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Keywords:

  • solitary fibrous tumor;
  • hemangiopericytoma;
  • Akt;
  • mammalian target of rapamycin (mTOR);
  • receptor tyrosine kinase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

BACKGROUND

Solitary fibrous tumors (SFTs) are soft tissue tumors of intermediate malignancy that rarely metastasize. Although unresectable SFTs are reported to have a poor prognosis, to the authors' knowledge there is currently no effective therapy. Molecular target therapy is a promising approach for patients with unresectable tumors, but knowledge of the molecular biology of SFTs is currently insufficient to support such therapy. The current study investigated the activation of receptor tyrosine kinases (RTKs) and the Akt-mammalian target of rapamycin (Akt-mTOR) pathway in SFTs as therapeutic targets.

METHODS

The phosphorylation statuses of Akt-mTOR pathway proteins (p-Akt, p-mTOR, phosphorylated 4E-binding protein [p-4EBP1], and phosphorylated S6 ribosomal protein [p-S6RP]) and RTKs (phosphorylated platelet-derived growth factor receptor-α [p-PDGFRα], p-PDGFRβ, p-c-met, and phosphorylated insulin-like growth factor-1 receptor-β [p-IGF-1Rβ]) were assessed by immunohistochemistry in 66 samples of SFTs, and the data were compared with clinicopathological and histopathological findings. The expression of phosphorylated proteins was assessed by Western blot analysis in 6 frozen samples.

RESULTS

The immunohistochemical results were as follows: p-Akt, 56.0% (nuclear and cytoplasmic staining); p-mTOR, 69.6% (nuclear and cytoplasmic staining); p-4EBP1, 80.3% (nuclear and cytoplasmic staining); p-S6RP, 69.6% (cytoplasmic staining); p-PDGFRα, 39.0% (cytoplasmic staining); p-PDGFRβ, 52.0% (cytoplasmic staining); p-c-met, 37.8% (nuclear staining) and 19.6% (cytoplasmic staining); and p-IGF-1Rβ, 16.6% (nuclear staining). Phosphorylation of the Akt-mTOR pathway proteins was correlated with one another except for p-Akt with S6RP. p-PDGFRβ and p-IGF-1Rβ were correlated with p-Akt. Moreover, significant relationships were noted between disease-free survival or overall survival and the presence of hypoglycemia, necrosis, cystic and myxoid degeneration, and atypical findings.

CONCLUSIONS

The Akt/mTOR pathway was activated in approximately 50% of the cases of SFTs and was associated with RTKs, which were phosphorylated at different rates. Thus, the Akt-mTOR pathway may be involved in the tumorigenesis of SFTs. Cancer 2014;120:864–876. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Solitary fibrous tumors (SFTs) are rare fibrous tumors characterized by several histopathological features, such as hemangiopericytomatous branching vessels, a patternless architecture, and a hyalinized vascular wall. The tumor cells are generally positive for CD34, which is not specific to SFTs but is one of the features distinguishing them from other spindle cell tumors. However, SFTs do not demonstrate consistent immunoreactivity for other markers, such as smooth muscle actin, desmin, S-100, and cytokeratins.

The molecular biology of SFT, especially the functions of various growth factors in SFT, has been examined. The expression of growth factors (platelet-derived growth factor subunit A [PDGF-A][1, 2] and insulin-like growth factor-2 [IGF-2][3]), and growth factor receptors (vascular endothelial growth factor receptor-2 [VEGFR-2],[4] platelet-derived growth factor receptor -α [PDGFRα]/β,[5] c-met,[4, 5] and IGF-1 receptor [IGF-1R][6]) has been noted, and 1 investigation demonstrated tyrosine kinase inhibitor-associated tumor regression.[2] Downstream from the receptor tyrosine kinases (RTKs), the Akt/mammalian target of rapamycin (mTOR) pathway, 1 of the signaling cascades located downstream from the RTK, can also be considered a candidate therapeutic target. The Akt-mTOR pathway plays an essential role in modulating cellular functions in response to extracellular signals, such as growth factors and cytokines.[6, 7] Phosphatidylinositide 3-kinase activates the downstream serine-threonine kinase, Akt. Akt in turn phosphorylates many downstream molecules involved in the regulation of cellular functions. mTOR, an Akt substrate, is a key effector of protein synthesis, and its downstream targets include ribosomal protein S6 (S6RP) kinase and the eukaryotic translation initiation factor 4E-binding protein (4EBP1).[8, 9] 4EBP1 phosphorylation upregulates protein translation by releasing eukaryotic translation initiation factor 4E. The activation of S6 kinase and the phosphorylation of its substrate S6 contribute to the regulation of cell size and cell proliferation.[10] The Akt/mTOR pathway has been shown to be highly activated in various malignant tumors. In a study investigating this pathway's involvement in a series of soft tissue sarcomas, Akt activation was consistently correlated with poor prognosis.[11] Moreover, Akt activation was correlated with a higher probability of metastasis.[12] The contribution of Akt activation to malignant behavior was also examined in patients with soft tissue leiomyosarcoma and malignant peripheral nerve sheath tumor series at the study institution.[13, 14]

