSNF5/INI1 gene seems to be critical to lethal biological behavior rather than dysadherin.

Prognostic significance of p14ARF, p15INK4b, and p16INK4a inactivation in malignant peripheral nerve sheath tumors

Purpose:p14ARF, p15INK4b, and p16INK4a are tumor suppressor genes that are located closely at 9p21 and are often coinactivated by genetic or epigenetic alterations. Malignant peripheral nerve sheath tumor (MPNST) is a rare sarcoma with poor prognosis. However, the prognostic implications of inactivation of p14ARF, p15INK4b, and p16INK4a in MPNSTs have not been adequately investigated. Here we carried out a genetic, epigenetic, and expression analysis of p14ARF, p15INK4b, and p16INK4a, and clarified the prognostic significance of their inactivation in MPNSTs. Experimental Design: p14ARF, p15INK4b, and p16INK4a protein expressions were assessed by immunohistochemistry in 129 formalin-fixed samples of MPNST including 85 primary tumors. Thirty-nine samples, for which frozen material was available, were also investigated by Western blotting and quantitative reverse transcription PCR (RT-PCR) to detect p14ARF, p15INK4b, and p16INK4a protein and mRNA expression, and by multiplex real-time PCR, PCR single strand conformation polymorphism and methylation-specific PCR to detect p14ARF, p15INK4b, and p16INK4a gene alterations. Results: Immunohistochemically decreased expressions of p14ARF, p15INK4b, and p16INK4a were observed in 48%, 54%, and 49% of primary MPNSTs, respectively, and were significantly correlated with their concordant mRNA levels. As for gene alterations, homozygous deletion of CDKN2A was detected in one third of the cases. Inactivation of p14ARF and p16INK4a was associated with poor prognosis by both univariate and multivariate analyses. Furthermore, cases with inactivation of all p14ARF, p15INK4b, and p16INK4a genes showed the worst prognosis in a combined prognostic assessment. Conclusion: A comprehensive analysis of p14ARF, p15INK4b, and p16INK4a inactivation status provides useful prognostic information in MPNSTs. Clin Cancer Res; 17(11); 3771–82. ©2011 AACR.

Detection of COL1A1-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibrosarcoma protuberans

Fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene has been described in dermatofibrosarcoma protuberans. The abnormal fusion transcripts probably cause PDGFB and its receptor (platelet-derived growth factor receptor beta, PDGFRB) autocrine stimulation and cell proliferation, which are responsible for the development of dermatofibrosarcoma protuberans. A reverse transcription–polymerase chain reaction assay was performed to detect the COL1A1-PDGFB fusion transcripts in 57 samples. In addition, the PDGFB gene amplification and PDGFB/PDGFRB mRNA levels were quantified by a real-time PCR system for the samples in which the fusion transcripts had been successfully detected. The fusion transcripts were detected in 42 of 57 samples. Various exons of the COL1A1 gene were fused in frame with the PDGFB gene; exons 7 and 25 were found to be slightly more frequently involved than the other exons. The PDGFB gene amplification levels varied from 0.6 to 8.3 (mean 2.4) in 42 tumor samples and from 0.4 to 3.0 (mean 1.2) in 20 adjacent normal tissue samples. In the 20 paired samples, the PDGFB gene amplification in the tumor was significantly higher than that in the normal tissue. The presence of PDGFB and PDGFRB mRNAs was demonstrated in 26 and 21 of 26 cases, respectively. The PDGFB and PDGFRB mRNA expression levels showed a good correlation (r=0.76, P<0.0001). These results indicate that the fusion protein, which is processed by the COL1A1-PDGFB transcripts, can serve as a functional ligand for PDGFRB.

Aberrant expression of CHFR in malignant peripheral nerve sheath tumors.

Mitotic checkpoint maintains genomic integrity before mitosis. Numerous observations have suggested that mitotic abnormalities produce chromosomal instability and aneuploidy. In MPNST, complex karyotypes showing numerical and structural aberrations have been described. ‘Checkpoint with forkhead-associated domain and ring finger’ (CHFR) was recently identified as defining a new early mitotic checkpoint. We examined the expression of CHFR in 96 cases of MPNST by immunohistochemical and molecular methods. We found reduced (score, ⩽3) expression of CHFR in 63 out of 96 (66%) cases of MPNST, and such alteration was significantly correlated with a high mitotic count, a high Ki-67-labeling index, and a poor prognosis. In addition, MPNST with normal karyotype showed a strong (score, =5) expression of CHFR. Our results support the assertion that CHFR functions as an inhibitor of tumor proliferation.

