Clin Exp Metastasis (2014) 31:715–725 DOI 10.1007/s10585-014-9662-5
RESEARCH PAPER
Hyaluronan expression as a significant prognostic factor in patients with malignant peripheral nerve sheath tumors Kunihiro Ikuta • Hiroshi Urakawa • Eiji Kozawa • Eisuke Arai Lisheng Zhuo • Naohisa Futamura • Shunsuke Hamada • Koji Kimata • Naoki Ishiguro • Yoshihiro Nishida
•
Received: 9 October 2013 / Accepted: 6 June 2014 / Published online: 24 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Hyaluronan (HA) regulates malignant tumor growth, invasion, and metastasis. However, few studies have focused on the roles of HA in tumorigenicity in malignant peripheral nerve sheath tumors (MPNST). In this study, we sought to clarify the prognostic value of HA in patients with MPNST. Specimens obtained from 15 patients with neurofibroma and 30 with MPNST were subjected to HA staining and scored as three grades. Protein expressions of HA synthase 1–3 were examined in the 22 MPNST tissue samples available. Statistically higher HA positivity was observed in MPNST as compared with neurofibroma (P = 0.020). The univariate analysis revealed that increased HA expression, age, neurofibromatosis type 1 (NF1) status, large tumor size, and histological grade were significantly associated with reduced overall survival of patients with MPNST; while increased HA expression, NF1 status, tumor size, and histological grade were correlated with disease-free survival. However, HA synthase 1–3 expression related to neither overall survival nor disease-free survival of these patients. In multivariate analysis, large tumor size (P = 0.022) was an independent prognostic factor for overall survival, and HA expression (P = 0.028) and tumor size (P = 0.002) were independent prognostic factors for disease-free survival. Statistically higher levels of HA in the human MPNST
cells were observed compared with neurofibroma cells in vitro. Our results demonstrate that HA expression can be a useful marker in differentiating MPNST from neurofibroma, and in identifying patients with a poor prognosis. Hyaluronan-targeting therapy for patients with MPNST may have potential as a therapeutic tool. Keywords Malignant peripheral nerve sheath tumors Hyaluronan Neurofibroma Prognostic factor Abbreviations MPNST Malignant peripheral nerve sheath tumors NF1 Neurfibromatosis type 1 HA Hyaluronan HAS Hyaluronan synthase BSA Bovine serum albumin PBS Phosphate buffered saline b-HABP Biotinylated hyaluronic acid binding protein DMEM Dulbecco’s modified Eagle’s medium VEGF Vascular endothelial growth factor HR Hazard ratio CI Confidence interval SD Standard deviation
Introduction K. Ikuta H. Urakawa E. Kozawa E. Arai N. Futamura S. Hamada N. Ishiguro Y. Nishida (&) Department of Orthopaedic Surgery, Nagoya University Graduate School and School of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan e-mail: [email protected] L. Zhuo K. Kimata Research Complex for the Medicine Frontiers, Aichi Medical University, Yazako, Nagakute, Aichi, Japan
Malignant peripheral nerve sheath tumors (MPNST) are uncommon tumors accounting for 5–10 % of all soft tissue sarcomas [1]. In 25–50 % of cases, MPNST are associated with neurofibromatosis type 1 (NF1), a tumor suppressor gene syndrome with an incidence of 1 in 3,000, whereas the remainder develop sporadically [1, 2]. NF1 is the most important known risk factor for developing MPNST, and
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MPNST is a leading cause of NF1-related mortality. It is known that MPNST in patients with NF1 arise within preexisting plexiform neurofibromas. Plexiform neurofibromas involving deep nerves have an increased risk for developing MPNST [3]. In contrast, cutaneous neurofibromas are superficial benign tumors, which have a low risk of malignant transformation. It is sometimes difficult to differentiate MPNST from neurofibroma [4, 5]. Because of their deep anatomic location, MPNST are frequently diagnosed at an advanced stage when primary tumor size may be larger and metastatic progression to the lungs has already occurred [6]. Surgical resection remains the mainstay of treatment for MPNST. However, MPNST are aggressive tumors known to show a poor response to standard chemotherapy and radiation therapy, and to have a high metastatic potential even after adequate surgical resection. The prognosis for patients with MPNST is generally poor with a 5-year survival rate of 20–50 % [7]. Therefore, it is important to diagnose MPNST accurately at an early stage, and novel markers are required to better predict the clinical course of these patients. Hyaluronan (HA) is a high-molecular weight linear glycosaminoglycan composed of repeating disaccharides of D-glucuronic acid and N-acetylglucosamine. HA is one of the major components of extracellular matrix and plays important roles in morphogenesis, wound healing, and embryogenesis [8, 9]. Previous studies reported that extracellular HA stimulates growth, migration, and invasion of various malignant tumors [10, 11], and that HA levels in tumor tissues correlate with an unfavorable prognosis of patients with malignancies such as ovarian, lung, thyroid, breast, colorectal and gastric cancers [12–17]. To our knowledge, no study has focused on the roles of HA in MPNST or its clinical relevance in patients with MPNST. HA is synthesized by three types of HA synthase (HAS1, HAS2, and HAS3) at the intracellular face of the plasma membrane and is extruded to the cell surface and extracellular matrices [18]. Determination of HAS1–3 expressions is essential for investigating the biological functions of HA. Although several studies have demonstrated that the manipulation of HAS genes in tumor cell lines alters its tumorigenic potential [19–22], few have examined HAS1–3 expression in human tumor tissues. In this study, we compared the expression of HA between neurofibroma and MPNST to investigate its potential to distinguish these tumors, and we assessed the correlation of the expression of HA and HAS1–3 with the clinical outcome of patients with MPNST.
Materials and methods Patient characteristics Between 1986 and 2011, 30 patients with MPNST and 15 patients with neurofibroma who were diagnosed and treated
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Clin Exp Metastasis (2014) 31:715–725 Table 1 Patient and tumor characteristics
Total
Neurofibroma
MPNST
15
30
7
14
Gender Female Male
8
16
40.8 (19–61)
44.5 (17–77)
Positive
7
16
Negative
8
14
Age (mean) NF1 status
Tumor site Extremity
10
16
Trunk Tumor size
5
14
B10 cm
8
19
[10 cm
3
8
Unknown
4
3
Subcutaneous
2
6
Deep tissues
11
20
Unknown
2
4
Low
–
11
High
–
19
Tumor depth
Histological grade
at our institution were enrolled in this study. The clinical data were reviewed on the basis of the patients’ records. Biopsy or surgical specimens unexposed to chemotherapeutic agents were obtained, and all diagnoses were confirmed by experienced pathologists. Sixteen patients with MPNST and seven with neurofibroma developed the disease in association with NF1. Eleven cases of low grade MPNST were also included in this study. Low grade MPNST was diagnosed when there was generalized nuclear atypia, increased cellularity, and typically low levels of mitotic activity. According to the Federation Nationale des Centres de Lutte Contre le Cancer grade system, grade 1 was defined as low grade in this study based on three parameters; tumor differentiation, mitotic index, and tumor necrosis. Because atypical neurofibromas (benign category) are occasionally difficult to differentiate from low grade MPNST [5], final pathological diagnosis was decided by our experienced pathologists after careful discussion. There were 16 males and 14 females with a mean age of 44.5 years (range 17–77 years) in patients with MPNST; and eight males and seven females with a mean age of 40.8 years (range 19–61 years) in those with neurofibroma, respectively. The 15 cases of neurofibroma included two cutaneous neurofibromas, 11 plexiform neurofibromas, and
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two neurofibromas unavailable for subtype data. The location of MPNST was an extremity in 16 cases (upper extremity in six cases, lower extremity in 10 cases) and the trunk in 14 cases (neck or back in seven cases, pelvic girdle in two cases, brachial plexus in three cases, retroperitoneum in one case, chest wall in one case). The location of neurofibroma was an extremity in ten cases (upper extremity in one, lower extremity in nine) and a trunk in five cases. The mean tumor size was 9.0 cm (range 2–22 cm) in patients with MPNST and 7.9 cm (range 2–17 cm) in those with neurofibroma. The mean length of follow-up for patients with MPNST was 46.8 months (range 2–104 months). Patient and tumor characteristics are listed in Table 1. Twenty-eight of 30 patients with MPNST were treated by either limb-sparing surgical resection or amputation aimed to achieve wide or marginal margins. If a resection margin was microscopically positive, the patient underwent radiotherapy after surgery. Patients were not routinely treated with chemotherapy. Generally, MPNST with large (more than 5 cm) and high grade tumors were administered chemotherapy in addition to surgical treatment. Two patients who underwent biopsy without surgery were also included in this study. Because both of them had already developed lung metastasis by the first visit, they received conservative therapy without surgical treatment for the primary lesions.