In the current study, we investigated the expression of growth factor receptors as well as the phosphorylation statuses of Akt, mTOR, S6, and 4EBP1 in a large series of solitary fibrous tumors, and evaluated the relationships between tyrosine kinase activation, Akt-mTOR pathway activation, and their clinical and histopathological features.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Materials

We used samples of solitary fibrous tumors that were registered from 1975 to 2011 in the files of the Department of Anatomic Pathology of the Graduate School of Medical Sciences at Kyushu University in Fukuoka, Japan. Each tumor was classified according to its histology by reference to the World Health Organization classification.[15] All the cases were reviewed based on histological examinations with hematoxylin and eosin staining and CD34 immunohistochemical analysis. A total of 66 formalin-fixed paraffin-embedded samples of primary SFTs from 66 patients were prepared for immunohistochemistry. Survival data were available for 53 cases (80%), with follow-up ranging from 6 to 327 months (median, 48 months). Clinical outcome was evaluated according to the history of disease recurrence or metastasis and tumor death. The blood sugar level of the patient was obtained right before surgery was used, and levels < 70 mg/dL were considered to indicate hypoglycemia. Western blot analysis was performed on 6 frozen samples of SFT, 4 of which were paired with normal tissue samples (skeletal muscle).

As in a previous study, the tumors were subdivided into 3 categories: 1) keloid-type, when the tumor consisted entirely of a hypocellular proliferation of spindle cells into a collagen-rich stroma; 2) mixed/conventional-type, when the tumor demonstrated both a keloid-type appearance and zones of hypercellularity; and 3) sarcoma-type, when the entire tumor demonstrated a proliferation of spindle cells.[5] Histologically malignant SFT was defined in 2 ways. The first definition required that at least 1 of the diagnostic criteria of malignant SFT proposed by England et al[16] be present. These criteria were defined as England's malignant SFT: 1) high cellularity; 2) > 4 mitotic figures per 10 high-power fields (HPFs); and 3) cellular pleomorphism.[16] The second definition required that at least 1 of the malignant features indicated by Vallat-Decouvelaere and Fletcher et al[17] be present. These features were defined as Fletcher's malignant SFT: 1) high cellularity; 2) > 4 mitotic figures per 10 HPFs; 3) more than intermediate nuclear atypia; and 4) tumor necrosis.[17]

Immunohistochemistry

Formalin-fixed paraffin-embedded tissue was cut at 3 μm. Antigen retrieval was performed by boiling the slides with 10 mM of sodium citrate (pH 6.0) or Target Retrieval Solution (Dako, Carpinteria, Calif). The primary antibodies are shown in the online supporting information. The immune complex was detected with the EnVision Detection System (Dako).

Coexistent endothelial cells (ECs) were evaluated as a positive internal control of phosphorylated (p)-Akt, p-mTOR, p-4EBP1, and p-S6RP, because it has been reported that the Akt/mTOR pathway is activated in ECs.[18] The percentage of immunoreactive cells and staining intensity compared with those of the ECs adjacent to the tumor cells were evaluated in the most representative areas, with reference to the evaluation method of Dobashi et al.[12] The percentage of significantly immunoreactive cells was scored from 0 to 2 as follows: 0, < 10%; 1, 10% to 50%; and 2, > 50%. Significant intensity was defined as definite staining with higher intensity than that observed in ECs adjacent to the tumor cells. When the score was 1 or 2, the samples were judged as positive, and thus p-Akt, p-mTOR, p-S6RP, or p-4EBP1 was defined as activated. Nuclear or cytoplasmic staining of tumor cells was judged as positive staining for anti–p-Akt, anti–p-mTOR, anti-p-4EBP1, and anti–p-c-met antibodies. Cytoplasmic staining was judged as positive staining for anti–p-S6RP antibodies. Nuclear staining was judged as positive staining for anti–p-IGF-1Rβ antibodies. With regard to for PDGF-B, PDGFRα, PDGFRβ, hepatocyte growth factor-α (HGFα), c-met, IGF-1, IGF-2, IGF-1Rα, IGF-2R, p-PDGFRα and p-PDGFRβ, cells were considered to be positive if staining for cytoplasm. The expression of these molecules, with the exception of Akt-mTOR pathway proteins (p-Akt, p-mTOR, p-4EBP1, and p-S6RP) was judged to be positive when at least 10% of the cells were positively stained; the cutoff was established by reference to the past report.[5]

