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Synovial sarcoma (SS) is regarded as a relatively chemosensitive sarcoma; when the disease is advanced, the prognosis remains devastating. Phosphoinositide 3-kinase (PI3K) and the downstream Akt/mammalian target of rapamycin (mTOR) pathway have essential roles in modulating cellular functions, activating molecules like ribosomal protein S6 kinase and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP1), which contribute to the regulation of cell size, proliferation, and survival.[1, 2] Therefore, the Akt/mTOR pathway is regarded as oncogenic and has the potential to be a therapeutic target. Several previous studies have demonstrated activation of the Akt/mTOR pathway and its contribution to cell survival and proliferation in SS cell lines[3-6] as well as the effects of the clinical application of therapeutic agents targeting mTOR in soft tissue sarcoma (STS). The prognostic impact of the activation of this pathway, however, remains to be clarified.
Although this pathway is activated in response to extracellular signals, such as growth factors and cytokines, aberration of phosphatase and tensin homolog (PTEN) is a potential activator of this pathway. In addition, activating mutations in PI3K catalytic subunit α (PIK3CA) and Akt1 have recently been recognized. PIK3CA mutations in human neoplasms have gained increasing attention since the landmark study of Samuels et al, and “hotspots” of mutations have been reported in exon 9 (E542, E545) and exon 20 (H1047). Akt1 mutation (E17K) has been reported as another activator. In this study, we investigated the phosphorylation status of Akt (a serine/threonine kinase also known as protein kinase B), mTOR, 4E-BP1, and S6 in a large series of SS and evaluated the relation between Akt/mTOR pathway activation and clinical and histopathologic features.
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It has been established that the Akt/mTOR pathway is highly activated in various malignant tumors. Several previous studies have demonstrated the activation of this pathway and its contribution to cell survival and proliferation in SS in vitro.[3, 5] However, the expression profiles of the molecules along the Akt/mTOR pathway have been analyzed in a relatively small number of clinical materials.[5, 13, 14] Some authors investigated Akt/mTOR pathway involvement in STS series and demonstrated a correlation between Akt activation and a worse prognosis or a greater probability of metastasis13; whereas, to our knowledge, no investigations have focused on SS as a single entity, and the prognostic impact of this pathway on SS has remained to be clarified.
In previous large studies, several definite adverse clinical prognostic factors were identified. We observed that frequent mitosis, sex (male), visceral location, and large tumor size were poor prognostic factors in multivariate analysis. High mitotic activity[16, 17] and large tumor size[16-19] were consistently reported as adverse prognostic factors for SS. To our knowledge, the prognostic implications of visceral locations have not been assessed; however, truncal/proximal tumors reportedly confer a worse prognoses than tumors of the distal extremities.[16, 18] We also observed that male sex was an unfavorable prognostic feature. This difference in survival according to sex also was observed in previous large studies.[17, 20] Takenaka et al documented that fewer women than men presented with metastasis. In our series, women tended to have lower AJCC stage than men (P = .0945), but no other parameters were correlated with sex.
Immunopositivity for pAkt, pmTOR, and p4E-BP1 also was identified as an adverse prognostic factor in this study, whereas immunopositivity for pS6 was not. The positive results for pAkt, pmTOR, and downstream p4E-BP1 and pS6 were correlated with each other, suggesting that these molecules are activated successively in a “pathway.” The univariate prognostic factors pmTOR positivity (+) and p4E-BP1 (+) were correlated with higher mitotic activity, and p4E-BP1 (+) was correlated with a larger extent of necrosis. This pathway may be associated with cellular proliferation through the phosphorylation of 4E-BP1. In our previous study, we demonstrated that these phosphorylated proteins were expressed in the cytoplasm of leiomyosarcoma cells, whereas strong nuclear staining was observed in a certain number of SS samples. Moreover, some tumors had extensive staining in the cytoplasm of epithelioid SS cells. This epithelioid-dominant staining pattern was mentioned in a previous study. However, its significance remains unclear, because this pattern had no correlation with other clinicopathologic parameters in the current study. Patients who had pAkt-positive tumors had a better clinical response to chemotherapy than those who had tumors that were negative for pAkt, and this tendency was not observed for the other molecular markers. This is probably because of the diverse functions of Akt that are not related to mTOR, such as cellular proliferation, survival, and antiapoptosis.
The expression of pAkt and pmTOR in tumor tissue also was demonstrated in Western blot analysis, although the correlation with immunohistochemistry results was not statistically significant, probably because of the small number of samples. Several patients had lower expression of p4E-BP1 and pS6 in tumors than in non-neoplastic tissues. This may correspond to the finding that the immunohistochemically positive ratios of these proteins were relatively low. Another explanation is that they may be activated by kinases other than mTOR[21, 22] in skeletal muscle tissue, which was used as a control in the current study. These proteins had lower expression in recurrent or metastatic lesions than in primary tumors according to our Western blot analysis; however, the cause of this phenomenon remains unknown.
Abnormalities in several molecules may be responsible for activation of the Akt/mTOR pathway. We observed somewhat increased PTEN expression in tumor tissue proportional to Akt expression. PTEN expression may be induced as a result of negative feedback from activation of the Akt pathway rather than being responsible for the activation. Another factor that may activate this pathway is a mutation in PIK3CA or in Akt1. We screened 35 SS samples but did not detect any mutations. These results are consistent with the recent findings of some investigators who reported only rare PIK3CA mutation in SS.[5, 23, 24]
Previous studies have indicated another potential factor in the Akt/mTOR pathway: the activation of receptor tyrosine kinases or cytokine receptors. Epidermal growth factor receptor (EGFR),[14, 25, 26] insulin-like growth factor-1 receptor (IGF1R),[14, 27] and platelet-derived growth factor receptor α (PDGFRα)[6, 28, 29] have been proven responsible for Akt/mTOR pathway activation in SS, and some preclinical studies have lent credence to targeting this pathway.[6, 29] Clinical trials of molecular targeting drugs also have been undertaken in STS. Ridaforolimus, an mTOR inhibitor, produced significantly prolonged progression-free survival in a clinical trial targeting advanced STS, although no significant difference was indicated for OS. Pazopanib, a multitargeted kinase inhibitor, also exhibited potential effectiveness against STS, especially in a group of patients with SS; and Hosaka et al demonstrated that this drug inhibited the growth of SS cells accompanied by the suppression of the Akt pathway. Moreover, Ho et al revealed that PDGFRα, a pazopanib target, can mediate intrinsic resistance to rapamycin in SS. These findings suggest that the activation mechanism of the Akt/mTOR pathway is multimodal and that we should seek a combination of drugs targeting not only mTOR but also the upstream receptors and the feedback loops.
At this point, we cannot conclude that any of the markers we studied are useful in making the decision whether targeting this pathway is the superior strategy for each patient. In the phase 2 study of ridaforolimus, pAkt, pS6, and 4E-BP1 were not identified as good predictors of a clinically beneficial response. Immunohistochemically “negative” patients were able to benefit from targeted therapies, because some extent of immunostaining (weaker than or equal to that in ECs) was observed in most tumor cells, even among the “negative” patients in our study. At the least, pS6 would not be suitable based on the results from our immunohistochemical analysis, in which no clinicopathologic parameters were correlated with pS6 positivity. The receipt of chemotherapy seemed to have little impact on this pathway, and targeted therapy against this pathway is a potential candidate for patients who have received conventional chemotherapy.
In conclusion, we have demonstrated that the Akt/mTOR pathway, including downstream 4E-BP1, is activated and is associated with aggressive clinical behavior in SS. These findings support the validity of molecular therapy targeting this pathway in patients with SS.