Immunohistochemical staining for HAS1, HAS2, and HAS3
HA staining
Evaluation of stainability
Tumor tissue samples unexposed to chemotherapeutic agents were subjected to HA staining. Informed consent was obtained from each patient for the use of tumor samples. The staining procedure was performed as described previously [23]. In brief, formalin-fixed, paraffin-embedded tumor sections of 5 lm thickness were deparaffinized with xylene, and rehydrated through graded ethanols. The slides were treated with 0.3 % H2O2 in methanol for 30 min to block the internal peroxidase activity, followed by incubation with 1 % bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at room temperature. The sections were then incubated with 2.0 lg/ml biotinylated HA-binding protein (b-HABP; Seikagaku, Tokyo, Japan) probe for 2 h at room temperature. Bound b-HABP was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidinecontaining substrate solution (Nichirei, Tokyo, Japan). The slides were counterstained with hematoxylin, dehydrated and then mounted. Stained sections incubated without b-HABP or pre-treated with 5 U/ml Streptomyces hyaluronidase for 1 h at 60 °C were used as negative controls.
Two observers (NF, KI) without any knowledge of the clinicopathological information evaluated the results of HA and HAS1–3 staining on the basis of a three-point scale: 0 % for positive stainable cell number (negative); 1–20 % (weak); 21–100 % (strong; Fig. 1a). On the basis of these criteria, both observers could analyze and grade the degree of positivity or negativity in all the samples. Since the specimens were heterogeneous in each case, three fields of magnification (magnification 4009) were randomly selected, and the mean positivity of these three fields was considered as representative of each case.
HAS1, HAS2, and HAS3 immunostaining was performed with 22 MPNST samples available since 1997. Not all tissues could be obtained for HAS1–3 immunohistochemistry because of the limited availability of MPNST tissues. Deparaffinized and rehydrated sections were treated with 0.3 % H2O2 in methanol for 30 min, followed by incubation with 1 % BSA in PBS for 1 h at room temperature. Thereafter, sections were incubated for 1 h with polyclonal antibodies against HAS1, HAS2, and HAS3 raised in rabbits by subcutaneous injection of the following synthetic peptides: VRRLCRRRSGGTRVGV, corresponding to amino acids 568–582 of HAS1; CGRRKKGQQYDMVLDV, corresponding to amino acids 537–552 of HAS2; and CGKKPEQYSLAFAEV, corresponding to amino acids 541–555 of HAS3, which had been coupled to keyhole limpet hemacyanin. These antibodies have been used in HAS immunostaining for human tissues in previous studies [24, 25]. Biotinylated anti-rabbit goat IgG (Nichirei, Tokyo, Japan) was applied as a secondary antibody for 30 min at room temperature, and antibody binding was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidine-containing substrate solution (Nichirei, Tokyo, Japan). The slides were counterstained with hematoxylin, dehydrated and then mounted. Stained sections incubated without primary antibodies were used as negative controls.
HA contents in tumor tissues To determine the levels of HA in tumor tissues, frozen samples from 20 patients with MPNST and six with neurofibroma, which were obtained at biopsy or resection and stored at -80 °C, were subjected to quantification with HA binding assay. The digestion of tumor tissues and HA extraction were performed according to the protocol described previously with a minor modification [26]. Briefly, each tissue sample was dried overnight at 60 °C to
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Fig. 1 a Representative images of staining grade for HA and HAS1-3 are shown (original magnification 9400). b Representative images of MPNST with strong HA expression and neurofibroma with negative HA expression (original magnification 9400). Bars depict 100 lm
determine the dry weight, and then dissolved in 2 ml of 0.15 M Tris–HCl, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM deferoxamine mesylate, pH 7.5, containing 40 U of protease for 8 h at 55 °C. Following incubation, all samples were heated at 100 °C for 15 min to inactivate protease activity and centrifuged at 15,000g for 30 min at 4 °C, and the supernatants were extracted. HA concentration was quantitatively determined with HA binding assay, as described previously [27]. Cell culture and quantification of HA The human neurofibroma cell line, Hs53T and the human MPNST cell line, sNF02.2 were obtained from the American
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Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. The cultures were maintained in a humidified atmosphere with 5 % CO2 at 37 °C. Following incubation for 12 and 24 h, HA was isolated according to the methods reported previously [28]. Briefly, the conditioned medium was collected and designated as ‘‘medium.’’ To remove the cell-surface associated HA, the cells were incubated for 10 min at 37 °C with trypsin–EDTA and washed with PBS. The trypsin solution and combined washes were designated as ‘‘pericellular.’’ After cell counts, the cells were placed in Protease K solution (0.15 M Tris– HCl, pH 7.5, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM
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deferoxamine mesylate containing 20 U of protease K) and incubated for 2 h at 55 °C and the solution was designated as ‘‘intracellular.’’ All samples were heated at 100 °C for 15 min to inactivate protease activity and centrifuged at 15,000g for 30 min at 4 °C, and the supernatants were subjected to the analyses for HA content. Because the cell size of sNF02.2 was larger than that of Hs53T, HA concentration was standardized by the amount of protein in each cell type. Following incubation for 12 and 24 h, total protein was extracted with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and protein concentration was determined with Protein Assay Reagent (Bio-Rad, Hercules, CA, USA).
719 Table 2 Expression of HA and HASs in relation to clinical outcome Positive cell fraction (%)
No. of cases
No. of patients with metastasis
No. of deceased patients
HA
30
12
7
Negative (0 %)
6
1
0
Weak (1–20 %)
15
4
3
Strong (21–100 %) HAS1 Negative (0 %) Weak (1–20 %) Strong (21–100 %) HAS2
Statistical methods SPSS 20.0 for Windows software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Fisher’s exact test was used to analyze the relationship of various clinicopathological variables to positivity of HA and HASs. Overall survival period was calculated from the date of the first visit to that of death or final follow-up. Disease-free survival period was calculated from the date of the first visit to that of first event recorded (local recurrence or metastasis). Prognosis of patients with MPNST was determined with Kaplan–Meier method according to the positivity of HA and HAS1–3, as well as each clinicopathological variable, and the differences were statistically analyzed with log-rank test. However, 3 of the 30 patients with MPNST were excluded from the analysis of diseasefree survival because of the presence of metastasis at the first visit. Similarly, 2 of 22 patients with MPNST evaluated for HAS1–3 expressions were excluded from the analysis of disease-free survival. Cox’s regression model was used for multivariate analysis. Quantitative experiments were performed more than three times, and analysis of variance followed by Bonferroni Dunn post hoc test was used to assess differences between means. Statistical comparison between two groups was analyzed with Student’s t test. P values \0.05 were considered statistically significant.