Western Blot Analysis

Protein was extracted from frozen samples using lysis buffer (PRO-PREP Protein Extraction Solution; iNtRON Biotechnology, Seongnam, South Korea), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 3% bovine serum albumin. Rabbit monoclonal antibodies for p-Akt (Ser473), p-mTOR (Ser2448), p-S6RP (Ser235/236), p–c-met (Y1234/Y1235), and p–IGF-1Rβ(Tyr1131)/insulin receptor β(Tyr1146), and goat polyclonal antibodies for p-PDGFRα (Tyr754) and p-PDGFRβ (Tyr720) were used as primary antibodies. As an internal control, antiactin (1:10,000) (MP Biomedicals, Irvine, Calif) was used. Membranes were next incubated with antirabbit immunoglobulin G (IgG) (Cell Signaling Technology, Danvers, Mass), antimouse IgG1, or antigoat IgG (Santa Cruz Biotechnology, Dallas, Tex). All membranes were observed using ECL Plus (GE Healthcare, Buckinghamshire, UK). Images were analyzed using an LAS-4000 Image Reader with MultiGauge software (version 3.0; Fujifilm, Tokyo, Japan). The extent of phosphorylation was calculated as follows, compared with normal tissue in each case: (p-protein [tumor]/actin [tumor])/(p-protein [normal]/actin [normal]).

Statistical Analysis

The Fisher exact test was used to evaluate differences between pairs of populations established according to each parameter. Thus, all the clinicopathological and histopathological parameters (age, size, location, hypoglycemia, recurrence/metastasis, tumor death, histology, necrosis, cystic degeneration, hyalinization, myxoid degeneration, hemorrhage, nuclear atypia, pleomorphic cells, mitosis, malignancy according to England et al16, and malignancy according to Vallat-Decouvelaere et al[17]) and immunohistochemical parameters (p-Akt, p-mTOR, p-4EBP1, p-S6RP, PDGFRα, PDGFRβ, c-met, IGF-1Rα, p-PDGFRα, p-PDGFRβ, p-c-met, p-IGF-1Rβ, IGF-2R, PDGF-B, HGFα, IGF-1, and IGF-2) were analyzed for their correlation to one another by using the Fisher exact test. The survival information was illustrated with Kaplan-Meier curves, and survival analyses were performed with the clinicopathological and immunohistochemical data using the log-rank test. A 2-sided P value of < .05 was considered to indicate statistical significance. The Cox proportional hazards model was adopted to validate the association between Kaplan-Meier curves and the items picked up in the above univariate analysis. The odds ratio (OR) was also described. Data analysis was conducted with the JMP statistical software package (version 9.0.2; SAS Institute Inc, Cary, NC.)

Informed consent was obtained from the subjects or guardians. This study was conducted in accordance with the principles embodied in the Declaration of Helsinki. The study was also approved by the Ethics Committee of Kyushu University (No.25-111, 25-143) and conducted according to the Ethical Guidelines for Epidemiological Research enacted by the Japanese Government.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Clinicopathological and Histopathological Findings

The clinicopathological findings in the current study corresponded approximately with the World Health Organization classification.[15] The clinicopathological data and representative histologies of the 66 primary tumors are presented in Figure 1 and Table 1.[16, 17] Twenty of the 66 tumors were pleural (including visceral pleura) and 46 were extrapleural; these were located in the trunk (14 cases), extremity (7 cases), head and neck (8 cases), retroperitoneum (8 cases), pelvis (5 cases), mediastinum (3 cases), and bone (1 case). The location had no definite association with disease recurrence/metastasis or tumor death. The majority of the tumors occurred in females (27 in males and 39 in females). The median tumor size was 6 cm (mean, 8.07 cm [range, 1 cm-27 cm]; standard deviation, 5.87), and 31 tumors measured < 8 cm. Hypoglycemia was clinically identified in 4 of the 35 available cases (11.4%), and local disease recurrence/metastasis was documented in 11 of the 51 available cases (21.5%). At the time of follow-up, 7 of the 53 patients (13.2%, including 2 unresectable cases) had died of the disease. The 2-year and 5-year survival rate was 97.5% and 84.5%, respectively, which was calculated by the Kaplan-Meier curve.

image

Figure 1. Microscopic features of solitary fibrous tumors are shown, demonstrating (a) conventional-type features, (b) keloid-type features, (c) cellular-type features, (d) mitosis, (e) hyalinization, (f) myxoid degeneration, (g) necrosis, (h) hemorrhage, (i) cystic degeneration, (j) intermediate nuclear atypia, and (k) nuclear pleomorphism.

