Nokitaka Setsu

Prognostic significance of AKT/mTOR and MAPK pathways and antitumor effect of mTOR inhibitor in NF1-related and sporadic malignant peripheral nerve sheath tumors

Purpose: Malignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma with poor prognosis. MPNSTs occur frequently in patients with neurofibromatosis type 1 (NF1), in which NF1 gene deficiency leads to Ras hyperactivation. Ras activation causes the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and regulates cellular functions. However, the activation profiles of the AKT/mTOR and MAPK pathways in MPNSTs are poorly understood. The purposes of this study are to examine the correlation between the activation of these pathways and clinicopathologic or prognostic factors and to identify candidate target molecules in MPNST. Moreover, we assessed the antitumor effects of the inhibitor of candidate target. Experimental Design: Immunohistochemistry was conducted to evaluate the activation profiles of AKT/mTOR and MAPK pathways using 135 tumor specimens. Immunohistochemical expressions were confirmed by Western blotting. Then, an in vitro study was conducted to examine the antitumor effect of the mTOR inhibitor on MPNST cell lines. Results: Phosphorylated-AKT (p-AKT), p-mTOR, p-S6RP, p-p70S6K, p-4E-BP1, p-MEK1/2, and p-ERK1/2 expressions were positive in 58.2%, 47.3%, 53.8%, 57.1%, 62.6%, 93.4%, and 81.3% of primary MPNSTs, respectively. Positivity for each factor showed no difference between NF1-related and sporadic MPNSTs. Univariate prognostic analysis revealed that p-AKT, p-mTOR, and p-S6RP expressions were associated with poor prognosis. Furthermore, activation of each p-mTOR and p-S6RP was an independent poor prognostic factor by multivariate analysis. mTOR inhibition by Everolimus showed antitumor activity on MPNST cell lines in vitro. Conclusion: mTOR inhibition is a potential treatment option for both NF1-related and sporadic MPNSTs. Clin Cancer Res; 19(2); 450–61. ©2012 AACR.

The Akt/mammalian target of rapamycin pathway is activated and associated with adverse prognosis in soft tissue leiomyosarcomas.

The Akt/mammalian target of rapamycin (mTOR) pathway mediates cell survival and proliferation and contributes to tumor progression. Soft tissue leiomyosarcoma continues to show poor prognosis, and little is known about its mechanisms of tumor progression. Here the authors investigated the significance of activation of the Akt/mTOR pathway in soft tissue leiomyosarcomas.

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.

Clinicopathological features of primary leiomyosarcoma of the gastrointestinal tract following recognition of gastrointestinal stromal tumours

We aimed to elucidate the clinicopathological and immunohistochemical features of leiomyosarcoma (LMS) of the gastrointestinal (GI) tract.

Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma

Background:Prognosis of osteosarcoma (OS) with distant metastasis and local recurrence is still poor. Y-box binding protein-1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and translation and its high expression of YB-1 protein was observed in OS, however, the role of YB-1 in OS remains unclear.Methods:Y-box binding protein-1 expression in OS cells was inhibited by specific small interfering RNAs to YB-1 (si-YB-1). The effects of si-YB-1 in cell proliferation and cell cycle transition in OS cells were analysed in vitro and in vivo. The association of nuclear expression of YB-1 and clinical prognosis was also investigated by immunohistochemistry.Results:Proliferation of OS cell was suppressed by si-YB-1 in vivo and in vitro. The expression of cyclin D1 and cyclin A were also decreased by si-YB-1. In addition, si-YB-1 induced G1/S arrest with decreased cyclin D1 and cyclin A in OS cell lines. Direct binding of YB-1 in OS cell lines was also observed. Finally, the nuclear expression of YB-1 was significantly related to the poorer overall survival in OS patients.Conclusion:Y-box binding protein-1 would regulate cell cycle progression at G1/S and tumour growth in human OS cells in vitro and in vivo. Nuclear expression of YB-1 was closely associated with the prognosis of OS, thus, YB-1 simultaneously could be a potent molecular target and prognostic biomarker for OS.

Prognostic Impact of the Activation Status of the Akt/mTOR Pathway in Synovial Sarcoma

The Akt/mammalian target of rapamycin (mTOR) pathway, downstream from phosphatidylinositol 3‐kinase (PI3K), mediates cell survival and proliferation. Although this pathway reportedly contributes to the progression of synovial sarcoma, its prognostic impact has not been clarified.

Osteosclerosis induced by denosumab

A 10-year-old boy, 144 cm tall, was referred to our hospital in October, 2012, with a 2 month history of persistent pain in the buttocks. He had altered gait due to pain, but no disturbance of bladder or bowels. Radiographs, CT, and MRI showed a large osteolytic lesion in the sacrum, and examination of a bone biopsy sample confi rmed a giant cell tumour of bone, which we considered to be unresectable because of the potential risk of neurological defi cit and massive bleeding. We obtained informed consent from the patient and his parents and the review board for off label use of denosumab, a potent inhibitor of osteoclastic bone resorption, to reduce the tumour mass. We gave subcutaneous denosumab 120 mg every 4 weeks, with loading doses on days 8 and 15 of the fi rst cycle. Due to the excellent clinical response and the obvious sclerotic changes along the growth plates (fi gure) we stopped treatment after fi ve cycles (seven injections). The sclerosing bands were seen in almost all the radiographs of metaphyses, most prominently in the distal radius and ulna, and also in the proximal humerus, proximal femur, and phalanges of the fi ngers. During the 5 months off treatment the tumour grew again, so we restarted treatment with denosumab for 4 months until the tumour had reduced enough in size for surgery to be safely carried out. Before surgery repeat radiographs showed double-layered sclerotic bands at the metaphysis (fi gure), refl ecting the longitudinal bone growth during the periods on and off denosumab. At last follow-up in March, 2014, the patient showed no signs of growth retardation (151 cm tall), was able to participate in sports without pain, and showed no evidence of tumour recurrence.