Results
9
7
4
22
10
5
5
3
1
6 11
0 7
0 4 5
22
10
Negative (0 %)
4
1
0
Weak (1–20 %)
5
3
2
Strong (21–100 %) HAS3
13
6
3
22
10
5
Negative (0 %)
7
1
0
Weak (1–20 %)
8
5
3
Strong (21–100 %)
7
4
2
(Fig. 1b). Although HA also accumulated in surrounding stromal tissues in most samples, we focused solely on the positivity associated with tumor cells and extracellular matrix around tumor cells. Staining grade for HA in MPNST samples was negative in six (20 %), weak in 15 (50 %), and strong in nine (30 %; Table 2), while that in neurofibroma samples was negative in four (27 %) and weak in 11 (73 %). No case of neurofibroma showed strong HA expression (Fig. 1b). Dividing three groups into two groups; negative to weak positive and strong positive group, HA positivity was statistically higher in MPNST tissues than in neurofibroma (P = 0.020; Table 3). In the analysis between MPNST and plexiform neurofibroma, HA positivity in MPNST tissues showed a tendency to increase as compared with that in plexiform neurofibroma tissues (P = 0.082). We evaluated the correlation of staining grade with clinicopathological variables in patients with MPNST. No significant correlations were noted between the intensity of HA staining and gender (P = 0.69), age (P = 0.69), NF1 status (P = 0.12), tumor site (P = 1.0), tumor size (P = 1.0), tumor depth (P = 0.13), or histological grade (P = 0.10; Table 4).
HA expression in neurofibroma and MPNST HASs expression in MPNST The distribution of clinicopathological variables did not differ significantly between patients with neurofibroma and those with MPNST. In 15 tissue samples collected from patients with neurofibroma and 30 samples from patients with MPNST, the expression levels of HA were diverse (Fig. 1a). HA positivity was observed in the extra- and pericellular matrix and/or intracytoplasm of tumor cells
HAS1–3 positivity was observed in the cytoplasm and/or on the surface of tumor cells. Of 22 MPNST tissue samples examined for HAS1–3 expression, 11 (50 %) showed strong HAS1 expression, 13 (59 %) strong HAS2 expression, and 7 (32 %) strong HAS3 expression. The relationship between HAS expression and clinical outcome is
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Table 3 Expression of HA in neurofibroma and MPNST HA positivity
P value
Negative–weak
Strong
Neurofibroma
15
0
MPNST
21
9
a
Table 5 Univariate analysis of overall and disease-free survival a
Variables
5-Year survival (%) 0.020
Negative–weak Total
30
21
Strong
74
\45
94
C45
69
0.69 14
9
5
Male
16
12
4
16
12
4
C45 NF1 status
14
9
5
Negative
14
12
2
Positive
16
9
7
Extremity
16
11
5
Trunk
14
10
4
Age
0.69
0.12
Tumor site
1.0
Tumor size
1.0
Negative
100
Positive
67
Extremity
81
Trunk
85
B10 cm
95
[10 cm
56
Subcutaneous
100
Low
100
High
72
[10 cm
8
6
2
Strong
59
Unknown
3
62
0.67
68 0.019
85
0.025
42 0.66
66
0.77
64 0.02
79
0.001
0 0.17
83
0.23
56 0.034
91
0.032
47 0.041
76
0.019
22
HAS1 expression 0.13
6
6
0
Negative– weak
90
Subcutaneous Deep tissues
20
12
8
Strong
80
0.33
70
0.26
40
HAS2 expression
4
Negative– weak
0.10 10
1
11
8
Fisher’s exact test
shown in Table 2. In the analyses of the correlation between the intensity of HAS1–3 expression and clinicopathological variables, HAS1 was associated with age (C45 years, P = 0.019), and HAS2 and HAS3 with histological grade (P = 0.041 and P = 0.040, respectively). Relationship between stainability and survival of patients with MPNST Of 30 patients with MPNST, three developed recurrent disease and 12 metastasis. At the last follow-up, 18 patients remained continuously disease-free or with no evidence of
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0.048
HA expression 91
19
0.77
63
Deep tissues 80 Histological grade
Negative– weak
High
68
Tumor depth
6
Unknown
0.21
Tumor size
13
Histological grade Low 11
P valuea
Tumor site
19
Tumor depth
a
P valuea
9
Female
B10 cm
5-Year survival (%)
NF1 status
Gender
\45
92
Male Age
Table 4 Correlation of HA expression with clinicopathological variables in patients with MPNST HA staining
P value
Disease-free survival a
Gender Female
Fisher’s exact test
No.
Overall survival
Strong
100
0.38
74
56
0.96
58
HAS3 expression Negative– weak Strong a
93 73
0.62
55
0.84
63
Log-rank test
disease, seven died of disease, and the remaining five were alive with disease. All patients who died of disease had developed distant metastasis. For patients with MPNST, overall survival and diseasefree survival at 5 years were 83 and 65 %, respectively. Strong HA expression was seen in four patients (57 %) with dead of disease and 7 (58 %) with distant metastasis. Overall survival at 5 years was 59 % in patients with
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Fig. 2 Kaplan–Meier survival curves of overall survival for HA and HAS 1–3 expressions in MPNST. a Subdivided according to HA expression (strong vs. negative-weak; N = 30). b Subdivided according to HAS1 expression (N = 22). c Subdivided according to HAS2 expression (N = 22). d Subdivided according to HAS3 expression (N = 22)
strong HA expression and 91 % in those with weaker HA expression, and disease-free survival at 5 years for these patients was 22 and 76 %, respectively. Univariate analysis revealed that strong HA expression was significantly associated with decreased overall survival (P = 0.041) and disease-free survival (P = 0.019) using log-rank test. However, HAS expression was related to neither overall survival nor disease-free survival in patients with MPNST (Table 5; Figs. 2, 3). In the multivariate analysis, strong HA expression (P = 0.028) was still an independent prognostic factor for disease-free survival. Strong HA expression and poor overall survival in patients with MPNST showed a trend to correlate (P = 0.089; Table 6). In the univariate analysis of clinicopathological variables, age (P = 0.048), NF1 status (P = 0.019), tumor size (P = 0.020), and histological grade (P = 0.034) were found to be significant prognostic factors for poorer overall survival, while NF1 status (P = 0.025), tumor size (P = 0.001), and histological grade (P = 0.032) were significantly correlated with disease-free survival. There was no statistically significant association between overall and disease-free survival and variables such as gender, tumor site, or tumor depth (Table 5). In the multivariate analysis, large tumor size was independently related to reduced overall survival and disease-free survival (P = 0.022 and P = 0.002, respectively; Table 6).
Quantification of HA We used the HA binding assay to measure HA levels (lg/g) in the extracts from neurofibroma and MPNST tissue samples. The mean HA concentrations in neurofibroma and MPNST tissues were 238.2 and 275.4 lg/g, respectively. There was no significant difference between the HA levels in neurofibroma tissues and those in MPNST tissues (P = 0.750; Fig. 4). The levels of HA in pericellular region and in medium in sNF02.2 cells incubated for 24 h were significantly higher than those in Hs53T cells (P = 0.002 and P = 0.001, respectively; Fig. 5a, b). The levels of intracellular HA in sNF02.2 cells were significantly higher than that in Hs53T cells at 12 and 24 h (P = 0.008 and P \ 0.001, respectively; Fig. 5c).
Discussion Our results demonstrated for the first time the differential expression of HA between MPNST and neurofibroma, and showed that increased HA expression in tissues correlates with unfavorable survival outcome in patients with MPNST. In contrast, HAS1–3 expressions were not related to the prognosis of patients with MPNST.