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Table 1. Clinicopathological Parameters and Immunohistochemical Data for 66 Primary Tumorsa
Parameters Case%p-Aktp-mTORp-4EBP1p-S6RPp-PDGFRαp-PDGFRβp-c-met (Nuclear Stain)p-c-met (Cytoplasmic Stain)p-IGF-1Rβ
  1. Abbreviations: −, absent; +, present; HPF, high-power field; int, intermediate nuclear atypia; NA, not available; NS, not significant; p-4EBP1, phosphorylated 4E-binding protein; p-Akt, phosphorylated Akt; p-c-met, phosphorylated c-met; p-IGF-1Rβ, phosphorylated insulin-like growth factor-1 receptor-β; p-mTOR, phosphorylated mammalian target of rapamycin; p-PDGFRα, phosphorylated platelet-derived growth factor receptor-α; p-PDGFRβ, phosphorylated platelet-derived growth factor receptor-β; p-S6RP, phosphorylated S6 ribosomal protein.

  2. a

    Difference in the percentage between 2 populations was tested by the Fisher exact test.

  3. b

    P< .05.

SexMale2740.9NSNSNSNSNSNSNSNSNS
Female3959.1
Age (range, 5-81; mean, 56), y<562639.4NSNSNSNSNSbNSNSNS
≥564060.6
Size (range, 1-27; mean, 8) cm<83156.4bNSNSNSNSNSNSNSNS
≥82443.6
NA11 
LocationExtrapleural4669.6NSNSNSNSNSNSNSNSNS
Pleuropulmonary2030.3
Hypoglycemia+411.4NSNSNSNSNSNSNSNSNS
3188.5
NA31 
Recurrence/metastasis+1121.6NSNSNSNSNSNSNSNSNS
4078.4
NA15 
Tumor death+713.2NSNSNSNSNSNSNSNSNS
4686.8
NA13 
HistologySarcoma913.6NSNSNSNSNSNSNSbNS
Others5786.4
Necrosis+46.1NSNSbNSNSNSNSNSNS
6293.9
Cystic degeneration+46.1NSNSNSNSNSNSNSNSNS
6293.9
Hyalinization+3248.5NSNSNSNSNSNSNSNSNS
3451.5
Myxoid degeneration+2537.9NSNSNSNSNSNSNSNSNS
4162.1
Hemorrhage+69.1NSNSNSNSNSNSNSNSNS
6090.9
Nuclear atypia>int2537.9NSNSNSNSNSNSNSNSNS
≤int4162.1
Pleomorphic cells+23NSNSNSNSNSNSNSNSNS
6497
Mitosis≥ 4/10 HPF1116.7NSNSNSNSNSNSNSNSNS
<4/10 HPF5583.3
Malignancy (England et al[16])Malignant1725.8NSNSNSNSNSNSNSNSNS
Benign4974.2
Malignancy (Vallat-Decouvelaere et al[17])Malignant2842.4NSNSNSNSNSNSNSNSNS
Benign3857.6

From a histopathological point of view, 52 cases were of the mixed/conventional-type (Fig. 1a), 5 were of the keloid-type (Fig. 1b), and 9 were of the sarcoma-type (Fig. 1c). Ten tumors had a high mitotic rate (> 4 mitoses/10 HPF) (Fig. 1d), 32 had hyalinization (Fig. 1e), and 25 had myxoid degeneration (Fig. 1f), whereas only 5 tumors demonstrated tumor necrosis (Fig. 1g), 6 demonstrated hemorrhage (Fig. 1h), 4 demonstrated cystic degeneration (Fig. 1i), and 25 demonstrated more than intermediate nuclear atypia (Fig. 1j); only 2 cases showed cellular pleomorphism (Fig. 1k). None of the cases exhibited a so-called “dedifferentiated” component.