Dysadherin expression as a significant prognostic factor and as a determinant of histologic features in synovial sarcoma: Special reference to its inverse relationship with E-cadherin expression

Dysadherin is a cancer-associated cell membrane glycoprotein, which down-regulates E-cadherin and promotes metastasis. Synovial sarcoma is a very rare mesenchymal tumor that exhibits an epithelial profile. To confirm the diagnosis of synovial sarcoma, we evaluated several immunohistochemical markers, or detected SYT-SSX fusion gene transcript. We studied the clinicopathologic features in 92 synovial sarcoma patients and also assessed the immunohistochemical expression of dysadherin and E-cadherin to examine their possible association with histologic subtype and biologic behavior. Moreover, among 30 patients, for whom frozen materials were available, dysadherin mRNA expression was examined by reverse transcription-polymerase chain reaction and real-time quantitative reverse transcription-polymerase chain reaction analysis. Dysadherin-positive expression was significantly correlated with E-cadherin–reduced expression (P=0.0004). Dysadherin-positive immunostaining was diffusely observed in the membranes of tumor cells in 30/68 (44%) patients with monophasic fibrous type and in 1/2 (50%) patients with poorly differentiated type. However, in biphasic tumors, dysadherin expression in the fibrous component was not diffusely observed, but often sporadically or focally observed [20/22 (91%) patients]. In addition, dysadherin mRNA expression in monophasic fibrous type was significantly higher than in biphasic type (P=0.0079). Synovial sarcoma patients with dysadherin expression survived for a significantly shorter time than those without dysadherin expression (P=0.0006). Patients with combined dysadherin-positive expression and E-cadherin–reduced expression had a significantly worse prognosis than those with other combinations of dysadherin and E-cadherin expression (P=0.0007). SYT-SSX fusion gene transcript was detected in 39 patients. In our series, SYT-SSX fusion type was found to have no correlation with histologic subtype, prognosis, or dysadherin expression. In multivariate analysis, dysadherin immunopositivity (P=0.0411) was an independent adverse prognostic factor, in addition to a high MIB-1 labeling index (≥10%). We conclude that E-cadherin dysfunction by dysadherin is associated with reduced E-cadherin expression and morphologic change from epithelioid to spindle phenotype. Dysadherin expression is considered to be one of the determinants of histologic subtype in synovial sarcoma. Moreover, dysadherin expression is an excellent and independent prognostic indicator.

Alterations of the p16INK4a/p14ARF pathway in clear cell sarcoma

Clear cell sarcoma (CCS) is a very rare soft tissue sarcoma with a poor prognosis. It has become apparent through immunohis‐tochemical, ultrastructural, and microarray analyses that CCS is a soft tissue melanocytic neoplasm. Alterations in the p16INK4a/ p14ARF gene are common in malignant melanoma, which is the prototypical melanocytic neoplasm. In the present study, we performed a clinicopathologic analysis and investigated p16 and cy‐clin D1 expression by immunohistochemistry in 14 cases. Furthermore, we investigated genetic changes of various tumor suppressor genes and an oncogene, including p16INK4a/p14ARF, p53, (‐catenin, and APC, in 11 cases. The 5‐year overall survival rate in all the patients was 33.3%. A high mitotic rate was a significant adverse prognostic factor (P=0.004). Decreased expression of p16 was observed in 4 (28.6%) of 14 cases. Overexpression of cyclin D1 was observed in 9 cases (64.3%). SSCP analysis followed by DNA direct sequencing revealed point mutations of the p16INK4a gene in 2 of 11 cases (18.2%). In addition, one case with the p14ARF mutation and 2 cases with the p53 mutation were observed. None of the cases harbored mutation of the (‐catenin or APC gene. Homozygous deletion of the p1ffNK4a/p14ARF gene was detected in one case. Methylation‐specific PCR did not reveal hy‐permethylation of the p16INK4a/p14ARF promoter region in any of the cases. Three cases harbored genetic alterations of the pl6INK4a/ p14ARF gene (27.3%). All tumors with genetic alterations of the p16INK4a/p14ARF or p53 gene showed a high mitotic rate or tumor necrosis. These alterations were considered to be influential in the poor prognosis of CCS patients.

Alterations of the RB1 gene in dedifferentiated liposarcoma

Dedifferentiated liposarcoma is a malignant adipocytic neoplasm containing a nonlipogenic sarcoma of variable histological grade that arises against the background of a pre-existing well-differentiated liposarcoma. The phenomenon of dedifferentiation is considered to be time-dependent, but the mechanism is not well known. The retinoblastoma protein, encoded by the RB1 gene located at 13q14, is a key regulator of proliferation, development, and differentiation of certain cell types, including adipocytes. In the current study, we investigated the genetic alterations of the RB1 gene, such as mutation (the essential promoter region and the protein-binding pocket domain; exons 20–24) and methylation of the promoter region, in addition to pRB expression and loss of heterozygosity (LOH) status, in two morphologically distinct areas (nonlipogenic dedifferentiated and well-differentiated components) in 27 patients. As a control, 11 undifferentiated high-grade pleomorphic sarcoma/pleomorphic malignant fibrous histiocytoma samples and 11 well-differentiated liposarcoma samples were also evaluated. Dedifferentiated components showed LOH (15/25; 60%) and abnormal retinoblastoma protein expression (18/27; 66.7%) more frequently than noted in the well-differentiated components (3/24; 12.5% and 9/27; 33.3%, respectively). Five and four out of the 27 dedifferentiated components harbored mutations and promoter methylation, respectively, whereas none of these alterations were seen in the well-differentiated components. These results suggest that retinoblastoma protein has a major role to play in dedifferentiation and that a ‘two-hit’ mechanism is involved in the altered retinoblastoma protein expression in dedifferentiated liposarcoma.