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Fig. 3 Kaplan–Meier survival curves of disease-free survival for HA and HAS 1–3 expressions in MPNST. a Subdivided according to HA expression (strong vs. negativeweak; N = 27). b Subdivided according to HAS1 expression (N = 20). c Subdivided according to HAS2 expression (N = 20). d Subdivided according to HAS3 expression (N = 20)
Table 6 Multivariate analysis of overall and disease-free survival
Variables
Overall survival HR
Disease-free survival a
HR
95 % CI
P value
1.35–46.8
0.022
7.94
0.79–26.9
0.089
5.65
95 % CI
P valuea
2.07–30.4
0.002
1.21–26.4
0.028
Tumor size B10 cm
1
[10 cm
7.96
1
HA expression
a
Cox’s regression model
Negative–weak
1
Strong
4.61
HA is known to play an important role in the tumorigenicity of malignant tumors. Previous studies revealed that higher expression of HA is significantly associated with poor prognosis in many types of human tumor [12–17], suggesting agreement regarding HA as a crucial prognostic factor. However, few reports have analyzed the significance of HA in mesenchymal malignancies [22]. On the other hand, few studies have reported the correlation between HAS expression in clinical tumor samples and poor patient prognosis. HAS1 expression is known to serve as a prognostic indicator in some tumors, including malignant mesothelioma and ovarian cancer [24, 29]. In specimens of colon cancer, Yamada et al. [30] examined mRNA levels of three HASs, and showed a positive correlation between an increased HAS1 mRNA level and poor patient prognosis. Nykopp et al. [31] investigated the hyaluronidase expression in addition to HASs in endometrioid endometrial
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1
cancer, and demonstrated that the accumulation of HA was influenced by not only HAS expression but also hyaluronidase expression. Complicated regulation mediated by both HASs and hyaluronidases acts to control HA accumulation in malignant tissues, but post-translational modification of HASs has not been clarified yet. The current study showed no association of HAS expression with survival of patients with MPNST possibly due to the yet-to-be-defined regulation mechanisms of HASs. Together, evaluation of HAS expression is complicated and not adequate by itself to identify patients having a poor prognosis. Evaluation of HA positivity in MPNST tissue samples is a readily available and useful modality to identify patients with poor survival. Recently, Slomiany et al. [32] investigated the roles of HA on MPNST in vitro and in vivo. In the study, HA oligomers, which inhibited HA production, suppressed HA secretion in MPNST cells, decreased doxorubicin resistance of MPNST,
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and suppressed the growth of MPNST xenografts in mice, which suggested the importance of HA in tumorigenicity of MPNST. This previous report supports our findings that increased HA expression in MPNST tissues correlated with poor prognosis in patients with MPNST. Mechanisms of malignant transformation of benign neurofibroma to MPNST are not fully understood at the molecular level. Because of the dismal prognosis of patients with MPNST, it is important to identify them at an early stage, especially patients with NF1, as well as those with localized MPNST who are at high risk for metastatic development. Occasionally, it is difficult pathologically to differentiate neurofibroma with atypical histological features from MPNST [5]. It is also difficult to predict the clinical course of patients with MPNST. Therefore, useful
markers are required to detect MPNST at an early stage so as to avoid delaying treatment, and to alter the treatment modality for patients with a poor prognosis. Previous reports have demonstrated that several proteins can differentiate patients with neurofibroma from those with MPNST. Wasa et al. [33] showed that positive immunostaining for vascular endothelial growth factor (VEGF) was significantly higher in MPNST tissues compared with those of neurofibroma. Zou et al. [34] reported that the mean staining intensity of p53 could significantly differentiate MPNST from neurofibroma in tissue microarray analysis. In our study, HA expression was significantly higher in patients with MPNST than in those with neurofibroma, and no patients with neurofibroma showed strong HA expression. This finding suggests that HA expression may be a useful tool to differentiate MPNST from neurofibroma, particularly low grade MPNST from neurofibroma. In vitro experiment of our study, the levels of intracellular, pericellular, and medium HA in human MPNST cells were significantly higher than those in human neurofibroma cells. This in vitro findings supports our results of HA positivity that can differentiate MPNST from neurofibroma. In the present study, however, we could obtain only Hs53T cells as commercially available neurofibroma cells. Some previous reports showed that HA content in plexiform neurofibroma tissues was increased compared to those in cutaneous neurofibroma tissues [35, 36]. However, there was no study evaluated HA content in MPNST tissues. In the current study, HA content in MPNST tissues was higher than that in plexiform neurofibroma tissues, but it was not statistically significant. One possible explanation was that tumorous tissues contain not only tumor cells but stromal cells, which may affect HA content. Other explanation is the small number of cases of neurofibroma tissues. A number of studies have analyzed possible prognostic factors of the clinical outcome of patients with MPNST [6, 33, 34, 37–41]. In these studies, clinicopathological factors
Fig. 5 Data presented are the HA contents in Hs53T and sNF02.2 cells standardized with the content of protein in these cells (ng/lg). HA contents were measured at 12 and 24 h time points of cell culture. a HA concentration in medium. b Pericellular HA concentration.
c Intracellular HA concentration. Each experiment was performed in triplicate, and bars indicate the mean ± SD (*P \ 0.01 and **P \ 0.001 compared with Hs53T cells as measured using Bonferroni–Dunn post hoc test)
Fig. 4 HA concentration in neurofibroma and MPNST tissues. The mean value HA concentration for neurofibroma is 238.2 lg/g (N = 6), and that for MPNST is 275.4 lg/g (N = 20). The level of HA in tissue samples did not differ significantly between neurofibroma and MPNST (P = 0.750, Student’s t test). Each experiment was measured in triplicate, and bars indicate the mean ± SD
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including older age, large tumor size, axial tumor site, inadequate surgical margin, NF1 status, local recurrence, previous radiotherapy, and tumor histological grade were linked with poor MPNST patient prognosis. In the current study, we reviewed the clinical records of 30 patients with MPNST to investigate the prognostic impact of various clinicopathological features. Univariate analysis indicated that age, NF1 status, tumor size and histological grade were prognostic factors for poor survival, while of these four factors, only tumor size remained an independent prognostic factor in multivariate analysis. Thus tumor size seems to be the most important risk factor for survival in MPNST, which is consistent with previous studies demonstrating that it was significantly associated with poor survival in MPNST. [6, 34, 37, 39–41]. Zou et al. [34] reported in a study of 140 patients that those with tumors larger than 10 cm carried a threefold increased risk of developing distant metastasis. Although the cutoff values of tumor size in these studies were variable, ranging from 5 to 10 cm, tumor size was found to be the most reliable and uncontroversial predictor of prognosis in patients with MPNST. However, a biomarker other than clinical variables would also be necessary to better predict the prognosis of patients with MPNST. There were several limitations in our study. First, the number of patients was small due to the rarity of MPNST, and it may have been underpowered to detect significant differences between groups. For instance, positivity of HA was significantly increased in MPNST compared with all neurofibromas, however, not significantly increased compared with plexiform neurofibroma (P = 0.082). Further studies with greater numbers of patients will be needed to better clarify the correlation of HA with patient prognosis. Second, there was a relatively high rate (37 %) of low grade MPNST in this study compared with that in previous studies (18–22 %) [6, 40]. In our institution, routine magnetic resonance imaging examination for neurofibroma in patients with NF1 is thought to facilitate the early detection of malignant transformation of the tumors as reflected by the high percentage of low grade MPNST detected. Third, the results of staining may be influenced by both the sensitivity of the antibody and protocol. However, the results of staining should be stable if performed with the same antibody and protocol in a single institution. In conclusion, HA expression in tissues of patients with MPNST is easily evaluated in the clinical setting using histochemical staining compared with the evaluation of multiple HAS expression. Our results indicate that HA expression may be a useful marker in differentiating MPNST from neurofibroma, and in identifying MPNST patients having a poor prognosis. HA-targeting therapy for patients with MPNST may have potential as a therapeutic tool, and accordingly warrants further investigation.
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Clin Exp Metastasis (2014) 31:715–725 Acknowledgments We thank Ms. Eri Ishihara for secretarial assistance regarding this study. We are grateful to Drs. Satoshi Tsukushi, and Junji Wasa for collection of samples and data. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid 20591751 for Scientific Research (C)], and by the Suzuken Memorial Foundation. Conflict of interest of interest.