Univariate analysis, which was validated by the Fisher exact test, revealed positive correlations between disease recurrence/metastasis and both hypoglycemia (OR, 37.5; P = .0084) and Fletcher's malignant SFT (OR, 8.35; P = .014), and between tumor death and hypoglycemia (OR, 40.5; P = .007), size > 8 cm (P = .0148), and Fletcher's malignant SFT (OR, 8.52; P = .043). The other clinical and histological factors shown in Table 1 were found to have no significant correlation with clinical outcome. In addition, disease-free survival, as assessed by the log-rank test, was found to be significantly related to differences in hypoglycemia (P < .0001), necrosis (P = .0021), cystic degeneration (P = .0102), myxoid degeneration (P = .0005), and Fletcher's malignant SFT (P = .0132). Overall survival was found to be significantly related to hypoglycemia (P < .0001) and myxoid degeneration (P = .0003) (Fig. 2).[17]

image

Figure 2. Kaplan-Meier survival curves for disease-free and overall survival are shown according to the results of clinicopathological and histopathological studies: disease-free survival with hypoglycemia, necrosis, cystic degeneration, myxoid degeneration, malignant solitary fibrous tumor according to the criteria of Vallat-Decouvelaere and Fletcher et al,[17] and overall survival with hypoglycemia and myxoid degeneration. The survival curves were compared using the log-rank test. + indicates presence; −, absence.

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Multivariate analysis, which was validated by the Cox proportional hazards model, revealed an independent association between disease recurrence/metastasis and hypoglycemia (P = .0005) and Fletcher's malignant SFT (P = .0376), and between tumor death and hypoglycemia (P = .0319). However, these must be considered reference values rather than definitive data because of the inadequate number of cases in each analyzed item.

Immunohistochemistry

The results of immunostaining for p-Akt, p-mTOR, p-S6RP, p-4EBP1, p-PDGFRα, p-PDGFRβ, p–c-met, p–IGF-1Rβ, PDGF-B, PDGFRα, PDGFRβ, HGFα, c-met, IGF-1, IGF-2, IGF-1Rα and IGF-2R are presented in Figure 3. Staining for p-Akt, p-mTOR, and p-4EBP1 was observed in both the nuclei and cytoplasm, whereas only cytoplasmic staining was observed for p-S6RP, p-PDGFRα, p-PDGFRβ, PDGF-B, PDGFRα, PDGFRβ, HGFα, c-met, IGF-1, IGF-2, and IGF-2R. p-IGF-1Rβ staining was located only in the nuclei, and p-c-met staining was present in the nuclei or cytoplasm. The associations between the phosphorylated proteins and the clinicopathological data and histological findings are summarized in Table 1. The associations between the phosphorylated proteins are summarized in Table 2. There was a positive correlation noted between p-4EBP1 and necrosis (P = .0485). In turn, significant associations were also observed between p-Akt and tumor size (P = .0499), and p-c-met and sarcoma-type histology (P = .0113). There was a positive correlation noted between p-Akt and p-mTOR (P = .0317); in the same way, associations were observed between Akt and p-4EBP1, p-mTOR and p-4EBP1, p-mTOR and p-S6RP, and p-4EBP1 and p-S6RP. Phosphorylation of the growth factor receptors was distantly related to the malignant histologies. Phosphorylation of Akt was associated with p-PDGFRβ (P = .0062) and p-IGF-1Rβ (P = .0171). Only PDGFRβ phosphorylation was found to have significant associations with the cophosphorylation of Akt and mTOR (P = .0465). With regard to the prognosis, a significant association between clinical outcome and the immunohistochemical result was not verified. In addition, hypoglycemia was found to have no association with the positivity of the growth factor ligands (PDGF-B, HGFα, IGF-1, and IGF-2).

image

Figure 3. The results of immunohistochemistry (IHC) are shown. Immunopositivity was observed for the following: phosphorylated (a) Akt (nuclear and cytoplasmic staining), (b) mammalian target of rapamycin (mTOR) (nuclear and cytoplasmic staining), (c) 4E-binding protein (4EBP1) (nuclear and cytoplasmic staining), (d) S6 ribosomal protein (S6RP) (cytoplasmic staining), (e) platelet-derived growth factor receptor-α (PDGFRα) (cytoplasmic staining), (f) PDGFRβ (cytoplasmic staining), (g) c-met (nuclear staining), (h) c-met (cytoplasmic staining), and (i) insulin-like growth factor-1 receptor-β (IGF-1Rβ) (nuclear staining). This was followed by total expression of the receptor and ligands for each of the following: (j) platelet-derived growth factor 5, subunit B (PDGF-B) (cytoplasmic staining); (k) PDGFRα (cytoplasmic staining), (l) PDGFRβ (cytoplasmic staining), (m) hepatocyte growth factor-α (HGFα) (cytoplasmic staining), (n) c-met (cytoplasmic staining), (o) IGF-1 (cytoplasmic staining), (p) IGF-2 (cytoplasmic staining), (q) IGF-1R (cytoplasmic staining), and (r) IGF-2R (cytoplasmic staining).