Conventional spindle cell–type malignant peripheral nerve sheath tumor arising in a sporadic schwannoma

Malignant peripheral nerve sheath tumor is a malignant tumor showing nerve sheath differentiation. Approximately one-half of malignant peripheral nerve sheath tumors arise from a benign peripheral nerve sheath tumor, which is commonly a neurofibroma in patients with neurofibromatosis type 1. Malignant peripheral nerve sheath tumor arising in a sporadic schwannoma of soft tissue is extremely rare. In this condition, malignant cells usually show epithelioid morphology, meeting the diagnostic criteria for epithelioid malignant peripheral nerve sheath tumor. Here, we present an extraordinary case of spindle cell-type malignant peripheral nerve sheath tumor arising in a schwannoma on the back of a 58-year-old woman without neurofibromatosis. The malignant component showed hypercellular spindle cell proliferation with high mitotic activities; in contrast, the benign component showed hypocellular spindle cell proliferation in a palisading pattern and with Verocay bodies. Immunohistochemical S-100 protein staining showed a clear contrast between the malignant (negative) and benign (positive) components, which was useful for differentiating cellular schwannoma. Recognizing this rare condition is helpful in the pathologic diagnosis of schwannoma showing cellular proliferation in part.

Phosphorylation of signal transducer and activator of transcription 3 in soft tissue leiomyosarcoma is associated with a better prognosis

Signal transducer and activator of transcription (STAT) 3 mediates a broad range of biological processes, including cell survival and proliferation, and STAT3 has generally been regarded as a pro‐oncogenic transcription factor. We investigated the phosphorylation status of STAT3 and the protein expression of the suppressor of cytokine signaling 3 (SOCS3) by immunohistochemistry in 145 formalin‐fixed, paraffin‐embedded samples of soft tissue leiomyosarcoma (LMS), including 129 primary tumors. Eight benign soft tissue smooth muscle tumors were also examined. Thirteen frozen LMS samples, which were paired with normal tissue samples, were assessed by Western blot analysis for the phosphorylation of STAT3 and SOCS3 expression. Immunohistochemical study showed that the phosphorylation of STAT3 was not a major event in LMS (37%). Moreover, phosphorylated STAT3 (pSTAT3) expression was significantly correlated with a better prognosis. Overexpression of SOCS3 was recognized in 52% of the cases and negatively correlated with pSTAT3 expression. Among the benign tumors, 63 and 25% were positive for pSTAT3 and SOCS3, respectively. Immunoblotting detected pSTAT3 in all tumor samples, but at lower levels than in non‐neoplastic tissue. SOCS3 was detected in 92% (12 out of 13) of tumor tissues, but in none of the normal tissues. Contrary to the previous investigations of many other malignant tumors, STAT3 was inactivated in most LMS cases, likely owing to SOCS3 overexpression. STAT3 might not contribute to the progression of soft tissue LMS, and the phosphorylation status of STAT3 has the potential to be a favorable prognostic marker of LMS.

Epithelial and cartilaginous differentiation in clear cell chondrosarcoma

Clear cell chondrosarcoma is a rare cartilaginous bone tumor, and little is known about its pathology. We investigated the immunohistochemical expression profiles of cytokeratins (CAM5.2, AE1/AE3, CK7, CK8, CK18, and CK20), epithelial membrane antigen, SRY (sex-determining region Y)-box 9, type II collagen, runt-related transcription factor 2, and osteocalcin in clear cell chondrosarcoma and compared them with those in chondroblastoma, conventional chondrosarcoma, and osteosarcoma. Of 5 cases of clear cell chondrosarcoma, 3 demonstrated positive staining for AE1/AE3 and some form of cytokeratin in the clear cell component. Of the 5 cases, 4 strongly expressed SRY (sex-determining region Y)-box 9 in the clear cell component but weakly expressed it in the cartilaginous component. Of the 5 cases of clear cell chondrosarcoma, 3 expressed runt-related transcription factor 2 in both the clear cell and cartilaginous components, but no expression of osteocalcin was detected. In chondroblastoma, 8 of 13 cases expressed AE1/AE3, and other cytokeratins, such as CK7 (4/13), CK8 (6/13), CK18 (8/13), and CK20 (3/13), demonstrated a similar staining extensity pattern between the cellular and cartilaginous components. Clear cell chondrosarcoma and chondroblastoma have similar immunohistochemical features in that they both express epithelial and chondrogenetic markers. On the other hand, tumor cells of clear cell chondrosarcoma have no osteoblastic immunohistochemical expression in comparison with chondroblastoma.