The authors declare that they have no conflict
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RESEARCH PAPER
Hyaluronan expression as a significant prognostic factor in patients with malignant peripheral nerve sheath tumors Kunihiro Ikuta • Hiroshi Urakawa • Eiji Kozawa • Eisuke Arai Lisheng Zhuo • Naohisa Futamura • Shunsuke Hamada • Koji Kimata • Naoki Ishiguro • Yoshihiro Nishida
•
Received: 9 October 2013 / Accepted: 6 June 2014 / Published online: 24 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Hyaluronan (HA) regulates malignant tumor growth, invasion, and metastasis. However, few studies have focused on the roles of HA in tumorigenicity in malignant peripheral nerve sheath tumors (MPNST). In this study, we sought to clarify the prognostic value of HA in patients with MPNST. Specimens obtained from 15 patients with neurofibroma and 30 with MPNST were subjected to HA staining and scored as three grades. Protein expressions of HA synthase 1–3 were examined in the 22 MPNST tissue samples available. Statistically higher HA positivity was observed in MPNST as compared with neurofibroma (P = 0.020). The univariate analysis revealed that increased HA expression, age, neurofibromatosis type 1 (NF1) status, large tumor size, and histological grade were significantly associated with reduced overall survival of patients with MPNST; while increased HA expression, NF1 status, tumor size, and histological grade were correlated with disease-free survival. However, HA synthase 1–3 expression related to neither overall survival nor disease-free survival of these patients. In multivariate analysis, large tumor size (P = 0.022) was an independent prognostic factor for overall survival, and HA expression (P = 0.028) and tumor size (P = 0.002) were independent prognostic factors for disease-free survival. Statistically higher levels of HA in the human MPNST
cells were observed compared with neurofibroma cells in vitro. Our results demonstrate that HA expression can be a useful marker in differentiating MPNST from neurofibroma, and in identifying patients with a poor prognosis. Hyaluronan-targeting therapy for patients with MPNST may have potential as a therapeutic tool. Keywords Malignant peripheral nerve sheath tumors Hyaluronan Neurofibroma Prognostic factor Abbreviations MPNST Malignant peripheral nerve sheath tumors NF1 Neurfibromatosis type 1 HA Hyaluronan HAS Hyaluronan synthase BSA Bovine serum albumin PBS Phosphate buffered saline b-HABP Biotinylated hyaluronic acid binding protein DMEM Dulbecco’s modified Eagle’s medium VEGF Vascular endothelial growth factor HR Hazard ratio CI Confidence interval SD Standard deviation
Introduction K. Ikuta H. Urakawa E. Kozawa E. Arai N. Futamura S. Hamada N. Ishiguro Y. Nishida (&) Department of Orthopaedic Surgery, Nagoya University Graduate School and School of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan e-mail: [email protected] L. Zhuo K. Kimata Research Complex for the Medicine Frontiers, Aichi Medical University, Yazako, Nagakute, Aichi, Japan
Malignant peripheral nerve sheath tumors (MPNST) are uncommon tumors accounting for 5–10 % of all soft tissue sarcomas [1]. In 25–50 % of cases, MPNST are associated with neurofibromatosis type 1 (NF1), a tumor suppressor gene syndrome with an incidence of 1 in 3,000, whereas the remainder develop sporadically [1, 2]. NF1 is the most important known risk factor for developing MPNST, and
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MPNST is a leading cause of NF1-related mortality. It is known that MPNST in patients with NF1 arise within preexisting plexiform neurofibromas. Plexiform neurofibromas involving deep nerves have an increased risk for developing MPNST [3]. In contrast, cutaneous neurofibromas are superficial benign tumors, which have a low risk of malignant transformation. It is sometimes difficult to differentiate MPNST from neurofibroma [4, 5]. Because of their deep anatomic location, MPNST are frequently diagnosed at an advanced stage when primary tumor size may be larger and metastatic progression to the lungs has already occurred [6]. Surgical resection remains the mainstay of treatment for MPNST. However, MPNST are aggressive tumors known to show a poor response to standard chemotherapy and radiation therapy, and to have a high metastatic potential even after adequate surgical resection. The prognosis for patients with MPNST is generally poor with a 5-year survival rate of 20–50 % [7]. Therefore, it is important to diagnose MPNST accurately at an early stage, and novel markers are required to better predict the clinical course of these patients. Hyaluronan (HA) is a high-molecular weight linear glycosaminoglycan composed of repeating disaccharides of D-glucuronic acid and N-acetylglucosamine. HA is one of the major components of extracellular matrix and plays important roles in morphogenesis, wound healing, and embryogenesis [8, 9]. Previous studies reported that extracellular HA stimulates growth, migration, and invasion of various malignant tumors [10, 11], and that HA levels in tumor tissues correlate with an unfavorable prognosis of patients with malignancies such as ovarian, lung, thyroid, breast, colorectal and gastric cancers [12–17]. To our knowledge, no study has focused on the roles of HA in MPNST or its clinical relevance in patients with MPNST. HA is synthesized by three types of HA synthase (HAS1, HAS2, and HAS3) at the intracellular face of the plasma membrane and is extruded to the cell surface and extracellular matrices [18]. Determination of HAS1–3 expressions is essential for investigating the biological functions of HA. Although several studies have demonstrated that the manipulation of HAS genes in tumor cell lines alters its tumorigenic potential [19–22], few have examined HAS1–3 expression in human tumor tissues. In this study, we compared the expression of HA between neurofibroma and MPNST to investigate its potential to distinguish these tumors, and we assessed the correlation of the expression of HA and HAS1–3 with the clinical outcome of patients with MPNST.
Materials and methods Patient characteristics Between 1986 and 2011, 30 patients with MPNST and 15 patients with neurofibroma who were diagnosed and treated
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Clin Exp Metastasis (2014) 31:715–725 Table 1 Patient and tumor characteristics
Total
Neurofibroma
MPNST
15
30
7
14
Gender Female Male
8
16
40.8 (19–61)
44.5 (17–77)
Positive
7
16
Negative
8
14
Age (mean) NF1 status
Tumor site Extremity
10
16
Trunk Tumor size
5
14
B10 cm
8
19
[10 cm
3
8
Unknown
4
3
Subcutaneous
2
6
Deep tissues
11
20
Unknown
2
4
Low
–
11
High
–
19
Tumor depth
Histological grade
at our institution were enrolled in this study. The clinical data were reviewed on the basis of the patients’ records. Biopsy or surgical specimens unexposed to chemotherapeutic agents were obtained, and all diagnoses were confirmed by experienced pathologists. Sixteen patients with MPNST and seven with neurofibroma developed the disease in association with NF1. Eleven cases of low grade MPNST were also included in this study. Low grade MPNST was diagnosed when there was generalized nuclear atypia, increased cellularity, and typically low levels of mitotic activity. According to the Federation Nationale des Centres de Lutte Contre le Cancer grade system, grade 1 was defined as low grade in this study based on three parameters; tumor differentiation, mitotic index, and tumor necrosis. Because atypical neurofibromas (benign category) are occasionally difficult to differentiate from low grade MPNST [5], final pathological diagnosis was decided by our experienced pathologists after careful discussion. There were 16 males and 14 females with a mean age of 44.5 years (range 17–77 years) in patients with MPNST; and eight males and seven females with a mean age of 40.8 years (range 19–61 years) in those with neurofibroma, respectively. The 15 cases of neurofibroma included two cutaneous neurofibromas, 11 plexiform neurofibromas, and
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two neurofibromas unavailable for subtype data. The location of MPNST was an extremity in 16 cases (upper extremity in six cases, lower extremity in 10 cases) and the trunk in 14 cases (neck or back in seven cases, pelvic girdle in two cases, brachial plexus in three cases, retroperitoneum in one case, chest wall in one case). The location of neurofibroma was an extremity in ten cases (upper extremity in one, lower extremity in nine) and a trunk in five cases. The mean tumor size was 9.0 cm (range 2–22 cm) in patients with MPNST and 7.9 cm (range 2–17 cm) in those with neurofibroma. The mean length of follow-up for patients with MPNST was 46.8 months (range 2–104 months). Patient and tumor characteristics are listed in Table 1. Twenty-eight of 30 patients with MPNST were treated by either limb-sparing surgical resection or amputation aimed to achieve wide or marginal margins. If a resection margin was microscopically positive, the patient underwent radiotherapy after surgery. Patients were not routinely treated with chemotherapy. Generally, MPNST with large (more than 5 cm) and high grade tumors were administered chemotherapy in addition to surgical treatment. Two patients who underwent biopsy without surgery were also included in this study. Because both of them had already developed lung metastasis by the first visit, they received conservative therapy without surgical treatment for the primary lesions.