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Table 2. Statistical Analysis of Phosphorylated Proteinsa
 Positive%p-Aktp-mTORp-4EBP1p-S6RP
  1. Abbreviations: NS, not significant; p-4EBP1, phosphorylated 4E-binding protein; p-Akt, phosphorylated Akt; p-c-met, phosphorylated c-met; p-IGF-1Rβ, phosphorylated insulin-like growth factor-1 receptor-β; p-mTOR, phosphorylated mammalian target of rapamycin; p-PDGFRα, phosphorylated platelet-derived growth factor receptor-α; p-PDGFRβ, phosphorylated platelet-derived growth factor receptor-β; p-S6RP, phosphorylated S6 ribosomal protein.

  2. a

    Difference in the percentage between 2 populations was tested by the Fisher exact test.

  3. b

    P<.05.

  4. c

    P<.001.

  5. d

    P<.01.

p-Akt37/6656.0
p-mTOR46/6669.7b
p-4EBP153/6680.3bc
p-S6RP46/6669.7NScc
p-PDGFRα25/6637.9NSNSNSNS
p-PDGFRβ34/6651.5dNSNSNS
p-c-met (nuclear stain)25/6637.8NSNSNSNS
p-c-met (cytoplasmic stain)13/6619.7NSNSNSNS
p-IGF-1Rβ11/6616.7bNSNSNS

Table 3 shows the intercorrelations between the immunohistochemical profiles of phospho-proteins (Akt-mTOR pathway proteins and receptor tyrosine kinases) and growth factor-associated antigens (receptors and ligands). The cases demonstrated receptor-ligand coexpression as follows: PDGFRβ and PDGF-B were coexpressed in 50 cases (75.8%), c-met and HGFα were coexpressed in 30 cases (45.5%), and IGF-1Rα and IGF-1 or 2 were coexpressed in 10 cases (15.2%). The cases positive for a receptor (PDGFRβ, c-met, and IGF-1R) and the corresponding ligand (PDGF-B, HGFα, IGF-1, and IGF-2) (eg, those coexpressing PDGF-B and PGDFRβ) were found to be significantly associated with phosphorylation of the receptor, except in the case of PDGFRα and PDGF-B. These results were as follows; PDGF-B–PDGFRβ coexpression and PDGFRβ phosphorylation: P = .0032; HGFα–c-met coexpression and c-met phosphorylation with cytoplasmic localization: P = .0003; and IGF-1- or IGF-2–IGF-1Rα coexpression and IGF-1Rβ phosphorylation: P < .0001. Moreover, there was a positive correlation noted between the coexpression of PDGF-B and PDGFRβ (P = .0079), and HGFα and c-met (P = .0448), with p-Akt (Table 3).

Table 3. Statistical Analysis of Growth Factor Receptors and Ligandsa
 Positive%p-Aktp-mTORp-4EBP1p-S6RPp-PDGFRαp-PDGFRβ
  1. Abbreviations: HGFα, hepatocyte growth factor-α; p-4EBP1, phosphorylated 4E-binding protein; p-Akt, phosphorylated Akt; p-IGF-1Rβ, phosphorylated insulin-like growth factor-1 receptor-β; p-mTOR, phosphorylated mammalian target of rapamycin; p-PDGFRα, phosphorylated platelet-derived growth factor receptor-α; p-PDGFRβ, phosphorylated platelet-derived growth factor receptor-β; p-S6RP, phosphorylated S6 ribosomal protein; PDGF-B, platelet-derived growth factor, subunit B.

  2. a

    Difference in the percentage between 2 populations was tested by the Fisher exact test.

  3. b

    Bold type indicates statistical significance.