Immunohistochemical staining for HAS1, HAS2, and HAS3
HA staining
Evaluation of stainability
Tumor tissue samples unexposed to chemotherapeutic agents were subjected to HA staining. Informed consent was obtained from each patient for the use of tumor samples. The staining procedure was performed as described previously [23]. In brief, formalin-fixed, paraffin-embedded tumor sections of 5 lm thickness were deparaffinized with xylene, and rehydrated through graded ethanols. The slides were treated with 0.3 % H2O2 in methanol for 30 min to block the internal peroxidase activity, followed by incubation with 1 % bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at room temperature. The sections were then incubated with 2.0 lg/ml biotinylated HA-binding protein (b-HABP; Seikagaku, Tokyo, Japan) probe for 2 h at room temperature. Bound b-HABP was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidinecontaining substrate solution (Nichirei, Tokyo, Japan). The slides were counterstained with hematoxylin, dehydrated and then mounted. Stained sections incubated without b-HABP or pre-treated with 5 U/ml Streptomyces hyaluronidase for 1 h at 60 °C were used as negative controls.
Two observers (NF, KI) without any knowledge of the clinicopathological information evaluated the results of HA and HAS1–3 staining on the basis of a three-point scale: 0 % for positive stainable cell number (negative); 1–20 % (weak); 21–100 % (strong; Fig. 1a). On the basis of these criteria, both observers could analyze and grade the degree of positivity or negativity in all the samples. Since the specimens were heterogeneous in each case, three fields of magnification (magnification 4009) were randomly selected, and the mean positivity of these three fields was considered as representative of each case.
HAS1, HAS2, and HAS3 immunostaining was performed with 22 MPNST samples available since 1997. Not all tissues could be obtained for HAS1–3 immunohistochemistry because of the limited availability of MPNST tissues. Deparaffinized and rehydrated sections were treated with 0.3 % H2O2 in methanol for 30 min, followed by incubation with 1 % BSA in PBS for 1 h at room temperature. Thereafter, sections were incubated for 1 h with polyclonal antibodies against HAS1, HAS2, and HAS3 raised in rabbits by subcutaneous injection of the following synthetic peptides: VRRLCRRRSGGTRVGV, corresponding to amino acids 568–582 of HAS1; CGRRKKGQQYDMVLDV, corresponding to amino acids 537–552 of HAS2; and CGKKPEQYSLAFAEV, corresponding to amino acids 541–555 of HAS3, which had been coupled to keyhole limpet hemacyanin. These antibodies have been used in HAS immunostaining for human tissues in previous studies [24, 25]. Biotinylated anti-rabbit goat IgG (Nichirei, Tokyo, Japan) was applied as a secondary antibody for 30 min at room temperature, and antibody binding was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidine-containing substrate solution (Nichirei, Tokyo, Japan). The slides were counterstained with hematoxylin, dehydrated and then mounted. Stained sections incubated without primary antibodies were used as negative controls.
HA contents in tumor tissues To determine the levels of HA in tumor tissues, frozen samples from 20 patients with MPNST and six with neurofibroma, which were obtained at biopsy or resection and stored at -80 °C, were subjected to quantification with HA binding assay. The digestion of tumor tissues and HA extraction were performed according to the protocol described previously with a minor modification [26]. Briefly, each tissue sample was dried overnight at 60 °C to
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Fig. 1 a Representative images of staining grade for HA and HAS1-3 are shown (original magnification 9400). b Representative images of MPNST with strong HA expression and neurofibroma with negative HA expression (original magnification 9400). Bars depict 100 lm
determine the dry weight, and then dissolved in 2 ml of 0.15 M Tris–HCl, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM deferoxamine mesylate, pH 7.5, containing 40 U of protease for 8 h at 55 °C. Following incubation, all samples were heated at 100 °C for 15 min to inactivate protease activity and centrifuged at 15,000g for 30 min at 4 °C, and the supernatants were extracted. HA concentration was quantitatively determined with HA binding assay, as described previously [27]. Cell culture and quantification of HA The human neurofibroma cell line, Hs53T and the human MPNST cell line, sNF02.2 were obtained from the American
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Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. The cultures were maintained in a humidified atmosphere with 5 % CO2 at 37 °C. Following incubation for 12 and 24 h, HA was isolated according to the methods reported previously [28]. Briefly, the conditioned medium was collected and designated as ‘‘medium.’’ To remove the cell-surface associated HA, the cells were incubated for 10 min at 37 °C with trypsin–EDTA and washed with PBS. The trypsin solution and combined washes were designated as ‘‘pericellular.’’ After cell counts, the cells were placed in Protease K solution (0.15 M Tris– HCl, pH 7.5, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM
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deferoxamine mesylate containing 20 U of protease K) and incubated for 2 h at 55 °C and the solution was designated as ‘‘intracellular.’’ All samples were heated at 100 °C for 15 min to inactivate protease activity and centrifuged at 15,000g for 30 min at 4 °C, and the supernatants were subjected to the analyses for HA content. Because the cell size of sNF02.2 was larger than that of Hs53T, HA concentration was standardized by the amount of protein in each cell type. Following incubation for 12 and 24 h, total protein was extracted with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and protein concentration was determined with Protein Assay Reagent (Bio-Rad, Hercules, CA, USA).
719 Table 2 Expression of HA and HASs in relation to clinical outcome Positive cell fraction (%)
No. of cases
No. of patients with metastasis
No. of deceased patients
HA
30
12
7
Negative (0 %)
6
1
0
Weak (1–20 %)
15
4
3
Strong (21–100 %) HAS1 Negative (0 %) Weak (1–20 %) Strong (21–100 %) HAS2
Statistical methods SPSS 20.0 for Windows software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Fisher’s exact test was used to analyze the relationship of various clinicopathological variables to positivity of HA and HASs. Overall survival period was calculated from the date of the first visit to that of death or final follow-up. Disease-free survival period was calculated from the date of the first visit to that of first event recorded (local recurrence or metastasis). Prognosis of patients with MPNST was determined with Kaplan–Meier method according to the positivity of HA and HAS1–3, as well as each clinicopathological variable, and the differences were statistically analyzed with log-rank test. However, 3 of the 30 patients with MPNST were excluded from the analysis of diseasefree survival because of the presence of metastasis at the first visit. Similarly, 2 of 22 patients with MPNST evaluated for HAS1–3 expressions were excluded from the analysis of disease-free survival. Cox’s regression model was used for multivariate analysis. Quantitative experiments were performed more than three times, and analysis of variance followed by Bonferroni Dunn post hoc test was used to assess differences between means. Statistical comparison between two groups was analyzed with Student’s t test. P values \0.05 were considered statistically significant.