PDGF-B52/6678.8.0332b.74491.1062
PDGFRα27/6640.9.0773.7865.2109.0756
PDGFRβ55/6683.3.1912.2867.6782.1016
PDGF-B and PDGFRα24/6636.4.005b.7828.3454.27011.4365
PDGF-B and PDGFRβ50/6675.8.0079b.2175.2765.014b.0769.0032b
         
 Positive%p-Aktp-mTORp-4EBP1p-S6RPp-c-met (Nuclear Stain)p-c-met (Cytoplasmic Stain)
HGFα38/6558.5.2053.1775.7596.7879
c-met58/6687.9.22741.6198.6666
HGFα and c-met30/6546.2.0448b.28631.7894.0003b1
         
 Positive%p-Aktp-mTORp-4EBP1p-S6RPp-IGF-1Rβ 
IGF-1Rα15/6423.4.1495.75881.5208 
IGF-2R41/6563.0387b.41251.7848 
IGF-118/6627.3.0498b.2287.4881 
IGF-236/6654.5.8043.42061.2941 
(IGF-1 or 2) and IGF-1Rα10/6415.6.1651.7116.38061<.0001b 

Western Blot Analysis

Western blot analysis for p-Akt, p-mTOR, p-4EBP1, p-S6RP, p-PDGFRα, p-PDGFRβ, and p-c-met confirmed the existence of the phospho-proteins examined by the immunohistochemical method, and the results are presented in Figure 4. p-IGF-1Rβ was excluded because all the cases used for Western blot analysis were immunohistochemically negative for p-IGF-1Rβ. These results demonstrate that the immunohistochemically positive proteins were phosphorylated to a higher extent in the tumor samples than in the nonneoplastic tissue based on Western blot analysis.

image

Figure 4. Results of Western blot analysis are shown for phosphorylated proteins in the tumor samples. Densitometric analysis was demonstrated in the cases that included normal tissue (N). The degree of phosphorylation was calculated as follows, compared with N in each case: (p-protein [tumor]/actin [tumor])/(p-protein [normal]/actin [normal]). The tumor tissue (T) tended to be phosphorylated more than N. M indicates male; F, female; IHC, immunohistochemistry; p-mTOR, phosphorylated mammalian target of rapamycin; p-4EBP1, phosphorylated 4E-binding protein; p-S6RP, phosphorylated S6 ribosomal protein; p-PDGFRα, phosphorylated platelet-derived growth factor receptor-α; p-PDGFRβ, phosphorylated platelet-derived growth factor receptor-β.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

With regard to the clinical aspect of SFTs, in the great majority of cases, SFT demonstrates favorable clinical behavior, but at least 10% to 15% of cases show more aggressive clinical outcomes that appear to be related to some of the morphologic features originally identified in reports by England et al[16] and Vallat-Decouvelaere et al.[17] Complete resection with tumor-free margins is the best predictor of good prognosis and is the mainstay of treatment, but local recurrences have also been well demonstrated in patients with histologically benign-appearing, completely resected SFTs.[19, 20] In addition, radiotherapy and chemotherapy, either singly or in combination, have to our knowledge never been proven clinically effective. For all these reasons, alternative treatments for unresectable tumors are urgently needed.

Continuing with the clinical considerations, we note that hypoglycemia, a classic symptom in SFT cases, has been reported to be related to poor prognosis, and that IGF-2 secretion has been reported to induce hypoglycemia.[21-24] In the current series, hypoglycemia was found to be a significant predictor of aggressive clinical outcome, although it which growth factor would induce hypoglycemia was not identified.

Statistical histopathological analysis confirmed that cystic degeneration and myxoid degeneration were prognostic factors. The data from the current study do not necessarily correspond with the criteria proposed by England et al,[16] most likely because the tumors with more than intermediate nuclear atypia but less than pleomorphic nuclei were not diagnosed as malignant. Cystic degeneration, a newly proposed feature based on the current study data, was found to be closely associated with necrosis (P = .0262); therefore, cystic degeneration may be formed by a necrosis-associated mechanism. If there is any degree of cystic or myxoid change in the tumor, necrosis or other malignant features should be closely reviewed.

The Akt-mTOR pathway appears to play a role in the development of various types of neoplasms. Several investigations into the Akt-mTOR pathway or its elements have been conducted, including analyses of their expression or significance in molecular target therapy.[11-13, 25-30] In the current study, 50% to 80% of SFT cases demonstrated immunohistochemical positivity for p-Akt, mTOR, 4EBP1, and S6RP, and there were also significant correlations noted among these factors, indicating the activation of the Akt-mTOR pathway in approximately one-half of the cases. In addition, activation of the Akt-mTOR pathway can affect the formation of malignancy-associated findings, especially with regard to necrosis and tumor size. In SFTs, tumor malignancy should be considered to consist of composite interactions of signal pathways, in part because there is only a marginal, rather than a definite, statistical relationship with clinical outcome in some molecules.