Results
9
7
4
22
10
5
5
3
1
6 11
0 7
0 4 5
22
10
Negative (0 %)
4
1
0
Weak (1–20 %)
5
3
2
Strong (21–100 %) HAS3
13
6
3
22
10
5
Negative (0 %)
7
1
0
Weak (1–20 %)
8
5
3
Strong (21–100 %)
7
4
2
(Fig. 1b). Although HA also accumulated in surrounding stromal tissues in most samples, we focused solely on the positivity associated with tumor cells and extracellular matrix around tumor cells. Staining grade for HA in MPNST samples was negative in six (20 %), weak in 15 (50 %), and strong in nine (30 %; Table 2), while that in neurofibroma samples was negative in four (27 %) and weak in 11 (73 %). No case of neurofibroma showed strong HA expression (Fig. 1b). Dividing three groups into two groups; negative to weak positive and strong positive group, HA positivity was statistically higher in MPNST tissues than in neurofibroma (P = 0.020; Table 3). In the analysis between MPNST and plexiform neurofibroma, HA positivity in MPNST tissues showed a tendency to increase as compared with that in plexiform neurofibroma tissues (P = 0.082). We evaluated the correlation of staining grade with clinicopathological variables in patients with MPNST. No significant correlations were noted between the intensity of HA staining and gender (P = 0.69), age (P = 0.69), NF1 status (P = 0.12), tumor site (P = 1.0), tumor size (P = 1.0), tumor depth (P = 0.13), or histological grade (P = 0.10; Table 4).
HA expression in neurofibroma and MPNST HASs expression in MPNST The distribution of clinicopathological variables did not differ significantly between patients with neurofibroma and those with MPNST. In 15 tissue samples collected from patients with neurofibroma and 30 samples from patients with MPNST, the expression levels of HA were diverse (Fig. 1a). HA positivity was observed in the extra- and pericellular matrix and/or intracytoplasm of tumor cells
HAS1–3 positivity was observed in the cytoplasm and/or on the surface of tumor cells. Of 22 MPNST tissue samples examined for HAS1–3 expression, 11 (50 %) showed strong HAS1 expression, 13 (59 %) strong HAS2 expression, and 7 (32 %) strong HAS3 expression. The relationship between HAS expression and clinical outcome is
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Table 3 Expression of HA in neurofibroma and MPNST HA positivity
P value
Negative–weak
Strong
Neurofibroma
15
0
MPNST
21
9
a
Table 5 Univariate analysis of overall and disease-free survival a
Variables
5-Year survival (%) 0.020
Negative–weak Total
30
21
Strong
74
\45
94
C45
69
0.69 14
9
5
Male
16
12
4
16
12
4
C45 NF1 status
14
9
5
Negative
14
12
2
Positive
16
9
7
Extremity
16
11
5
Trunk
14
10
4
Age
0.69
0.12
Tumor site
1.0
Tumor size
1.0
Negative
100
Positive
67
Extremity
81
Trunk
85
B10 cm
95
[10 cm
56
Subcutaneous
100
Low
100
High
72
[10 cm
8
6
2
Strong
59
Unknown
3
62
0.67
68 0.019
85
0.025
42 0.66
66
0.77
64 0.02
79
0.001
0 0.17
83
0.23
56 0.034
91
0.032
47 0.041
76
0.019
22
HAS1 expression 0.13
6
6
0
Negative– weak
90
Subcutaneous Deep tissues
20
12
8
Strong
80
0.33
70
0.26
40
HAS2 expression
4
Negative– weak
0.10 10
1
11
8
Fisher’s exact test
shown in Table 2. In the analyses of the correlation between the intensity of HAS1–3 expression and clinicopathological variables, HAS1 was associated with age (C45 years, P = 0.019), and HAS2 and HAS3 with histological grade (P = 0.041 and P = 0.040, respectively). Relationship between stainability and survival of patients with MPNST Of 30 patients with MPNST, three developed recurrent disease and 12 metastasis. At the last follow-up, 18 patients remained continuously disease-free or with no evidence of
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0.048
HA expression 91
19
0.77
63
Deep tissues 80 Histological grade
Negative– weak
High
68
Tumor depth
6
Unknown
0.21
Tumor size
13
Histological grade Low 11
P valuea
Tumor site
19
Tumor depth
a
P valuea
9
Female
B10 cm
5-Year survival (%)
NF1 status
Gender
\45
92
Male Age
Table 4 Correlation of HA expression with clinicopathological variables in patients with MPNST HA staining
P value
Disease-free survival a
Gender Female
Fisher’s exact test
No.
Overall survival
Strong
100
0.38
74
56
0.96
58
HAS3 expression Negative– weak Strong a
93 73
0.62
55
0.84
63
Log-rank test
disease, seven died of disease, and the remaining five were alive with disease. All patients who died of disease had developed distant metastasis. For patients with MPNST, overall survival and diseasefree survival at 5 years were 83 and 65 %, respectively. Strong HA expression was seen in four patients (57 %) with dead of disease and 7 (58 %) with distant metastasis. Overall survival at 5 years was 59 % in patients with
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Fig. 2 Kaplan–Meier survival curves of overall survival for HA and HAS 1–3 expressions in MPNST. a Subdivided according to HA expression (strong vs. negative-weak; N = 30). b Subdivided according to HAS1 expression (N = 22). c Subdivided according to HAS2 expression (N = 22). d Subdivided according to HAS3 expression (N = 22)
strong HA expression and 91 % in those with weaker HA expression, and disease-free survival at 5 years for these patients was 22 and 76 %, respectively. Univariate analysis revealed that strong HA expression was significantly associated with decreased overall survival (P = 0.041) and disease-free survival (P = 0.019) using log-rank test. However, HAS expression was related to neither overall survival nor disease-free survival in patients with MPNST (Table 5; Figs. 2, 3). In the multivariate analysis, strong HA expression (P = 0.028) was still an independent prognostic factor for disease-free survival. Strong HA expression and poor overall survival in patients with MPNST showed a trend to correlate (P = 0.089; Table 6). In the univariate analysis of clinicopathological variables, age (P = 0.048), NF1 status (P = 0.019), tumor size (P = 0.020), and histological grade (P = 0.034) were found to be significant prognostic factors for poorer overall survival, while NF1 status (P = 0.025), tumor size (P = 0.001), and histological grade (P = 0.032) were significantly correlated with disease-free survival. There was no statistically significant association between overall and disease-free survival and variables such as gender, tumor site, or tumor depth (Table 5). In the multivariate analysis, large tumor size was independently related to reduced overall survival and disease-free survival (P = 0.022 and P = 0.002, respectively; Table 6).
Quantification of HA We used the HA binding assay to measure HA levels (lg/g) in the extracts from neurofibroma and MPNST tissue samples. The mean HA concentrations in neurofibroma and MPNST tissues were 238.2 and 275.4 lg/g, respectively. There was no significant difference between the HA levels in neurofibroma tissues and those in MPNST tissues (P = 0.750; Fig. 4). The levels of HA in pericellular region and in medium in sNF02.2 cells incubated for 24 h were significantly higher than those in Hs53T cells (P = 0.002 and P = 0.001, respectively; Fig. 5a, b). The levels of intracellular HA in sNF02.2 cells were significantly higher than that in Hs53T cells at 12 and 24 h (P = 0.008 and P \ 0.001, respectively; Fig. 5c).
Discussion Our results demonstrated for the first time the differential expression of HA between MPNST and neurofibroma, and showed that increased HA expression in tissues correlates with unfavorable survival outcome in patients with MPNST. In contrast, HAS1–3 expressions were not related to the prognosis of patients with MPNST.
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Clin Exp Metastasis (2014) 31:715–725
Fig. 3 Kaplan–Meier survival curves of disease-free survival for HA and HAS 1–3 expressions in MPNST. a Subdivided according to HA expression (strong vs. negativeweak; N = 27). b Subdivided according to HAS1 expression (N = 20). c Subdivided according to HAS2 expression (N = 20). d Subdivided according to HAS3 expression (N = 20)
Table 6 Multivariate analysis of overall and disease-free survival
Variables
Overall survival HR
Disease-free survival a
HR
95 % CI
P value
1.35–46.8
0.022
7.94
0.79–26.9
0.089
5.65
95 % CI
P valuea
2.07–30.4
0.002
1.21–26.4
0.028
Tumor size B10 cm
1
[10 cm
7.96
1
HA expression
a
Cox’s regression model
Negative–weak
1
Strong
4.61
HA is known to play an important role in the tumorigenicity of malignant tumors. Previous studies revealed that higher expression of HA is significantly associated with poor prognosis in many types of human tumor [12–17], suggesting agreement regarding HA as a crucial prognostic factor. However, few reports have analyzed the significance of HA in mesenchymal malignancies [22]. On the other hand, few studies have reported the correlation between HAS expression in clinical tumor samples and poor patient prognosis. HAS1 expression is known to serve as a prognostic indicator in some tumors, including malignant mesothelioma and ovarian cancer [24, 29]. In specimens of colon cancer, Yamada et al. [30] examined mRNA levels of three HASs, and showed a positive correlation between an increased HAS1 mRNA level and poor patient prognosis. Nykopp et al. [31] investigated the hyaluronidase expression in addition to HASs in endometrioid endometrial
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1
cancer, and demonstrated that the accumulation of HA was influenced by not only HAS expression but also hyaluronidase expression. Complicated regulation mediated by both HASs and hyaluronidases acts to control HA accumulation in malignant tissues, but post-translational modification of HASs has not been clarified yet. The current study showed no association of HAS expression with survival of patients with MPNST possibly due to the yet-to-be-defined regulation mechanisms of HASs. Together, evaluation of HAS expression is complicated and not adequate by itself to identify patients having a poor prognosis. Evaluation of HA positivity in MPNST tissue samples is a readily available and useful modality to identify patients with poor survival. Recently, Slomiany et al. [32] investigated the roles of HA on MPNST in vitro and in vivo. In the study, HA oligomers, which inhibited HA production, suppressed HA secretion in MPNST cells, decreased doxorubicin resistance of MPNST,
Clin Exp Metastasis (2014) 31:715–725
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and suppressed the growth of MPNST xenografts in mice, which suggested the importance of HA in tumorigenicity of MPNST. This previous report supports our findings that increased HA expression in MPNST tissues correlated with poor prognosis in patients with MPNST. Mechanisms of malignant transformation of benign neurofibroma to MPNST are not fully understood at the molecular level. Because of the dismal prognosis of patients with MPNST, it is important to identify them at an early stage, especially patients with NF1, as well as those with localized MPNST who are at high risk for metastatic development. Occasionally, it is difficult pathologically to differentiate neurofibroma with atypical histological features from MPNST [5]. It is also difficult to predict the clinical course of patients with MPNST. Therefore, useful
markers are required to detect MPNST at an early stage so as to avoid delaying treatment, and to alter the treatment modality for patients with a poor prognosis. Previous reports have demonstrated that several proteins can differentiate patients with neurofibroma from those with MPNST. Wasa et al. [33] showed that positive immunostaining for vascular endothelial growth factor (VEGF) was significantly higher in MPNST tissues compared with those of neurofibroma. Zou et al. [34] reported that the mean staining intensity of p53 could significantly differentiate MPNST from neurofibroma in tissue microarray analysis. In our study, HA expression was significantly higher in patients with MPNST than in those with neurofibroma, and no patients with neurofibroma showed strong HA expression. This finding suggests that HA expression may be a useful tool to differentiate MPNST from neurofibroma, particularly low grade MPNST from neurofibroma. In vitro experiment of our study, the levels of intracellular, pericellular, and medium HA in human MPNST cells were significantly higher than those in human neurofibroma cells. This in vitro findings supports our results of HA positivity that can differentiate MPNST from neurofibroma. In the present study, however, we could obtain only Hs53T cells as commercially available neurofibroma cells. Some previous reports showed that HA content in plexiform neurofibroma tissues was increased compared to those in cutaneous neurofibroma tissues [35, 36]. However, there was no study evaluated HA content in MPNST tissues. In the current study, HA content in MPNST tissues was higher than that in plexiform neurofibroma tissues, but it was not statistically significant. One possible explanation was that tumorous tissues contain not only tumor cells but stromal cells, which may affect HA content. Other explanation is the small number of cases of neurofibroma tissues. A number of studies have analyzed possible prognostic factors of the clinical outcome of patients with MPNST [6, 33, 34, 37–41]. In these studies, clinicopathological factors
Fig. 5 Data presented are the HA contents in Hs53T and sNF02.2 cells standardized with the content of protein in these cells (ng/lg). HA contents were measured at 12 and 24 h time points of cell culture. a HA concentration in medium. b Pericellular HA concentration.
c Intracellular HA concentration. Each experiment was performed in triplicate, and bars indicate the mean ± SD (*P \ 0.01 and **P \ 0.001 compared with Hs53T cells as measured using Bonferroni–Dunn post hoc test)
Fig. 4 HA concentration in neurofibroma and MPNST tissues. The mean value HA concentration for neurofibroma is 238.2 lg/g (N = 6), and that for MPNST is 275.4 lg/g (N = 20). The level of HA in tissue samples did not differ significantly between neurofibroma and MPNST (P = 0.750, Student’s t test). Each experiment was measured in triplicate, and bars indicate the mean ± SD
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including older age, large tumor size, axial tumor site, inadequate surgical margin, NF1 status, local recurrence, previous radiotherapy, and tumor histological grade were linked with poor MPNST patient prognosis. In the current study, we reviewed the clinical records of 30 patients with MPNST to investigate the prognostic impact of various clinicopathological features. Univariate analysis indicated that age, NF1 status, tumor size and histological grade were prognostic factors for poor survival, while of these four factors, only tumor size remained an independent prognostic factor in multivariate analysis. Thus tumor size seems to be the most important risk factor for survival in MPNST, which is consistent with previous studies demonstrating that it was significantly associated with poor survival in MPNST. [6, 34, 37, 39–41]. Zou et al. [34] reported in a study of 140 patients that those with tumors larger than 10 cm carried a threefold increased risk of developing distant metastasis. Although the cutoff values of tumor size in these studies were variable, ranging from 5 to 10 cm, tumor size was found to be the most reliable and uncontroversial predictor of prognosis in patients with MPNST. However, a biomarker other than clinical variables would also be necessary to better predict the prognosis of patients with MPNST. There were several limitations in our study. First, the number of patients was small due to the rarity of MPNST, and it may have been underpowered to detect significant differences between groups. For instance, positivity of HA was significantly increased in MPNST compared with all neurofibromas, however, not significantly increased compared with plexiform neurofibroma (P = 0.082). Further studies with greater numbers of patients will be needed to better clarify the correlation of HA with patient prognosis. Second, there was a relatively high rate (37 %) of low grade MPNST in this study compared with that in previous studies (18–22 %) [6, 40]. In our institution, routine magnetic resonance imaging examination for neurofibroma in patients with NF1 is thought to facilitate the early detection of malignant transformation of the tumors as reflected by the high percentage of low grade MPNST detected. Third, the results of staining may be influenced by both the sensitivity of the antibody and protocol. However, the results of staining should be stable if performed with the same antibody and protocol in a single institution. In conclusion, HA expression in tissues of patients with MPNST is easily evaluated in the clinical setting using histochemical staining compared with the evaluation of multiple HAS expression. Our results indicate that HA expression may be a useful marker in differentiating MPNST from neurofibroma, and in identifying MPNST patients having a poor prognosis. HA-targeting therapy for patients with MPNST may have potential as a therapeutic tool, and accordingly warrants further investigation.
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Clin Exp Metastasis (2014) 31:715–725 Acknowledgments We thank Ms. Eri Ishihara for secretarial assistance regarding this study. We are grateful to Drs. Satoshi Tsukushi, and Junji Wasa for collection of samples and data. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid 20591751 for Scientific Research (C)], and by the Suzuken Memorial Foundation. Conflict of interest of interest.
The authors declare that they have no conflict
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