The Akt-mTOR pathway is also increasingly recognized as a prognostic predictor in some soft tissue sarcomas, such as leiomyosarcoma and malignant peripheral nerve sheath tumor.[11-14] This study demonstrated an association between activation of the Akt-mTOR pathway protein and malignant features such as tumor necrosis, whereas the association with actual clinical outcome could not be directly established. In addition, the insufficient number of cases with an aggressive clinical course and distinctive malignant histological features could have resulted in a large range of error, and therefore a larger study with more clinical data will be needed.

The expression of growth factor receptors has been assessed in some reports,[1-6] but the significance of growth factors in the clinical behavior or tumorigenesis of SFTs has to the best of our knowledge never been demonstrated. The growth factor receptors examined in the current study belong to RTKs, which were phosphorylated in varied percentages in the SFT cases in the current study. The RTKs have been shown to present different localizations with either a nuclear or cytoplasmic pattern predominating from tumor to tumor,[31-33] although in the current study the localization of RTKs appeared to have no definite association with biological behavior. With regard to the relationship between RTKs and the Akt-mTOR pathway, PDGFRβ and IGF-1R may be able to deliver signals to the Akt-mTOR pathway. Conversely, PDGFRα and c-met appear to be remotely relevant to the activation of the Akt-mTOR pathway. We speculate that these RTK signaling pathways also link to other signaling systems such as the mitogen-activated protein kinases (MAPK) cascade or the JAK/STAT pathway, making it difficult to precisely analyze their effects.

The finding that growth factor ligands and receptors were coexpressed lends support to the idea that a paracrine-autocrine system was operating in these tumors. Indeed, the results of the current study indicate that ligand-receptor coexpression was significantly associated with phosphorylation of corresponding growth factor receptors. Both receptor-ligand coexpression and receptor-ligand coexpression-associated phosphorylation of the receptors even more convincingly indicates the paracrine-autocrine system, especially concerning the PDGF-B–PDGFRβ, HGF–c-met, and IGF–IGF-1R systems in the current study. Considering the association with the Akt-mTOR pathway, the PDGF-B–PDGFRβ system was only 1 growth factor system fulfilling all the conditions sustaining the existence of a functioning paracrine-autocrine system such as receptor-ligand coexpression-associated phosphorylation of the receptor, receptor-associated Akt phosphorylation, and receptor-ligand coexpression-associated phosphorylation of Akt. The data suggests that a multiple growth factor paracrine-autocrine system and interaction of those systems in SFTs may contribute to the formation of histopathological features of malignancy.

Growth factor receptors are known to share a downstream pathway, and a variety of signaling pathways are affected by multiple growth factor signals.[34] Although the analyzed signaling process appears not to affect poor prognosis, p-IGF-1Rβ was found to have a marginally significant relationship to tumor death, which indicates the necessity of a more broad-range molecular analysis, considering the interaction among growth factor receptors.

Soft tissue tumors associated with RTKs have attracted increasing attention, as represented by gastrointestinal stromal tumor or dermatofibrosarcoma protuberans, both of which are remarkably controlled by molecular target therapy using imatinib, a tyrosine kinase receptor inhibitor.[35] The efficacy of the therapeutic targeting of growth factor signaling pathways has been examined in various tumors, such as lung, breast, ovarian, kidney, colon, thyroid, liver, and gastric cancers; indeed, several clinical trials are currently in progress.[33, 36-38] The current study concentrated on analysis performed in clinical samples, and we could not find an available SFT cell line. The establishment of cell lines from SFT would allow further investigation into the roles and mechanisms of RTKs and into Akt/mTOR pathway activation. Despite this limitation, the results of the current study may have significant clinical and therapeutic implications in light of the potential of RTKs and the Akt/mTOR pathway as therapeutic targets. Thus, the results of the current study encourage the application of such inhibitors to the treatment of patients with SFT.

The NAB2-STAT6 fusion gene, a crucial tool for the diagnosis of SFT, was introduced by Chmielecki et al and Robinson et al in 2013[39, 40]. We have already prepared the molecular genetic data of their fusion transcripts in another investigation concerning its biological function.

The results of the current study demonstrate the activation of the Akt/mTOR pathway, the existence of paracrine-autocrine system–associated phosphorylation of RTKs, and some histological features as candidate prognostic factors. Although the relationship between the growth factor receptors and the definite signaling tracts under the RTKs remains to be elucidated, the RTKs and the Akt/mTOR pathway have the potential to be therapeutic targets.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Supported by a Grant-in-Aid for Scientific Research (B) (No. 2139107) from the Japan Society for the Promotion of Science, by the National Cancer Center Research and Development Fund (12-B-12), and by the Scholarship of Takeda Science Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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